Process of feed batch fermentation at high cell density for recombinant protein production

FIELD: food industry.

SUBSTANCE: described are version methods for production of recombinant meningococcal Proteins-2086 by way of feed batch fermentation with a bacterial culture with continuous supply of an inducer in a total quantity from approximately 7 g/l to approximately 15 g/l after the threshold parameter has been reached with continuous supply of a carbon source such as supply at a constant rate with subsequent meningococcal protein extraction from the bacterial culture. The cultivation method may be implemented using a bacterial host cell containing an expression vector encoding recombinant protein under control of an induced promoter or by means of a nucleic acid sequence with continuous supply of a carbon source (such as glucose) and an inducer (such as arabinose). Described are compositions for meningococcal protein extraction from the bacterial culture, produced by the said methods and noted for high Protein-2086 density. The protein may be lipidised or non-lipidised.

EFFECT: meningococcal Proteins-2086 yield improvement.

79 cl, 20 dwg, 7 tbl, 3 ex

 

Related applications

[0001] This application claims priority under provisional patent application U.S. serial number 60/833,479, entitled PROCESS PERIODIC fermentation of FEED WITH a HIGH DENSITY of CELLS TO OBTAIN a RECOMBINANT PROTEIN, filed July 27, 2006, the description of which is fully incorporated into this application by reference.

The scope of the invention

[0002] the Present invention relates in General to new ways of periodic fermentation feed, which provides increased expression of the protein in bacterial systems, as well as to compositions with a high density of proteins and compositions, which are used in new ways periodic fermentation with water.

The level of technology

[0003] Various strategies fermentation is used to obtain proteins in sufficient quantities for laboratory, clinical or commercial use. Periodic fermentation injection is used to provide an increased yield of protein compared with those, which provide simple ways of periodic fermentation. Periodic fermentation injection is a process in which the initial periodic phase follows the phase in which one or more nutrients are fed into the culture by feeding.

[0004] Usually, in rematerializes phase, cells are first grown to the desired concentration. In this phase the growth of cells increases, and, usually, the target protein is not produced until then, until you add an inductor, such as arabinose, lactose or isopropyl-beta-D-thiogalactoside (IPTG), depending on the promoter, or is produced, if there is some incomplete blocking of the promoter. During phase with water, a carbon source and other required nutrients is usually served in a fermenter in a relatively concentrated stream of fluid at a certain feed rate. Once achieved the target cell density, feed type inductor or inductor and other nutrients. In this phase the focus is on production of the protein produced by the cells. The substrate (namely nutrients and inductor), which is fed into the fermenter at this stage, apply, as a rule, for cell growth and synthesis of the product. Cell growth is controlled by the feed speed for optimal cell growth and production of the protein. During the stage production of the protein need to add an inductor to recombinant organisms.

[0005] expression of the protein in the environment, including a common source of carbon, such as glucose, or other source of carbon-based sugars and the inductor, satisfactory to the occurrence limiting at the conditions at the end of phase with water. Examples of limiting conditions include reduced concentration of oxygen, the reduction of nutrients, such as vitamins, carbon, nitrogen, and accumulation of toxic compounds in the growth environment.

[0006] the strategy of periodic fermentation feed often include various forms of regulation with feedback, including indirect and direct feedback to control the supply of nutrients. One such method periodic fermentation feed includes applying a control algorithm with feedback through the supply of nutrients to support the process parameter at a certain specified value. For example, a direct ow control can be based on measurement of the concentration of nutrients. Regulation with feedback then directly related to the activity of the cells during fermentation. The control parameters that were used for the regulation of fermentation with feedback, include pH value, measured in real-time cell density or pressure of dissolved oxygen (DOT).

[0007] However, the use of algorithms feedback accompanied by a number of disadvantages. One such drawback is the dependence of the rate of feed from the current process parameters. Any violation of the process can affect this parameter, thereby altering the soon the be the submission and the final yield of protein. These shortcomings increase when scaling process to obtain high amounts of protein.

[0008] Another disadvantage of the previously used strategy with makeup is that when using the regulation with feedback specific growth rate it is impossible to predict or control, which leads to suboptimal output processes in which the formation of the product depends on growth.

[0009] Additionally, when the flow of carbon (for example, when high concentrations of glucose) in the main metabolic pathway exceeds the maximum power of the tricarboxylic acid cycle (TCA)can be accumulated by-products. The accumulation of by-products may inhibit the growth of cells and production of protein in the fermentation process.

[0010] Additionally, various shortcomings of the periodic fermentation with water often lead to inefficient use of components of the nutrients. For this reason, these methods can be economically disadvantageous, in particular for large-scale commercial production of the protein.

[0011] previously Used approaches to recombinant protein expression through periodic fermentation with water as described above have various disadvantages. Given the importance of cost-effective obtain a sufficient amount is in protein for a variety of purposes, there is a need for an effective method periodic fermentation with water, which would lead to a higher cell growth, increased formation of product (i.e. a higher yield of protein) and reduced the accumulation of by-product.

Brief description of the invention

[0012] the Present invention relates to new ways of periodic fermentation with water to obtain an unexpectedly high yield of recombinant protein.

[0013] a Certain embodiment of the present invention provides a method of obtaining a recombinant protein, comprising culturing the recombinant bacterial cells for the expression of recombinant proteins, including continuous addition of carbon source in the culture containing the indicated recombinant bacterial cells, and the continuous addition of the inducer in the specified culture after culture has reached a threshold parameter, and the selection of the indicated recombinant protein from cell culture.

[0014] an additional embodiment of the present invention provides a method of obtaining a recombinant protein, comprising: (a) introducing into a bacterial cell by the host expression vector encoding a recombinant protein under the control of the inducible promoter, to obtain a recombinant bacterial CL the TCA; (b) the introduction of the indicated recombinant bacterial cells in culture medium to obtain a cell culture; (C) adding a carbon source into the specified cell culture in the form of a continuous feed; (a) monitoring the achievement of the growth of the cells in said cell culture threshold optical density (OD600); (e) adding an inductor specified inducible promoter in the indicated cell culture in the form of a continuous feed, once reached the specified threshold optical density (OD600); and (f) obtaining a recombinant protein from cell culture.

[0015] Another additional embodiment of the present invention provides a method of obtaining a recombinant protein, comprising culturing the recombinant bacterial cells for expression of recombinant protein by continuous addition of inducer to the culture, including specified bacterial cells after the indicated culture reached a threshold parameter, characterized in that the said bacterial cell comprises a sequence of nucleic acids, the corresponding gene from N. meningitidis sero-group C.

[0016] According to a similar additional implementation variant, the present invention provides a method of obtaining a recombinant protein 2086 (rP2086), including: (a) introduction to bacterial the th cell-host expression vector, encoding recombinant meningococcal 2086 protein under the control of the inducible promoter for the production of recombinant bacterial cells; (b) the introduction of the indicated recombinant bacterial cells in culture medium to obtain a culture; (C) adding a carbon source to the culture; (d) monitoring the achievement of the growth of the cells in the specified culture threshold optical density (OD); (e) continuous addition of inducer specified inducible promoter in the indicated culture, as soon as the density of cells in a given culture reached an optical density of approximately from 70 to 110; and (f) the receipt of the indicated recombinant meningococcal 2086 protein from the specified culture through the period of time from about 3 hours to about 6 hours after the start of continuous addition of inducer.

[0017] According to another implementation variant, the present invention provides a composition comprising a bacterial culture comprising a recombinant protein 2086 (rP2086) at a density equal to at least about 1.5 g/l in the total amount specified bacterial culture.

[0018] According to still another implementation variant, the present invention provides a composition comprising a bacterial culture medium, including recombinant meningococcal protein 2086 (rP2086), p is obtained according to the methods of the present invention.

Brief description of drawings

[0019] Figure 1 - periodic fermentation with water at various constant flow rates without induction.

[0020] Figure 2 - periodic fermentation with water at various constant flow rates without induction.

[0021] Figure 3 - induction at different optical densities.

[0022] Figure 4 - induction of different levels of arabinose.

[0023] Figure 5 - influence of the method of adding arabinose to the output rLP2086.

[0024]. Figure 6 - influence of feed rate arabinose products rLP2086.

[0025] Figure 7 - influence of time of induction on the expression.

[0026] Figure 8 is a periodic fermentation with water to obtain rLP2086 subfamily Century

[0027] Figures 9A and 9B - analysis by electrophoresis in polyacrylamide gel with sodium dodecyl sulfate (SDS page/LTOs) and analysis by Western-blot induction rLP2086 subfamily b respectively.

[0028] Figure 10 is a periodic fermentation with water to obtain rLP2086 subfamily A.

[0029] Figures 11A and 11B - PAG/LTOs and analysis Western blot induction rLP2086 subfamily a, respectively.

[0030] Figure 12A, 12B and 12C - simultaneous supply of glucose and arabinose in the process of induction.

[0031] Figure 13A - periodic fermentation of E. coli with water to obtain rLP2086 subfamily In at 100 HP

[0032] Figure 13B is a periodic fermentation of E. coli with p is detcog to get rLP2086 subfamily And at 100 HP

[0033] Figure 14A - periodic fermentation of E. coli with water to obtain rIP2086 subfamilies In with the simultaneous supply of glucose and arabinose at 100 HP

[0034] Figure 14B is a periodic fermentation of E. coli with water to obtain rIP2086 subfamilies And with simultaneous supply of glucose and arabinose at 100 HP

Detailed description of the invention

[0035] These methods of the present invention is based on the unexpected discovery that was unexpectedly obtained a high yield of protein using periodic fermentation of feed in the continuous supply of the inductor in the process of induction culture medium. Perhaps the source of carbon is continuously applied before and/or during the continuous feed of the inductor. Influenced by arabinose received approximately 2-3 g/l recombinant lipoprotein 2086 (rLP2086) (which is expressed by the microorganism and has the sequence corresponding to the 2086 gene from N. meningitidis sero-group b) in accordance with some variant of realization of the present invention. This represents approximately 2-3-fold increase in the yield of rLP2086 using periodic fermentation of feed for both subfamilies a and b specified 2086 protein compared with the comparative process periodic fermentation. Moreover, these methods of the present invention can easily consider in order to beat to obtain on a commercial scale of these and other proteins.

[0036] To improve understanding of these implementation options described in this application will be made with reference to various implementations and will be used specific language to describe this. The terminology used in this application is solely for the purpose of describing a specific implementation options and is not intended to limit the scope of the present invention. As used throughout this description, all singular forms include reference to the plural form, unless the context clearly indicates to the contrary. Similarly, singular form of terms such as "environment"include reference to the plural form of "medium", and Vice versa. Thus, for example, reference to "a certain cultural environment" includes many of these environments, as well as one medium; and a reference to "culture medium" includes a wide range of environments, as well as one environment.

[0037] the Term "coil"as used in this application, refers to any agent that induces, enhances or induces the expression of a recombinant protein, resulting in the expression of genes under the control of the inducible promoter can be directly controlled via the concentration of this agent.

[0038] the Term "carbon source", as used in this application refers to the source of carbon and energy for the cells.

[0039] the Terms "pitch", "is", "feeding" or "continuous addition", as used interchangeably in this application, refer to adding substances continuously for some period of time, rather than adding the entire amount at one time. These terms imply a single beginning and/or end or the set of points of the start and/or stop for the continuous addition of the indicated substances in the fermentation process.

[0040] the Term "recombinant protein"as used in this application, refers to any protein or biologically active portion thereof (e.g., the part which retains the biological activity of the whole protein), which is not a reporter or marker gene (e.g., green fluorescent protein), expressed from recombinant genetic material that encodes amino acids, including peptides, polypeptides, proteins, oligopotent and/or fused proteins. Recombinant protein product may include therapeutic, prophylactic or diagnostic product.

The methods of the present invention

[0041] These methods of the present invention provide unexpectedly high yield of protein through a new process fermentation of feed, including continuous addition of inducer, such as arabinose in the culture medium after TRG is, as this culture reached a threshold parameter. A carbon source such as glucose, are usually added to the culture comprising a recombinant bacterial cell, before phase induction. Specified source of carbon can be submitted together with the inductor. The specified inductor can also serve as a secondary carbon source.

[0042] the carbon Source such as glucose, continuously add to the specified culture medium before and/or during the continuous feed of the specified inducer in the culture medium, in accordance with some variant of realization of the present invention. Thus, the continuous supply of the carbon source is covered with a continuous flow of inductor, according to some implementation variant. The continuous supply of the carbon source may continue during the entire process continuous feed inductor, or only during part (s) of this process. In another implementation, the continuous supply of carbon source does not overlap with a continuous flow of the inductor. According to some implementation variant of the present invention, the specified inductor and/or a carbon source can be supplied to the culture at a constant speed.

[0043] the process of fermentation with water involves several steps, resulting in the production of the necessary is imago protein in accordance with some variant of realization of the present invention. At the initial stage get the expression vector, which encodes a recombinant protein under the control of the inducible promoter, and then injected into the bacterial cell host. Specified bacterial cell host is injected into the culture medium. The inductor inducible promoter serves in culture (namely, the inductor is added in culture continuously for some period of time). The specified inductor can be fed to the culture at a constant speed. Then the indicated recombinant protein product is collected from the specified culture. Recombinant protein obtained in this way can then be cleaned as necessary and/or to use any suitable manner, such as in prophylactic, therapeutic or diagnostic pharmaceutical form.

[0044] the High density of cells and increased protein yield were unexpectedly achieved through periodic fermentation with water at a constant feed rate of the inductor, which provides the output of the recombinant protein product is approximately 2-3 times higher compared with periodic fermentation, as illustrated in the examples below. The methods of the present invention is applicable to large-scale fermentation, as well as small-scale fermentation. "Large-scale fermentation, as used in the Noi application refers to fermentation in the fermenter, which is at least approximately 1000 l volume capacity, namely the working volume, leaving enough space for the availability of space above the product. "Small-scale" fermentation is usually to fermentation in a fermenter, which usually has not more than approximately 100 l volume capacity, such as 5 l, 10 l, 50 l or 100 l of Proven advantage of this fermentation process with makeup is that it can be used to obtain the recombinant protein product in the scale fermentor 5-10 l and can be scaled to any size, for example, 100 l, 150 l, 250 l, 500 l, 1000 l or more, without restrictions.

Inductors

[0045] the Methods described in this application relate to the production of recombinant protein, wherein expression of the indicated recombinant protein is under the transcriptional control of the inducible promoter, resulting in expression of genes under control of the specified inducible promoter is directly regulated by the concentration of the inducer present in the culture medium. The specified inductor is fed continuously to the culture medium, possibly at a constant speed. The specified inductor added to the culture medium, as soon as you reach the threshold parameter. N the example, recombinant protein may be under the control of the araB promoter (for example, ParaB), which can directly regulate the concentration of arabinose, which is added at constant speed in the culture medium. Suitable inductors for use in accordance with the present invention are well known to specialists in this field. Examples of inductors according to the present invention are given below, without restriction.

The promoterThe inductor
Arabinosyl promoter, such as ParaBArabinose
Inhibitor-1 plasminogen activator human Hpai-1The tumor necrosis factor,
TNF
Cytochrome P-450Toxins
CYP1A1 element responsible for the induction of glucocorticoid metal, MOE glandHeavy metals virus breast tumor mouse
CollagenaseHerbology ether
StromelysinHerbology ether
SV40Formolo the initial broadcast
ProliferinHerbology ether
α-2-macroglobulinIL-6
MX gene mouse NewcastleInterferon, a virus disease
VimentinSerum
Gene thyrostimulin hormone αThyroid hormone
HSP70Ela, large T-antigen SV40
The tumor necrosis factorFMA
InterferonViral infection, dnrc
SomatostatinCyclic AMP
FibronectinCyclic AMP
lac promoter/operatorIPTG

The carbon source

[0046] Any suitable carbon source such as glycerol, succinate, lactate, or a source of carbon-based sugars, such as glucose, lactose, sucrose and fructose, is provided for use in the present invention, which should be obvious to a person skilled in this field. For example, the R, sources of carbon-based sugars that can be used in the present invention include, without limitation, branched or unbranched polysaccharides which comprise monomers of sugars D-mannose, D - and L-galactose, fucose, fructose, D-xylose, L-arabinose, D-glucuronic acid, sialic acid, D-galacturonic acid, D-mannurone acid (for example, polymannuronic acid, or alginic acid), D-glucosamine, D-galactosamine, D-glucose and Narimanovo acid, including homopolysaccharides and heteropolysaccharides such as lactose, amylopectin, starch, hydroxyethyloxy starch, amylose, dextran sulfate, dextran, dextrins, glycogen, or the polysaccharide subunit of acid mucopolysaccharides, e.g. hyaluronic acid; polymers of sugar alcohols such as Polysorbate and pelimannit; heparin or heparin; or any combination thereof, without limitation. Glucose is the primary source of carbon according to some implementation variant of the present invention. Arabinose, if used as an inductor, can also serve as a secondary carbon source, although it can also be a primary source of carbon. According to some implementation variant, these carbon sources include any of D-glucose, L-arabinose, sucrose, I-Inositol, D-mannitol, β-D-fructose, α-L RA is nosy, D-xylose, cellulose, or any combination of them. One or more than one carbon source can be used in the present invention.

Bacterial expression system and plasmids

[0047] the Present invention also provides a recombinant bacterial cell comprising the expression vector such as a plasmid comprising a sequence controlling the expression with promoter sequence and initiation sequence and a nucleotide sequence that encodes the desired polypeptide, the nucleotide sequence is located 3' side of the promoter and initiation sequences. Provided by any suitable sequence controlling the expression, and a host cell/vector genetic material that should be obvious to the person skilled in the art based on the description given in this application.

[0048] Suitable sequence controlling the expression, and the combination of host cells/carriers of genetic material well known in this field and is described as an example in Sambrook, J., E.F.Fritsch, and .Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, cold spring Harbor, new York. Generally, methods of recombinant DNA include obtaining through synthesis or selection consistently the t DNA which encodes the recombinant protein of interest, and its introduction into a suitable expression system vector/a host cell where it is expressed, preferably under the control of the induced arabinose promoter. Any of these methods for inserting DNA into an expression vector, can be used for ligating the promoter and other regulatory elements at specific sites within the selected recombinant vector. Suitable cell host and then transform, infect, transducers or transferout such vectors or plasmids using traditional methods.

[0049] Many systems a host cell-vector (plasmid) can be used for expression of the recombinant protein of interest. The specified vector system, such as, for example, the system comprising a promoter induced by arabinose compatible with your cell host. DNA encoding interested recombinant protein product was inserted into the expression system, and a promoter (preferably a promoter induced by arabinose) and other regulatory elements ligated into specific sites within the specified vector in such a way that when the vector is introduced into a cell of the host by transformation, transduction or transfection, depending on the system used a host cell - vector), DNA coding for the maintenance of interest recombinant protein product is expressed by the cell-master.

[0050] the Vector may be selected from one of the viral vectors or non-viral vectors described above, but it must be compatible with the cell owner. Recombinant DNA vector can be entered in the appropriate cell hosts (bacteria, virus, yeast, mammalian cells or the like) by transformation, transduction or transfection and so on (depending on system vector/cell - host). System host-vector include, but are not limited to the above, the bacterium transformed by the DNA of the bacteriophage, plasmid DNA or kosmidou DNA.

[0051] the Expression in prokaryotes of interest recombinant protein product can be maintained in any suitable form or strain of bacteria, such as E. coli, using vectors comprising constitutive or inducible promoters directing the expression of either merged or Nikitich proteins.

[0052] the Hybrid vectors (for slit proteins) add a number of amino acids coded to them protein, amino or carboxyl end of the recombinant protein. Such hybrid vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to facilitate purification of recombinant protein by acting as a ligand ol the affinity purification. Often, a hybrid expression vectors enter the site of proteolytic cleavage at the junction of the recombinant protein and merged with it in molecule to facilitate separation of the recombinant protein from the slit with him molecules after cleaning the slit protein. Such enzymes and their sequence cognatio recognition include factor XA, thrombin and enterokinase.

[0053] a Typical hybrid expression vectors include Pgex (Pharmacia Biotech Inc; Smith and Johnson, 1988), Pmal (New England Biolabs, Beverly, mA) and Prit5 (Pharmacia, Piscataway, new Jersey), which attach to the glutathione-6-transferase (GST), maltose E binding protein, or protein a, respectively, to the target recombinant protein.

[0054] Examples of suitable inducible non-hybrid expression vectors in E. coli include pTrc (Amann and others (1988) Tightly regulated tac promoter vectors useful for the expression of unfused and fused proteins in Escherichia coli, Gene, 69, 301-315)and Pet lid (Studier and others (1990) Use of T7 RNA polymerase to direct expression of cloned genes, Methods in Enzymology, 185, 60-89). The expression of the target gene from the pTrc vector relies on transcription by RNA polymerase of the host with a hybrid trp-lac promoter fused. The expression of the target gene with the vector Pet lid relies on transcription from a T7 gn1 0-merged lac promoter, mediated jointly expressed viral RNA polymerase J7 gnl. This viral polymerase is provided by the host strains BL21 (DE3) or HMS I 74(DE3) present in them n is ofage, carrying the gene for T7 gnl under the transcriptional control of the lacUV 5 promoter.

[0055] the Regulatory sequence of the vector construct is induced promoter, according to some implementation variant. The use of inducible promoter will allow the cell to produce activated protein on a low basic level, in the ordinary course of cultivation and breeding. Subsequently, cells can be induced for expression of large quantities of the desired protein in the process of production or screening. Inducible promoter can be isolated from the genome of the cell or virus.

[0056] Inducible promoters that are regulated by exogenously applied compounds include, without limitation, arabinosyl promoter, the zinc-inducible promoter of metallothionein sheep (MT), dexamethasone (Dex)-inducible promoter of the virus mammary tumor mice (MMTV), promotor system T7 polymerase (WO 98/10088); the promoter ecdysone insects (No and others, 1996 Proc. Natl. Acad. Sci. USA, 93:3346-3351), tetracycline-repressed system (Gossen and others, 1992 Proc. Natl. Acad. Sci. USA, 89:5547-5551), the tetracycline-inducible system (Gossen and others, 1995 Science 268:1766-1769; see also Harvey and others, 1998 Curr. Opin. Chem Biol, 2:512-518), RU486-inducible system (Wang and others, 1997 Nat. Biotech., 15:239-243 and Wang and others, 1997, Gene Ther., 4:432-441) and the rapamycin-inducible system (Magari and others, 1997, J. Clin. Invest., 100:2865-2872). According to some implementation variant to the present invention, specified by the promoter is a promoter induced by arabinose.

[0057] Any suitable bacterial a host cell is provided for use within the present invention that should be obvious to the person skilled in the art based on the description given in this application. For example, suitable bacteria for these purposes include Escherichia, Enterobacter, Azotobacter, Erwinia, Bacillus, Pseudomonas, Klebsiella, Proteus, Salmonella, Serratia, Shigella, Rhizobia, Vitreoscilla, and Paracoccus, or combination thereof, without limitation. Any suitable strain of any such right bacteria also intended by the present invention. Additionally, the use of suitable mutant cells, which should be obvious to a person skilled in this field, it is also intended by the present invention. The person skilled in the art will easily be able to select a suitable cell host for use in specific circumstances, based on the guidance provided in this application.

[0058] Examples of suitable inducible expression vectors in E. coli include, without limitation, pTrc (Amann and others, 1988, Gene, 69:301-315), arabinose expression vectors (for example, Pbad18, Guzman and others, 1995 J. Bacteriol., 177:4121-4130) and pETIId (Studier and others, 1990 Methods in Enzymology, 185:60-89). The expression of the target gene with the specified pTrc vector relies on transcription by RNA polymerase of the host with a hybrid trp-lac promoter fused. Expre is this the target gene with the specified pETIId vector relies on transcription from a T7 gn10-lac promoter fused, mediated jointly expressed by the viral RNA polymerase, T7 gn1. This viral polymerase is provided by the host strains BL21 (DE3) or HMS I 74(DE3) present in them prophage carrying the gene for T7 gn1 under the transcriptional control of the lacUV5 promoter. PBADthe system relies on induced arabinosyl a promoter that is regulated by the araC gene. The specified promoter is induced in the presence of arabinose.

[0059] In other embodiments of the present invention are adjustable arabinose expression vectors or vectors in which expression of the recombinant protein of interest is under the control of arabinose promoter such as the promoter for arabinose operon of E. coli, PBADor PARAwithout limitation.

[0060] the Sequence of nucleic acids (nucleotides)encoding any desired protein provided by the present invention. The nucleotide sequence may be fully or partially naturally occurring nucleotide sequence or a fully or partially modified nucleotide sequence, or any sequence that's hybrid it under strict conditions. References in this application to nucleic acid sequences that correspond to a gene, refers to any sequence of nukleinovihkh, expressed in the form of necessary protein.

[0061] for Example, such modified nucleic acid sequences include a deletion, substitution, including transition and transverse, or insertion of one nucleotide, and wherein these changes may occur in the 5'- or 3'-terminal positions of the reference of a nucleotide sequence or anywhere between those terminal positions, scattered either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence. The number of modified nucleotides is determined by multiplying the total number of nucleotides in any order by the numerical percent of the respective percent identity (divided by 100) and subtracting this work from the specified total number of nucleotides in the sequence.

[0062] for Example, the present invention assumes the use of a nucleotide sequence which is at least 70% identical to a sequence of nucleic acids, vyrozhdennom its variant or fragment, wherein the specified sequence may include up to nnchanges of nucleic acids across the polynucleotide segment sequence of nucleic acids, etc is that n nrepresents the maximum number of alterations and is calculated by the formula:

nn=xn(xn·y)

in which xnrepresents the total number of nucleic acids of any sequence and has a value of 0.70, wherein any non-integer product of xnand y is rounded to the nearest integer value before subtraction of the work of xn. Of course, we can also have a value of 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.94 94%, 0.95 for 95%, 0.96 to 96%, 0.97 for 97%, 0.98 for 98% or 0.99 for 99%, etc. Changes in sequence can lead to nonsense, missense mutations or mutations shift the reading frame in this coding sequence and thereby change the polypeptide encoded by the specified polynucleotide, after such changes.

[0063] In the present invention assumes the use of degenerate variants, or fragments. As defined in this application, "degenerate variant" is polynucleotide, which differs from the sequence of nucleotides (and its fragments) due to the degeneracy of the genetic code, but still encodes the same protein.

[0064] the Specified nucleic acid may include DNA, chromosomal DNA, cDNA and RNA, and may additionally include heterologous nucleotides. According the different variants of implementation, the specified nucleic acid's hybrid with some of nucleic acid, complementary to her, with her degenerate variant, or fragment, under highly stringent conditions of hybridization. In still other implementations, the specified polynucleotide's hybrid under medium stringent conditions of hybridization.

[0065] it Should be obvious that these nucleic acids can be obtained from natural, synthetic or semi-synthetic sources; moreover, the nucleotide sequence may be a naturally occurring sequence, or it may be related due to mutation, including single or multiple substitutions, deletions, insertions and inversions grounds, in respect of such naturally occurring sequence. This molecule is a nucleic acid can be RNA, DNA, single-stranded or double, linear or covalently closed circular form.

[0066] Examples of the conditions of severity are shown in Table Conditions rigor below: highly stringent conditions are those conditions that are at least as stringent as, for example, conditions A-F; stringent conditions are at least as stringent as, for example, conditions G-L; and the conditions of reduced stringency at least as stringent as, for example, conditions M-R.

CONDITIONS RIGOR

Condition severityPolynucleotide hybridThe length of the hybrid (BP)ITemperature and bufferIIfor hybridizationTemperature and bufferIIfor washing
AndDNA:DNA>5065°C; 1×SSC or 42°C; 1×SSC, 50% formamide65°C; 0.3×SSC
InDNA:DNA<50TB; 1×SSCTB; 1×SSC
DNA:RNA>5067°C; 1×SSC or 45°C; 1×SSC, 50% formamide67°C; 0.3×SSC
DDNA:RNA<50TD; 1×SSCTD; 1×SSC
ERNA:RNA>5070°C; 1×SSC or 50°C; 1×SSC, 50% formamide70°C; 0.3×SSC
FRNA:RNA <50TF; 1xSSCTF; 1×SSC
GDNA:DNA>5065°C; 4×SSC or 42°C; 4×SSC, 50% formamide65°C; 1×SSC
NDNA:DNA<50TN; 4×SSCTN; 4×SSC
IDNA:RNA>5067°C; 4×SSC or 45°C; 4×SSC, 50% formamide67°C; 1×SSC
JDNA:RNA<50TJ; 4xSSCTJ; 4×SSC
KRNA:RNA>5070°C; 4×SSC or 50°C; 4×SSC, 50% formamide67°C; 1×SSC
LRNA:RNA<50TL; 2×SSCTL; 2×SSC
MDNA:DNA>50 50°C; 4×SSC or 40°C; 6×SSC, 50% formamide50°C; 2×SSC
NDNA:DNA<50TN; 6×SSCTN; 6×SSC
ODNA:RNA>5055°C; 4×SSC or 42°C; 6×SSC, 50% formamide55°C; 2×SSC
PDNA:RNA<50TP; 6×SSCTP; 6×SSC
QRNA:RNA>5060°C; 4×SSC or 45°C; 6×SSC, 50% formamide60°C; 2xSSC
RRNA:RNA<50TR; 4×SSCTR; 4×SSC

[0067] BPI: Length of the hybrid is the expected length hybridizing plot(s) for hybridization of polynucleotides. When hybridization polynucleotide with the target polynucleotide unknown sequence length hybrid expected length for hybridization of polynucleotide. When hybridization is olignucleotides with the known sequence length of the hybrid can be identified by sequence alignment of these polynucleotides and definition of the parcel or parcels with optimal complementary sequences.

[0068] the BufferH: SSPE (1×SSPE is a 0.15 M NaCl, 10 mm NaH2PO4and 1.25 mm EDTA, pH 7.4) can be substituted for SSC (1×SSC is a 0.15 M NaCl and 15 mm sodium citrate) in the buffers for hybridization and washing; washing was performed for 15 minutes after hybridization was complete.

[0069] From TInto TR: the hybridization temperature for hybrids anticipated length less than 50 base pairs should be 5-10°C. less than the melting temperature (Tm) of the hybrid, where Tmdetermined according to the following equations. For hybrids less than 18 base pairs in length, Tm(°C) = 2 (number of A+T bases) + 4 (number of G+C bases). For hybrids between 18 and 49 base pairs in length, Tm(°C)=81.5+16.6(log10[Na+])+0.41 (%G+C)-(600/N), where N represents the number of bases in the hybrid, and [Na+] represents the concentration of sodium ions in the hybridization buffer ([Na+] for 1×SSC=0.165 M).

[0070] Additional examples of conditions of stringency for hybridization polynucleotides described in Sambrook, J., E.F.Fritsch, and T.Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, cold spring Harbor, N.Y., chapters 9 and 11, and Current Protocols in Molecular Biology, 1995, F.M.Ausubel and others, editor, John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4, incorporated in this application by reference.

[0071] In the present invention assumes the use of polynuclear is the Chida, which are fully complementary to these polynucleotides, as well as antisense sequences. These antisense sequences, also called antimyeloma the oligonucleotides include as having an internal origin and externally administered sequences that inhibit the expression of polynucleotides encoding the polypeptides according to the present invention. Antisense sequences according to the present invention include, for example, approximately 15-20 base pairs, without restrictions. Antisense sequences can be developed, for example, so that they inhibit transcription by preventing promoter binding transcription factors, hybridizes with a promoter located in front of the untranslated sequence, or by preventing translation of the transcript encoding the polypeptide according to the present invention, by preventing binding of ribosomes.

[0072] These polynucleotide can be prepared or obtained by any suitable method (e.g., by chemical synthesis from a DNA library from the organism itself) and can take various forms (such as single-stranded, denitive, vectors, probes, primers), which should be obvious to a person skilled in this field. The term "polynucleotide" includes the DNA and RNA, as well as their analogues, including such which have a modified backbone. According to additional implementations of the present invention, these polynucleotide include a DNA library such as a cDNA library.

Expression system, protein 2086

[0073] Provides a recombinant microorganism capable of Express 2086 polypeptide from Neisseria meningitidis sero-group, in accordance with some variant of realization of the present invention. Specified recombinant microorganism includes a sequence controlling the expression with promoter sequence and initiation sequence, and a nucleotide sequence which encodes a polypeptide 2086, the nucleotide sequence is located 3' side of the promoter and initiation sequences. In an additional aspect, is provided a host cell comprising the recombinant polynucleotide 2086, as described in this application, and in WO 03/063766, and WO 04/094596, which are fully incorporated into this application by reference. For this reason, the present invention provides a method of obtaining a recombinant protein 2086, as described, for example, in WO 03/063766 and WO 04/094596, without restrictions.

[0074] once constructed cell-hosts expressing the necessary protein or polypeptide according to the present from which retenu, by transformation, transferowania or infetsirovaniya host cells with plasmids containing the corresponding polynucleotide 2086, these host cells are cultured under such conditions that the polypeptides expressibility in accordance with the methods of the present invention. The specified polypeptide can then be identified so that it was essentially free from contaminating components of the host cell using techniques well known to specialists in this field.

Threshold settings

[0075] Some of the parameters can be used for tracking and monitoring the development of culture in the context of cell growth and expression of recombinant protein. Such parameters include, but are not limited to the above, the optical density (OD), dissolved oxygen (DO), pH, nutrient intake/energy (such as a carbon source), the accumulation of by-products of metabolism (e.g., acetic acid), a collection of cells and temperature. Any suitable parameter or combination of parameters is provided for use in the present invention, which should be obvious to a person skilled in this field, based on the guidance provided in this application.

[0076] the Threshold parameter set to determine the point at which you need to continuously add an inductor in the decree of the bacterial culture (namely to serve in culture over time). The threshold parameter is a predefined parameter. Some suitable threshold parameter, such as a predetermined optical density, is easily determined by the person skilled in the art based on the guidance provided in this application is, in accordance with various implementations of the present invention. You can use one threshold parameter, or a combination of threshold parameters.

[0077] the Parameter or combination of parameters can be monitored by any appropriate intervals in the specified culture. For example, OD600and the concentration of glucose can be monitored at intervals of one hour, half hour or quarter of an hour, without restrictions.

[0078] the Optical density is used as a threshold parameter to start the continuous feed of the inductor, in accordance with some variant of realization of the present invention. When the density of cells in a given culture reaches a predefined threshold parameter, such as an optical density equal to from about 70 to about 110, the inductor begin to file in culture, as described in this application. You can install a more narrow range for a given threshold parameter. For example, in the present invention, it is assumed that it is possible to start the continuous addition of an inductor in Kul is ur, when the density of cells in a given culture reached an optical density from about 70 to about 105, from about 75 to about 100, from about 75 to about 95, from about 75 to about 85, from about 76 to about 84, from about 78 to about 82, or about 80, in accordance with the variants of implementation of the present invention.

[0079] the Present invention also involves the use of threshold parameters to signal the beginning and/or ending supply of carbon source.

[0080] Any suitable device or combination of devices intended for monitoring the threshold parameter(s)that should be obvious to a person skilled in this field. For example, a sensor or combination of sensors for measuring the threshold parameter can be attached to the device for fermentation ("fermenter") by any suitable means, without limitation.

The constant feed speed

[0081] a Constant feed rate refers to the speed at which the inductor(s) and/or source(s) of carbon added to the culture. The inductor was added to the culture after the threshold parameter has been reached. The carbon source can also be added to the culture after the threshold parameter has been reached (and similarly, adding a source of carbon which may be completed when the threshold parameter). These threshold parameters include, without limitation, the optical density (OD), dissolved oxygen (DO), pH, concentration of nutrients in the culture medium, the total concentration of the first carbon source is added to the culture medium, or any combination of them.

[0082] Using any suitable constant speed for continuous addition of the inductor and/or a carbon source in the culture that should be obvious to the person skilled in the art based on the guidance provided in this application. In accordance with various implementations of the present invention, a suitable constant speed determined by the system DO-stat, as described in the examples below. For example, the feed rate equivalent to the DO-stat controller can be selected by adding a sufficient amount of glucose to reach concentrations of up to 15 and 24 g/l every hour, without restrictions.

[0083] for Example, the inductor and/or a carbon source can be added to the specified culture at constant speed up until a certain amount of inductor and/or a carbon source, such as from about 4 g/l to about 40 g/l, such as, 4 g/l, 5 g/l, 6 g/l, 7 g/l, 8 g/l, 9 g/l, 9.5 g/l, 9.75 g/l, 10 g/l, 10.25 g/l, 10.5 g/l 11 g/l, 12 g/l 13 g/l 14 g/l, 15 g/l, 16 g/l 17 g/l 18 g/l 19 g/l, 20 g/l 21 g/l 22 g/l, 23 g/l 24 g/l, 25 g/l 26 g/l 27 g/is, 28 g/l 29 g/l, 30 g/l 31 g/l 32 g/l 33 g/l, 34 g/l, 35 g/l 36 g/l, 37 g/l 38 g/l 39 g/l, 40 g/l, based on the total volume of culture was not added to the specified culture, without restrictions. According to various implementations, the total number of inductor and/or a carbon source, which was filed in the specified culture is from about 5 g/l to about 20 g/l, 7 g/l to about 15 g/l, 8 g/l to about 14 g/l, 9 g/l to about 11 g/l, or about 10 g/L.

[0084] the Total amount of the inductor, which must be added to the specified culture can be offset by the total amount of carbon source added to the culture. For example, when a carbon source is glucose and the inductor is arabinose, the number of added inductor can be reduced by adding glucose. For example, in some implementation, a total of 10 g/l of the inductor (namely, such as arabinose) was added to the culture was added 11 g/l of a carbon source such as glucose. The yield of protein obtained in this way approximates the output when using the total amount of 20 g/l arabinose (namely only 20000 g or 20 kg arabinose in 1000 l of culture) and no glucose. Thus, the offset provided by these methods is best, whereas high what kind of price arabinose relative to glucose.

[0085] According to various implementations of the present invention, the constant speed at which the inductor and/or a carbon source is added in a specified culture, can be set in the range of from about 1.5 g/l to about 24 g/l every hour. For example, when a carbon source is glucose, the constant speed of adding glucose may include, without limitation, 1.8 g glucose/l/h, 3.3 g of glucose/l/h, 6.7 g of glucose/l/h, 15 g glucose/l/h 16.4 g glucose/l/h, 18 g of glucose/l/h, 24 g of glucose/l/h, etc. According to various implementations, some inductor, such as arabinose, add at constant speed equal to from about 1.5 g/l/h to about 16 g/l/h

[0086] According to various implementations, as soon as you reach the threshold setting, the supply of the carbon source can continue, stop or temporarily stop. The supply of the carbon source can be interrupted, in this case, the flow will begin again at constant speed, as soon as you reach the threshold to initiate enrollment. Thus, according to some implementation variant, the initial threshold and a stopping threshold can be used to regulate the flow of the carbon source in the specified culture. According to another implementation variant, as glucose and arabinose were applied at constant speed, on the basis of p is horn option without stop or start feeding again.

[0087] a Suitable total amount of carbon source that you want to add in any specific culture, can be easily determined by the person skilled in the art based on the guidance provided in this application. The total number of carbon source, which was added to the indicated cultures can vary from about 1 g/l to about 100 g/l (based on the total volume of culture in liters), according to some implementation variant of the present invention. For example, according to some implementation variant, 50 g/l glucose was added during the growth phase, starting with 10 g/l in the environment, starting supply of glucose at a constant speed, when the glucose level reaches zero, and continuing the supply of glucose at a constant speed until, while OD has not reached 80, at this time, approximately 40 g/l glucose will be served in addition to the original 10 g/l glucose. According to some implementation variant, the total number of carbon source is delivered in a concentrated form to facilitate scalability. This number is easily converted to the total weight of the carbon source that you want to use under certain circumstances. For example, when you want to add 10 g/l of carbon source in 1000 l of culture, the total number of light source, the CA carbon, I want to add, is easily defined as 10 g/l × 1000 l = 10000 grams (or 10 kg) total carbon source. The total added amount of the carbon source can serve as a threshold parameter, in accordance with various implementations, as described in this application.

[0088] a Suitable total amount of coil, you need to add to any particular culture, can be easily determined by the person skilled in the art based on the guidance provided in this application. The total amount of inducer added to the specified culture can vary from about 4 g/l to about 40 g/l (based on the total volume of culture in litres) in accordance with various implementations. According to various implementations, the total number of carbon source, which was added in the specified culture is from about 5 g/l to about 20 g/l, 7 g/l to about 15 g/l, 8 g/l to about 14 g/l, 9 g/l to about 11 g/l, or about 10 g/l, based on the total volume of this culture. According to some implementation variant, the total number of inductor is supplied in a concentrated form to facilitate scalability. This number is easily converted to the total mass of the inductor to be used when measuring the certain circumstance. For example, when 10 g/l of the inductor should be added to 1000 l of culture, the total number of inductor that you want to add, it is easy to determine as only 10 g/l × 1000 l = 10,000 g (10 kg) of the inductor.

[0089] the Fresh culture medium will typically contain the starting number of the first carbon source during the inoculation of the host-cell, thus creating a culture. This initial concentration can be monitored and the concentration of the first carbon source to use as a threshold parameter.

[0090] Any suitable additive or nutrients, in addition to the carbon source can also be submitted in the specified culture in suitable quantities. You can keep track of other nutrients or additives, and set the appropriate thresholds. Additives, such as sources of nitrogen, or inorganic phosphate, it has been proposed for use in the present invention. Non-limiting examples of compounds that are anticipated for use in the methods of the present invention include KH2PO4, K2HPO4, sodium citrate, dihydrate, (NH4)2SO4, MgSO4, (Na)2SO4, CaCl2, FeSO4, chloramphenicol or any combination of them. The use of an additional source or sources of carbon it is also assumed.

Optical PLO is the ability and the logarithmic phase of growth

[0091] the Introduction of bacterial host cells in fresh culture medium allows you to create a culture that typically passes through four in varying degrees, different phases of growth: (i) latent phase, (ii) log (logarithmic or exponential) phase, (iii) stationary phase and (iv) the phase of decline (death). Itself logarithmic phase can be further divided into different phases, such as the early logarithmic growth phase, the average logarithmic growth phase and late logarithmic growth phase. The optical density is related to the phase of logarithmic growth. Logarithmic growth phase and the optical density can also be used as a threshold parameter to signal the start and/or stop continuous supply of carbon source and/or inductor.

[0092] for Example, inducing, or continuous addition of the inducer, you can start in the early logarithmic growth phase, the average logarithmic growth phase and late logarithmic growth phase. Late logarithmic growth phase can go with OD equal to from about 70 to about 110. In a variant of realization of the present invention, a constant feed rate of the inductor will begin in late logarithmic growth phase specified culture medium or OD equal to from about 70 to about 110, from CA is approximately 70 to about 105, from about 75 to about 85, or about 80, in accordance with various implementations.

[0093] OD can be measured at different wavelengths, which are typically used specialists in this area. As a rule, OD600use as a measure of cell growth and density of cells in culture. Unless otherwise specified, "OD", as used in this application refers to OD600.

Dissolved oxygen

[0094] Another parameter that can serve as a switch for starting and/or stopping of the controller supply is dissolved oxygen (DO) (namely periodic fermentation with makeup DO-stat control). DO can be controlled by adjusting the shaking, air flow, added oxygen and pressure in the vessel for holding the culture medium. The threshold DO can be set in the range from 5% to 80% DO, such as 20%, 40% or 80%. Once the threshold has been reached, the controller of the filing of the carbon source or inductor may be included as long as you reach the threshold, which indicates the end of a feed. The threshold for the stop may be a different threshold or DO another parameter, such as the number of carbon source or inductor. For example, in all cases, when DO rise above 30% or 40% in the culture medium, the controller supply may not start until the eye DO not fall to 20%, or, alternatively, up until 0.5 g/l or 1 g/l of a carbon source or inductor will not be newly added, in accordance with various implementations of the present invention.

pH

[0095] Another parameter that can serve as a switch for starting and/or stopping of the controller supply is pH (namely periodic fermentation with water with a pH-stat control). the pH can be controlled by adding a base or acid to the culture medium. The threshold pH can be set in the range from 6.8 to 7.2, such as 7.0. Once the threshold has been reached, the controller of the filing of the carbon source or inductor may be included as long as you reach the threshold, which indicates the end of a feed. The threshold for the stop may be a different threshold pH or other parameters such as the number of carbon source or inductor. For example, in all cases, when the pH rises to 6.97 in the culture medium, the controller being fed may start up until the pH drops to 6.95, or, alternatively, until such time as 1 g/l carbon source or inductor will not be newly added, in accordance with various implementations of the present invention.

The collection of cells

[0096] the collection of cells represents the amount of time that passes after the original is th induction or adding an inductor. Any time collection of cells provided by the present invention. The collection of cells can vary from about 2 hours to about 10 hours, from about 2 hours to about 8 hours, from about 2.5 hours to about 7 hours, from about 3 hours to about 6 hours, etc. in accordance with various implementations of the present invention. Using a constant feed rate, the collection of cells and the total number of inductor, the average of the experts in this field will understand how you can adjust each parameter to achieve the desired results. Specialists in this field will understand when you need to collect cells on the basis of submitted number of arabinose, because they can easily determine filed number based on the speed and time of submission. Thus, it is possible to reach the final concentrations of the specified inductor equal to 5, 10, 20, 30 and 40 g/l, which was filed within 3 hours, as an example without limitation.

The concentration of inducer

[0097] Some inductor in any suitable concentration provided by the present invention. The concentration of the inducer, applicable for the induction of host cells, can vary from about 0.00001% to about 20% (volume ratio, v/v), without restrictions.

Tempera is ur

[0098] Culture, according to real options implementation, can be incubated at any temperature that allows the growth of cells. Different temperatures at which must be incubated abundantly growing culture include, without limitation, 22°C, 28°C, 37°C, or any combination of them.

Device for fermentation

[0099] Any suitable device for fermentation (namely "fermenter") is provided for use in the present invention, which should be obvious to a person skilled in this field. For example, the fermenter can contain any number of impellers (such as Rushton impellers), intakes and/or probes. In accordance with some alternative implementations, the specified fermenter configured with three Rushton impeller and the annular or radial bubbler to introduce air into the fermenter. In the present invention assumes the use of manual and/or based on the application of computer systems. For this reason, the fermentation system can be linked to a computerized system for tracking and controlling fermentation. Thus, this system may be fully or partially automated, in accordance with the variants of implementation of the present invention.

Compositions according to the present invention

[0100] Compositions comprising recombinant the tree, such as those that are obtained in accordance with the methods of the present invention, provided in this application in accordance with the variants of implementation of the present invention. Compositions according to the present invention include recombinant protein at high density in culture, such as recombinant proteins, obtained in accordance with the methods of the present invention, not intended to limit.

[0101] this composition includes some culture with the recombinant protein at a density equal to at least about 1.5 g/l, based on the total volume of this culture. The density of the indicated recombinant protein of at least about 1.7 g/l, based on the total volume of the culture, according to an additional variant of implementation of the present invention. The density of the indicated recombinant protein of at least about 2.0 g/l, based on the total volume of the culture, according to another implementation variant of the present invention. The density of the indicated recombinant protein is equal to at least about 3.0 g/l, based on the total volume of the culture, according to another implementation variant of the present invention.

[0102] Some composition comprising a recombinant protein 2086, provided a case of " the present invention. This protein 2086, as used in this application, is a protein expressed from polynucleotide, which corresponds to the 2086 gene in N. meningitidis sero group, including any fragment, derivative or mutation. Non-limiting examples of proteins and polynucleotides 2086 described in WO 03/063766 and WO 04/094596.

[0103] the Composition with recombinant 2086 protein includes a recombinant protein 2086 in culture, characterized in that the recombinant protein 2086 contained at a density equal to at least about 1.5 g/l, based on the total volume of the culture. The density of the recombinant protein 2086 equal to at least about 1.7 g/l, based on the total volume of the culture, according to an additional variant of implementation of the present invention. The density of the recombinant protein 2086 equal to at least about 2.0 g/l, based on the total volume of the culture, according to another implementation variant of the present invention. The density of the recombinant protein 2086 equal to at least about 3.0 g/l, based on the total volume of the culture, according to another implementation variant of the present invention.

[0104] the Compositions according to the present invention can include any protein, such as protein obtained in accordance with the method of the present invention. Recombinant proteins can be the ü limitirovanie or nelimitirovannoe proteins. In a variant of realization of the present invention, indicated recombinant protein is a recombinant protein 2086 or libidinously or Neopalimovsky. Recombinant 2086 protein can be protein 2086 subfamily a or subfamilies, or their combination. Compositions according to the present invention may include one protein or more than one protein. These proteins may be related or unrelated proteins. For example, a composition according to the present invention may include 2086 protein, corresponding to one or more strains of the subfamily and/or one or more strains of the subfamily Century

[0105] Compositions comprising a substance for use in implementing methods of the present invention, also provided in this application. Such compositions include the necessary components for cultivation, including recombinant cells and nutrients, in accordance with the variants of implementation of the present invention. Various compositions may be provided together in a kit, in accordance with some variant of realization of the present invention. For example, components for conducting culture can be conveniently pre-Packed in the required quantities to facilitate laboratory or industrial use, without restrictions. This set is can also include labels, indicators and guidelines to facilitate the implementation of each component and method of combining components, in accordance with various implementations of the present invention.

[0106] the Following examples are included to demonstrate different ways of implementing the present invention. For professionals in this field should be obvious that the methods described in the examples that follow represent techniques that the authors of the present invention found that works well when implementing the present invention, and thus, they can be considered components of different models for its implementation. However, for specialists in this field, in view of the present description, it should be obvious that you can make a lot of changes in specific embodiments of which are described and still obtain a like or similar result without going beyond the nature and scope of the present invention.

EXAMPLES

Example 1: Periodic fermentation with water at a constant feed speed

[0107] E. coli (pPW62) subfamily In used as a model strain for the fermentation process with water. Based on the results, this process will also apply to the subfamily And E. coli (pPW102).

[0108] the Environment and the solution to recharge for periodic fermentation contribution was received, using the components listed in the following tables.

The environment and the solution to feed:

Table 1
The primary environment
ComponentQuantity per liter
Dextrose, anhydrous10 g
KH2PO43 g
K2HPO47 g
(NH4)2SO41 g
Sodium citrate, dihydrate1 g
MgSO4·7H2O1 g
(Na)2SO40.58 g
CaCl2·2H2O0.075 g
FeSO4·7H2O0.09 g
1000x concentrated solution of trace metals1 ml
(Table 6)
Haram is enical 15 mg

Table 2
1000× concentrated solution of trace metals
ComponentQuantity per liter
ZnSO4·7H2O30 g
CuSO4·5H2O9 g
MnSO4·H2O4.2 g
CCl2·6H2O0.6 g
Molybdenum acid, ammonium salt, tetrahydrate1.5 g
Table 3
A concentrated solution of glucose to fuel
ComponentQuantity per liter
Glucose500/700 g
KH2PO43 g
Ksub> 2HPO45 g
(NH4)2SO42 g
Table 4
A concentrated solution of arabinose to recharge
ComponentQuantity per liter
Arabinose250/500 g and varies from experiment
KH2PO43 g
K2HPO45 g
(NH4)2SO42 g

Methods

[0109] Periodic fermentation with water at a constant feed rate was used to achieve a high density of cells in fermentation of E. coli. The initial glucose concentration was 10 g/l in the environment. The concentration of (NH4)2SO4was increased to 3 g/l in the medium for fermentation, but kept at a level of 1 g/l in the seed culture medium. To determine the feed speed, first performed periodic fermentation with water in the system DO-stat. At over DO supported by 20% using a cascade controller, which increased the rate of shaking up to a maximum and then added oxygen. When glucose was exhausted, DO sharply increased (above 40%), and was added to the concentrate glucose to a final concentration of 1 g/l in the fermenter. After each addition of glucose, the pump was switched off for a set period of time before being allowed to make the following addition. Maximum OD of approximately 160 was achieved when performed periodic fermentation with water in the system DO-stat. Constant speed then chose the equivalent DO-stat controller, adding a sufficient amount of glucose to reach concentrations of up to 18 g/l 24 g/l every hour. During periodic fermentation feed when feeding at constant speed, the supply of glucose was included at the required constant speed, when there was a sharp rise in DO up to 40%.

[0110] crops initiated using one vial of E. coli (pPW62) per liter of the primary environment +15 μg/ml chloramphenicol in 2800 ml flask of Fernbach. Flasks were incubated at 32°C, 150 rpm overnight (~16 hours). End OD600was usually ~3. 10% of the size of the inoculum, which was used for inoculation of each fermenter. Each fermenter had 3 Rushton impeller and ring bubbler. The initial set value: temperature: 36°C, pH: 7.00±0.05 (kontrolirovat and using 7.4 N NH 4OH), air flow: ~1 l of air per 1 liter of medium per minute (~1 vvm), DO: 20%. DO was controlled by cascade shaking (min: 150 rpm, max: 1000 rpm) and Appendix O2through the gas mixing device. Foaming was controlled, if necessary, by adding manually PPG-2000. 0.35 ml/l AF was added to the medium before sterilization. In the process of fermentation, samples were taken hourly for tracking glucose, pH and OD600outside the system. Supernatant was obtained from 1 ml samples and stored for further analysis of organic acids by liquid chromatography high resolution (HPLC).

Results

Periodic fermentation with water when feeding at constant speed

[0111] In the Figure 1 illustrates the dynamics of the OD, the consumption of glucose and accumulation of acetic acid at a constant feed speed. Maximum OD equal to 158 and 150 received at a constant feed rate, equal to 24 g glucose/l/h and 18 g of glucose/l/h, respectively. A large amount of glucose, such as 28 g/l was accumulated during operation, when used feed rate equal to 24 g/l/h Glucose was accumulated up to 12 g/l, when used, the feed rate of 18 g/l/h a Small amount of acetic acid (namely, less than 1.5 g/l) were formed in both cases. The exponential growth phase ended close to OD 100. Characteristic near the industry growth was approximately equal to 0.60 (h -1) in both cases.

[0112] To reduce the accumulation of glucose were investigated low constant feed speed equal to 16.4 g/l/h and 15 g/l/h On Figure 2 shows the dynamics of the OD, the consumption of glucose and the accumulation of acetic acid at constant flow rates mentioned above. The maximum OD was equal to 142 and 147, respectively. As in the previous runs, the culture with the most rapid feed rate accumulated more glucose, although the number of accumulated glucose was much less than in the previous experiments. Approximately 8 g/l of glucose was accumulated during the fermentation with feed rate of 16.4 g/l/h and 5.4 g/l of glucose by fermentation with a feed rate of 15 g/l/h a Small amount of acetic acid (such as less than 1.5 g/l) were formed in both cases (see Figure 2). The typical growth rate was approximately equal to 0.60 (h-1) in both cases. Thus, the typical growth rate was not influenced by the feed rate of between 15 g/l/h and 24 g/l/h

Induction at different OD growth

[0113] a Constant feed rate of 15 g glucose/l/h, was used to study the induction of arabinose, because it led to a high density of cells and a small accumulation of glucose and acetic acid. In this experiment, the induction with the average logarithmic growth phase, OD ~55, and late logarithmic phase p is a hundred, OD ~80 was comparable. The culture was induced by simple replacement of the supply of glucose to submit arabinose, and gave the arabinose at a constant speed, equal to 13.4 g/l/h in Just 40 g/l arabinose was added to each culture for 3 hours. After induction, samples were taken every hour for analysis on rLP2086 by SDS page/LTOs, analysis of organic acid and arabinose by HPLC.

[0114] In the Figure 3 shows the dynamics of the OD and products rLP2086, when the induction is when OD ~55 and ~80. As the maximum OD and output rLP2086 were higher when cells were induced at OD ~80 (maximum OD: 101 vs. 84; maximum output: 1.8 g/l vs. 1.2 g/l).

Induction at different levels of arabinose

[0115] the Purpose of the following experiments was to estimate the total amounts of arabinose, filed in culture, and to explore whether even lead to a decrease in the total number of arabinose, filed in culture to high expression rLP2086. Concentrate arabinose was applied in 4 different cultures, each with different flow rates, within 3 hours, which resulted in final concentrations of arabinose, equal to 10, 20, 30 and 40 g/L. All cultures induced at OD600~80. The Figure 4 shows the dynamics of the OD and products rLP2086. Table 5 summarizes the OD and the output rLP2086 for each of the four conditions. It shows the maximum output rLP2086 equal to: .2 g/l to 10 g/l of the total quantity of added arabinose; 1.6 g/l to 20 g/l total quantity added arabinose; 1.7 g/l to 30 g/l total quantity added arabinose; 2.0 g/l to 40 g/l total quantity added arabinose. Submission of arabinose in amounts of between 20 g/l and 40 g/l led to similar output rLP2086, however, 10 g/l arabinose resulted in the production of a much smaller number of rLP2086 (namely 1.2 g/l). These results suggest that the total number of arabinose was added for induction, can be reduced from 40 g/l to 20 g/l without compromising performance rLP2086. Thus, the decline in the use of arabinose will be the most economically feasible, especially given the high price of arabinose (approximately US $ 500 per kg).

Table 5
Induction with different levels of arabinose
PartyThe total number of submitted arabinose (g/l)Maximum rLP2086 (g/l)
X-BRN05-027101.2
X-BRN05-024201.6
X-BRN05-025 301.7
X-BRN10-114402.0

Comparison of methods for adding arabinose

[0116] the Following experiment was conducted to investigate whether the strategy of continuous supply better than the simple strategy of the periodic addition of the induction by arabinose. When you run X-BRN05-039, 20 g/l arabinose at a time was added to the fermenter, instead of filing in the course of time, when OD was approximately 80. The Figure 5 shows the dynamics of the OD, the consumption of glucose and arabinose, and products rLP2086. Received the maximum number of rLP2086 equal to 1.3 g/l Periodic addition of arabinose, although operationally simpler allowed to get fewer rLP2086 than in continuous flow. Thus, the strategy of continuous supply arabinose better than simple periodic add.

[0117] in Order to examine whether it is more efficient to use the arabinose by reducing the feed speed of arabinose, compared feedrate is equal to 3.3, arabinose/l/h and 6.7, arabinose/l/h On the Figure 6 shows the dynamics of the OD and products rLP2086. Concentrate arabinose was served in one culture at a feed rate equal to 6.7 g/l/h for 3 hours, and the second culture was applied at a speed equal to 3.3 g/l/h for 6 hours. For beichler total added arabinose was equal to 20 g/L. As shown in Figure 6, in both conditions were producirovanie the same maximum number of rLP2086 (namely, 2.2 g/l), but there were differences in the kinetics products. A higher feed rate resulted in a higher production speed. The maximum level of rLP2086 was reached after 3 hours and 6 hours after induction at a feed rate equal to 6.7 g/l/h and 3.3 g/l/h, respectively. The advantage of using a higher feed rate (namely, 6.7 g/l/h) is the product price (e.g., energy costs) will be lower than when using a higher feed rate than when using a lower feed speed.

The influence of time of induction to the output expression rLP2086

[0118] to determine the optimal time for collection of cells, normal profile feed (20 g/l arabinose was filed within 3 hours) protovale to 40 g/l, filed within 6 hours. When you launch X-BRN05-028 and X-BRN05-029 cells induced at OD ~55 and OD ~80 respectively. The Figure 7 shows the dynamics of the OD and products rLP2086. Although filing arabinose was extended from 3 hours to 6 hours, it is noteworthy that the peak titer was obtained in approximately 3 hours after induction. The title product was slightly higher in the culture, which is induced at higher OD. The maximum yield of rLP2086 induction at OD ~55 was equal to 2.0 g/l (X-BRN05-028), while he was Raven g/l at induction at OD ~80 (X-BRN05-029). This result suggests that cells should be collected 3 hours after induction.

Comparison of solutions for feeding with and without adding salt

[0119] To investigate whether the added salt is needed in solutions of glucose and arabinose for recharge, recharge glucose and arabinose without additives were compared with standard fertilization glucose + salt (namely, K2HPO4/KH2PO4+(NH4)2SO4) and arabinose + salt. For both cultures, 20 g/l arabinose was filed within 3 hours. Profiles of growth and production of rLP2086 were very similar. The maximum yield of rLP2086 was equal to 1.8 g/l, when the salts were added to the fertilization, and 2.0 g/l, when the feeding of glucose and arabinose were prepared without salt. These results suggest that there is no need to add salt in glucose and arabinose to feed.

Periodic fermentation with water at a constant feed rate for the strain of the subfamily

[0120] crops were started by inoculation of 1 l main medium containing 15 μg/ml chloramphenicol, 1 ml of the thawed working seed. The cultures were grown in 2.8-liter flask of Fernbach and incubated for approximately 16 hours at 32°C and 150 rpm End OD600was equal to ~3.0. 350 ml of the crop is aseptically transferred to 3.15 l cos who ate environment, containing 3 g/l (NH4)2SO4without chloramphenicol. The fermentation was conducted at a controlled pH 7.0±0.05 by 7.4 N NH4OH, at a temperature of 36°C, DO 20% and the air flow of 1 vvm. DO was controlled by cascade shaking (min: 150 rpm, max: 1000 rpm) and add oxygen. Antifoam PPG-2000 automatically added to control foaming. In the process of fermentation, samples were taken every hour to monitor glucose and OD outside the system. After inoculation, the DO dropped from ~100% to 20% and then maintained at 20%. When was the sharp increase in the level DO from 20% to over 40% (usually after 6 hours of actual duration of fermentation (EFT)), were included feed pump glucose (without salt) at a speed of 15 g/l/h As shown in Figure 8, the glucose was completely exhausted after 6 hours EFT, which led to a sharp rise DO. Samples were taken every half hour, when OD reached ~40. The supply of glucose off when OD 90 and filing arabinose was included at a speed of 13.4 g/l/h After 3 hours of filing arabinose (without salt) (namely, the total adding 20 g/l arabinose) supply arabinose off, and allowed to continue fermentation for another one hour. As shown in Figure 8, was obtained OD equal to 102, and expressionalism 2.0 g/l MnB rLP2086, based on SDS page/LTOs (see Figure 9). The peak was 3 hours after induction (namely h the RES ~12 hours EFT). Page/LTOs and analysis by the method of Western blot showed that the expressed protein was, without a doubt, protein subfamily In rLP2086.

The use of the fermentation process with water for MnB rLP2086 strain of the subfamily And

[0121] to test whether the fermentation process with water, used for rLP2086 subfamily B, applicable to rLP2086 subfamily A, the specified process was performed by applying the procedure set for the strain of the subfamily Cent. On the Figure 10 shows the OD, consumption of glucose and arabinose and products rLP2086. Profiles of growth and production of rLP2086 to subfamily And were similar to those obtained for the subfamily (compared to Figure 8). Page/LTOs and analysis by the method of Western blot showed that the expressed protein was, without a doubt, rLP2086 subfamily a (see Figure 11). Table 6 lists the maximum OD and output expression rLP2086 for six different runs for the subfamily A. Range maximum output expression rLP2086 was 1.5-2.1 g/l (average maximum output: 1.8±0.2 g/l), which was similar to the results for periodic fermentation with water, applied to obtain rLP2086 subfamily C. Thus, periodic fermentation with feed, designed for strain subfamily, also suitable for strain subfamily A.

Table 6
The maximum yield expression and OD rLP2086 subfamily And
PartyThe maximum level of rLP2086 (g/l)Maximum OD
X-BRN10-1181.9104
X-BRN10-1191.9104
X-BRN10-1201.6100
X-BRN05-0421.5100
X-BRN05-0432.077
X-BRN10-1212.192

Joint filing of glucose and arabinose in the process of inducing arabinose

[0122] To reduce the required number of arabinose without reducing production rLP2086 was investigated simultaneous supply of glucose and arabinose during induction. As part of this strategy was the implementation of the delivery of 10 g/l arabinose for 3 hours (half the normal amount), continuing supply of glucose during the phase of induction at 25% (3.75 g/l/h), 50% (7.5 g/l/h)and 100% (15 g/l/h) from standartveikalos supply of glucose. All of fertilization, glucose and arabinose were prepared without additives.

[0123] In Figures 12A, 12B and 12C shows the dynamics of cell growth subfamily B, concentrations of glucose, arabinose, acetic acid and the products of rLP2086. All three run were induced at OD ~80. As shown in Figure 12A, OD when you start with a 100% supply of glucose continued to rise after induction, having a peak at 117, whereas if you start with 50% of the peak flow was at 106 (Figure 12B) and when you start with 25% stayed about 100 (Figure 12C) after induction. When applying 100% glucose began the accumulation of glucose and arabinose in 3 hours after induction. When run from the submission of 50% glucose was only a small amount of glucose in the last sample (reading = 0.21 g/l). Accumulation of glucose was not at all when run from the filing of 25% glucose. None of the three runs was not accumulated arabinose. If it is launched to supply 100% (Figure 12A) and 50% (Figure 12B) glucose was producirovanie ~1.5 and 1.7 g/l rLP2086 respectively, whereas when you start feeding 25% (Figure 12C) producirovanie more than 2.1 g/l Peak production when you start feeding 100% of the glucose was before arabinose ended, suggesting that the expression of rLP2086 can be suppressed when the accumulation of glucose and acetic acid. Peak production when run from the submission of 50% glucose was approximately simultaneously with the exhaust of arabinose, and the IR output when run from the filing of 25% was after as arabinose ended, suggesting that the expression of 2086 may not be suppressed until such time as the glucose concentration is maintained at a minimum level (glucose is not accumulated, as seen in Figures 12B and 12C). Although only 10 g/l arabinose was applied to these crops, their products rLP2086 was similar to that obtained when applying 20 g/L. Simultaneous feeding glucose and arabinose can reduce the consumption of arabinose by 50% and still achieve the same output rLP2086, when the glucose concentration is maintained at a low level during induction. Thus, the price of chemicals can be greatly reduced.

[0124] in Order to examine whether it is possible to further reduce the consumption of arabinose and to increase the output of rLP2086, have been investigated by various feed rate of glucose, total number of submitted arabinose and OD induction. Table 7 lists the various combinations of these conditions. It seems that the feed rate of glucose between 2.25 and 7.5 g/l/h and the feed rate of arabinose between 1.7 and 6.7 g/l/h will not significantly affect the output rLP2086. OD induction between 80 and 105 lead to similar output rLP2086.

Table 7
Maximum OD and rLP2086 at different flow rates of glucose, different number is two added arabinose and induction at different OD
The batch numberThe feed rate of glucose (g/l/h)The feed rate of arabinose (g/l/h)Number filed arabinoseOD inductionMaximum ODMaximum rLP2086 (g/l)
X-BRN10-1273.751.75 g/l in 3 hours74861.6
X-BRN05-0563.753.320 g/l in 3 hours791222.8
X-BRN05-0583.751.710 g/l for 6 hours941223.0
X-BRN10-1293.756.720 g/l in 3 hours1101242.7
X-BRN05-059 3.320 g/l for 6 hours1051262.9
X-BRN05-0615.253.320 g/l for 6 hours1021122.6
X-BRN10-1302.253.320 g/l for 6 hours931082.4

Example 3: Periodic fermentation with feed in scale, increased to 100 l

[0125] Seed culture was started by inoculation of 2×1 l main medium containing 15 μg/ml chloramphenicol, and 1 ml (namely, 1 vialou) thawed working seed. Culture in 2.8-liter flask of Fernbach incubated for approximately 16 hours at 32°C and 150 rpm in a rotary shaker.

[0126] Two night crops in a 1-l flask of Fernbach aseptically transferred into a 150-liter fermenter containing 70 l of the primary environment without chloramphenicol. The fermentation is conducted in 150 l of the primary environment with controlled pH 7.0±0.05 using a 7.4 N NH4OH, the temperature of 36°C, DO 20% and the air flow of 1 vvm. DO controlled posledstviya shaking and adding oxygen. Antifoam PPG-2000 automatically added to control foaming. In the fermentation process DO fell from ~100% to 20% and was maintained at 20%. When was the sharp increase in the level DO from 20% to over 40% (usually, when OD ~20), indicating that the depletion of glucose, then the feed pump concentrated glucose (namely 500 g/l) at a speed of 15 g glucose/l content of the fermenter/h In the fermentation samples were taken hourly for tracking glucose and OD outside the system. Samples were taken every half hour, when OD reached ~40. As soon as the OD reached ~80, the supply of glucose was stopped and the supply of arabinose was started (for example, 500 g/l of concentrate arabinose) at a speed of 6.7 garbanati/l content of the fermenter/h for 3 hours.

[0127] Figure 13A shows the dynamics of cell growth of the subfamily In the consumption of glucose and accumulation of acetic acid and the product rLP2086 at 100 HP Profile fermentation scale (100 l) was similar to those for small-scale fermentation. Received maximum OD equal to 99, and the maximum yield of rLP2086 equal to 1.9 g/L. These results indicate that periodic fermentation injection is scalable.

[0128] Figure 13B shows the dynamics of cell growth subfamily A, consumption of glucose and accumulation of acetic acid and the product rLP2086 in 100 liters of the fermentation Profile at 100 l b is l similar to those for small-scale fermentation. Received maximum OD equal to 96, and the maximum yield of rLP2086 equal to 2.0 g/L. These results indicate that periodic fermentation injection is scalable and sustainable.

[0129] Figure 14A shows the dynamics of the density of the cells of the subfamily, concentrations of glucose, arabinose, acetic acid and output rLP2086 while filing glucose and arabinose at 100 HP In the process inducing the feed rate was maintained at 3.75 and 1.67 g/l/h to supply glucose and arabinose, respectively. Both arabinose and glucose were applied for 5 hours. Received maximum OD equal to 90, and the maximum yield of rLP2086 equal to 1.8 g/l Maximum output rLP2086 was at 4-hour induction. Average maximum OD and the average maximum output rLP2086 to subfamily In values were 84.8±6.8 and 1.6±0.3 g/l, respectively. These results show that the periodic fermentation injection with simultaneous supply of glucose and arabinose during the phase of induction is scalable.

[0130] Figure 14B shows the dynamics of cell growth subfamily A, consumption of glucose and accumulation of acetic acid and the product rLP2086 in 100 liters of the Fermentation is conducted under the same conditions specified in paragraph [0129] the cells were induced for 6 hours. Received maximum OD equal to 89, and the maximum output is rLP2086, equal to 1.8 g/l Maximum output rLP2086 was at 4-hour induction. Average maximum OD was 87.9±10.5 and an average maximum output rLP2086 was 1.8±0.2 g/l for the subfamily A. These results show that the periodic fermentation injection is scalable and sustainable.

[0131] Although the present invention has been described with reference to various implementations and examples for professionals in this field should be obvious that various modifications of the present invention, is not beyond the bounds of its nature and scope.

1. A method of obtaining a recombinant protein, comprising:
cultivation of recombinant bacterial cells for the expression of recombinant proteins, including continuous addition of carbon source in a culture comprising a recombinant bacterial cell, and the continuous addition of the inducer in the specified culture after culture has reached a threshold parameter, and the selection of the indicated recombinant protein from a specific culture, while the total amount of inducer added to the culture in the process of filing inductor is equal to from about 7 g/l to about 15 g/L.

2. The method according to claim 1, wherein the specified threshold parameter represents the optical density (OD), dissolved oxygen (DO), concentration Pete is positive substances in the culture medium, the total concentration of the carbon source added to the culture medium, or any combination of them.

3. The method according to claim 2, characterized in that the specified threshold parameter represents the optical density (OD) of the culture.

4. The method according to claim 3, comprising a continuous addition of inducer to the culture, when the density of cells in a given culture reaches OD600equal to from about 70 to about 110.

5. The method according to claim 4, comprising continuously adding an inductor in a specified culture, when the density of cells in a given culture reaches OD600equal to from about 70 to about 105.

6. The method according to claim 5, comprising a continuous addition of inducer to the culture, when the density of cells in a given culture reaches OD600equal to from about 75 to about 85.

7. The method according to claim 6, comprising a continuous addition of inducer to the culture, when the density of cells in a given culture reaches OD600approximately 80.

8. The method according to claim 1, including the addition of inducer to the culture at a constant speed, the addition of inducer to the culture through the DO-stat feeding or adding an inducer to the culture by pH-stat feeding.

9. The method according to claim 1, including the addition of inducer to the culture at a constant speed equal to from bring the flax to 3.35 g/l/h to about 16 g/l/h, the addition of inducer to the culture through the DO-stat feeding or by pH-stat feeding.

10. The method according to claim 1, characterized in that the total amount of inducer added to the culture, is from about 4 g/l to about 40 g/L.

11. The method according to claim 10, characterized in that the total amount of inducer added to the culture in the process of filing inductor is approximately 10 g/L.

12. The method according to claim 1, comprising continuously adding an inductor in culture for approximately 2 to approximately 8 h after the beginning of the specified method.

13. The method according to item 12, which includes the continuous addition of inducer to the culture within approximately 3 to approximately 6 h after the start.

14. The method according to claim 1, including the selection of the indicated recombinant protein through the period of time from approximately 2 to approximately 8 h after the start of addition of inducer to the culture.

15. The method according to 14, including the selection of the indicated recombinant protein through the period of time from approximately 3 to approximately 6 h after the start of addition of inducer to the culture.

16. The method according to claim 1, characterized in that the inductor represents the arabinose.

17. The method according to claim 1, comprising continuously adding a carbon source in the culture before induction.

18. The method according to claim 1, comprising n the continuous addition of carbon source in the culture before and during induction.

19. The method according to claim 1, comprising continuously adding a carbon source in the culture at that time as specified inductor continuously add in the specified culture.

20. The method according to claim 1, comprising continuously adding a carbon source in the culture up until the density of the cells in this culture reaches OD600equal to from about 70 to about 110.

21. The method according to claim 1, characterized in that the carbon source is a source of carbon-based sugars.

22. The method according to item 21, wherein the specified source of carbon-based sugars represents glucose.

23. The method according to item 22, characterized in that the inductor represents the arabinose.

24. The method according to item 23, which includes the continuous addition of glucose to the culture medium while the arabinose continuously added to the culture medium.

25. The method according to item 23, which includes the continuous addition of glucose as long as the density of cells in a given culture reaches OD600approximately 80.

26. A method of obtaining a recombinant protein, comprising:
a) introducing into a bacterial cell by the host expression vector encoding a recombinant protein under the control of the inducible promoter, to obtain a recombinant bacterial cell;
b) introduction indicated the recombinant bacterial cells in a culture medium, to obtain a cell culture;
c) adding a carbon source into the specified cell culture in the form of a continuous feed;
d) monitoring the growth of cells in culture threshold optical density (OD600);
e) adding an inductor specified inducible promoter in the indicated cell culture in the form of a continuous feed, as soon as the threshold optical density (OD600) is reached, the total amount of inducer added to the culture in the process of filing inductor is equal to from about 7 g/l to about 15 g/l; and
f) isolation of the indicated recombinant protein from the specified cell culture.

27. The method according to p, including continuous addition of inducer to the culture, when the density of cells in a given culture reaches OD600equal to from about 70 to about 110.

28. The method according to item 27, which includes the continuous addition of inducer to the culture, when the density of cells in a given culture reaches OD600equal to from about 70 to about 105.

29. The method according to p, including continuous addition of inducer to the culture, when the density of cells in a given culture reaches OD600;
equal to from about 75 to about 85.

30. The method according to clause 29, which includes the continuous addition of inducer to the culture, when platnost the cells in this culture reaches OD 600approximately 80.

31. The method according to p, characterized in that the inductor is added to the culture at a constant speed, the specified inductor is added to the culture through the DO-stat feeding or the specified inductor is added to the culture through a pH-stat feeding.

32. The method according to p, characterized in that the inductor is added to the culture at a constant speed equal to from about to 3.35 g/l/h to about 16 g/l/h, this inductor is added to the culture through the DO-stat feeding or the specified inductor is added to the culture through a pH-stat feeding.

33. The method according to p, characterized in that the total amount of inducer added to the culture, is from about 4 g/l to about 40 g/L.

34. The method according to p, characterized in that the total amount of inducer added to the culture, equal to approximately 10 g/L.

35. The method according to p, including the selection of the indicated recombinant protein from the specified crops after the interval of time from about 2 h to about 8 h after the start of supply of the inductor.

36. The method according to p, including the selection of the indicated recombinant protein from the specified crops after the interval of time from about 3 h to about 6 h after the start of supply of the inductor.

37. The method according to p, characterized in that the specified source pirodomast in the specified cell culture at the time of filing a constant speed as a carbon source, and the inductor, in the process of DO-stat feeding as carbon source and inducer, or during pH-stat feeding as carbon source and inducer.

38. The method according to p, characterized in that the inductor represents the arabinose.

39. The method according to p, characterized in that the carbon source is a source of carbon-based sugars.

40. The method according to § 39, characterized in that the carbon source on the basis of sugar is a glucose.

41. The method according to p, characterized in that the carbon source is a glucose and the inductor represents the arabinose.

42. The method according to paragraph 41, including continuous addition of glucose to the cell culture, while the arabinose continuously added to the specified cell culture.

43. The method according to paragraph 41, including continuous addition of glucose to the cell culture up until the density of the cells in this culture reaches OD600approximately 80.

44. A method of obtaining a recombinant protein, comprising:
cultivation of recombinant bacterial cells for expression of recombinant protein using continuous addition of inducer to the culture, including specified bacterial cells after the culture reached a threshold parameter, otlichalis the same time, that said bacterial cell comprises a sequence of nucleic acids, the corresponding gene from N. meningitidis serological group b, while the total amount of inducer added to the culture in the process of filing inductor is equal to from about 7 g/l to about 15 g/L.

45. The method according to item 44, wherein the specified threshold parameter represents the optical density (OD), dissolved oxygen (DO), pH, concentration of nutrients in the culture medium, the total concentration of the carbon source added to the culture medium, or any combination of them.

46. The method according to item 45, wherein the specified threshold parameter represents the optical density (OD) of the culture.

47. The method according to item 46, wherein the continuous supply of the inductor begins when the density of cells in culture reaches OD600equal to from about 70 to about 110.

48. The method according to p, characterized in that the continuous supply of the inductor begins when the density of cells in culture reaches OD600equal to from about 70 to about 105.

49. The method according to p, characterized in that the continuous supply of the inductor begins when the density of cells in culture reaches OD600equal to from about 75 to about 85.

50. JV is own by § 49, characterized in that the continuous supply of the inductor begins when the density of cells in culture reaches OD600approximately 80.

51. The method according to item 44, wherein the inductor is added in the specified culture at a constant speed equal to from about to 3.35 g/l/h to about 16 g/l/h, the specified inductor type by DO-stat feeding or the specified inductor type by pH-stat feeding.

52. The method according to item 44, wherein the total amount of inducer added to the culture in the process of induction, equal to approximately 10 g/L.

53. The method according to item 44, wherein the supply of the inductor continues for from about 2 h to about 8 h after the beginning of the specified method.

54. The method according to item 53, wherein the supply of the inductor continues for from about 3 h to about 6 h after the beginning of the specified method.

55. The method according to item 44, characterized in that the recombinant protein is collected through the period of time from about 2 h to about 8 h after the start of supply of the inductor.

56. The method according to § 55, characterized in that the recombinant protein product emit through the period of time from about 3 h to about 6 h after the start of supply of the inductor.

57. The method according to item 44, wherein the feed is a carbon source in the culture medium is continued in the process of filing inducer in the culture.

58. The method according to item 44, characterized in that the inductor represents the arabinose.

59. The method according to item 44, characterized in that the carbon source is a source of carbon-based sugars.

60. The method according to p, characterized in that the carbon source on the basis of sugar is a glucose.

61. The method according to p, characterized in that the inductor represents the arabinose.

62. The method according to p, wherein glucose is fed to the culture at a constant feeding speed at the time of filing with constant speed arabinose in the culture through the DO-stat feeding as glucose and inductor, or by pH-stat feeding as glucose and inducer.

63. The method according to p, wherein glucose is fed to the culture at a constant feeding speed at the time of filing with constant speed arabinose in the culture through the DO-stat feeding as glucose and inductor as long as the density of cells in culture has not reached OD600approximately 80, or by pH-stat feeding as glucose and inductor as long as the density of cells in culture has not reached OD600approximately 80.

64. The method according to p, characterized in that the recombinant meningococcal protein 2086 includes lipoprotein (rLP2086).

65. The method according to p characterized in that that this protein includes Neopalimovsky protein.

66. The method according to p, characterized in that the protein includes meningococcal protein 2086 subfamily A.

67. The method according to p, characterized in that the protein includes meningococcal protein 2086 subfamily Century

68. A method of obtaining a recombinant meningococcal protein 2086 (R), including:
(a) introducing into a bacterial cell by the host expression vector encoding a recombinant protein 2086 under the control of the inducible promoter, to obtain a recombinant bacterial cell;
(b) the introduction of the indicated recombinant bacterial cells in culture medium to obtain a culture;
(c) continuous addition of a carbon source in the specified culture;
(d) monitoring the growth of cells in a given culture threshold optical density (OD);
(e) continuous addition of inducer specified inducible promoter in culture, as soon as the density of cells in a given culture reached an optical density of approximately from 70 to 110, while the total amount of inducer added to the culture in the process of filing inductor is equal to from about 7 g/l to about 15 g/l; and
(f) the allocation of the indicated recombinant meningococcal 2086 protein from the specified crops after the interval of time from the AP is sustained fashion 3 h to about 6 h after the start of continuous addition of inducer.

69. Composition for the isolation of recombinant meningococcal protein 2086, including: bacterial culture comprising a recombinant meningococcal protein 2086 at a density equal to at least about 1.5 g/l in total volume specified bacterial culture.

70. The composition according to p, characterized in that the recombinant meningococcal protein 2086 is libidinously protein.

71. The composition according to p, characterized in that the recombinant meningococcal protein 2086 is Neopalimovsky protein.

72. The composition according to p, characterized in that the recombinant meningococcal 2086 protein is a protein 2086 subfamily A.

73. The composition according to p, characterized in that the recombinant protein is a protein 2086 subfamily Century

74. The composition according to p, characterized in that the said culture comprises a second recombinant meningococcal protein 2086.

75. The composition according to p, characterized in that the said second recombinant meningococcal 2086 protein is a protein 2086 subfamily A.

76. The composition according to p, characterized in that the density of the indicated recombinant meningococcal protein 2086 equal to at least about 1.7 g/l in total volume specified bacterial culture.

77. Com is azizia on p, characterized in that the density of the indicated recombinant meningococcal protein 2086 equal to at least about 2.0 g/l in total volume specified bacterial culture.

78. The composition according to p, characterized in that the density of the indicated recombinant meningococcal protein 2086 equal to at least about 3.0 g/l in total volume specified bacterial culture.

79. Composition for the isolation of recombinant meningococcal protein 2086, including:
bacterial culture comprising a recombinant meningococcal 2086 protein obtained according to the methods according to any one of claims 1 to 75.



 

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SUBSTANCE: there are described versions of compositions of human glypican 3 IgGl antibodies. Each composition contains a mixture of human glypican 3 antibodies in an effective amount. The mixture consists of the antibodies of standard structure and either more than 20 % of the fucose-free antibodies, or the antibodies having bisectional N-acetylglucosamide. Each antibody is characterised by a certain amino acid sequence. One version of the composition provides the antibody containing three CDR light and three CDR heavy chains. According to the other version, each antibody is characterised by the presence of variable areas of heavy and light chains. There are described versions of the method for preparing glypican 3 antibody with using a cell with the reduced ability of fucose attachment to sugar chains into which the gene coding the antibody from the composition is introduced. What is offered is an anticancer drug on the basis of the antibody composition.

EFFECT: using the invention provides higher ADCC cytotoxic activity that can find application in cancer therapy.

10 cl, 7 dwg, 2 tbl, 9 ex

FIELD: medicine.

SUBSTANCE: what is offered is a compound of structure (I) presented in the patent claim, referred to labyrinthopeptines. There are also offered a method for preparing the compound of formula (I) and the pharmaceutical composition containing the compound of formula (I).

EFFECT: compound can be applied for preparing the drug for bacterial infections caused by gram-positive bacteria or for neuropathic pain or pain initiated by inflammations.

19 cl, 7 tbl, 18 ex

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1 dwg, 4 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: there are presented versions of antibodies specific to claudin 18A2 produced by immunisation by a related amino acid sequence or a nucleic acid or a host cell expressing said peptide. The antibodies possess an ability to mediate elimination of cancer cells expressing claudin 18A2. There are disclosed versions of antibody-producing hybridomas. What is described is a conjugate or a pharmaceutical composition on the basis of antibodies or conjugates for elimination and/or inhibition of a cancer cell expressing claudin 18A2. There are disclosed versions of the method for growth inhibition and/or elimination of the cancer cell, as well as for treating or preventing a disease or a disorder involving cancer cells expressing claudin 18A2 with using the antibodies, conjugate and pharmaceutical composition under the invention.

EFFECT: use of the invention can find further application in medicine for treating cancer cells expressing claudin 18A2.

39 cl, 33 dwg, 5 tbl, 10 ex

FIELD: biosynthesis.

SUBSTANCE: invention refers to isolation of nucleic acid molecules that mediate enzymes required for biosynthesis route of lantibiotic 107891 and its homologs. NC contains nucleotide sequence (NS) selected out of a) mlb gene cluster with a certain sequence or b) sequence that corresponds to the sequence a) due to genetic code degeneracy and mediates the same polypeptides, or c) any of ORF NS that mediate polypeptides with the determined amino acid sequence or their combinations. Recombinant vector for transformation of a host cell capable of producing lantibiotic 107891, its homologs and precursors were discovered in the invention. Lantibiotic 107891 or its derivatives are received by culture of host cell transformed by the vector in conditions suitable for cell growth, gene expression and production of the lantibiotic ot its derivative. The isolated polypeptide involved in biosynthesis of lantibiotic 107891 and its homologs is described. Lantibiotic 107891 is beneficial as food supplement and for antibacterial agent preparation.

EFFECT: antimicrobial activity, particularly, against gram-positive bacteria, including methicillin- and vancomycin-resistant strains.

34 cl, 4 dwg, 1 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: construction of recombinant plasmid DNAs containing a structured gene of human interferon alpha-2b under control of a sensitive promoter enables producing Escherichia coli strain to enable synthesis in the form of an insoluble precursor protein of mature methionine-free human interferon alpha-2b. In the precursor protein, a sequence of mature methionine-free human interferon alpha-2b is fused with a sequence of SUMO (SMT3) Saccharomyces cerevisiae yeast protein. What is produced is the Escherichia coli strain providing biosynthesis of ULP275 proteinase for enzymatic processing of the precursor protein of methionine-free interferon. What is presented is a method for producing methionine-free interferon which provides carrying out the coupled processes of renaturation, disulphide link formation and enzymatic segregation of methionine-free human interferon alpha-2b from the precursor protein under ULP275 proteinase.

EFFECT: invention provides the range of products for preparing human methionine-free IFN-α2.

3 cl, 14 ex

FIELD: medicine.

SUBSTANCE: described is method of purification of recombinant granulocyte colony-stimulating factor (filgrastim) of human. Method characteristics: at the stage of purification from coarse pollutions applied is sorbent SP-Sepharose Fast Flow, elution is carried out by gradient 0.5 M NaCl in acetate buffer pH 5-5.5, stage of fine purification is carried out by ion-changeable chromatography on sorbent YMC BioPro S30, at the stage of purified product concentration applied is sorbent CM-Sepharose Fast Flow, and elution is carried out by gradient 0.5 M NaCl in acetate buffer pH 5-5.5, additionally carried out is stage of gel-filtration by sorbent Sephadex G25, elution is carried out by 13.3 mM sodium-acetate buffer, pH 3.9.

EFFECT: invention makes it possible to considerably reduce waste of protein during purification.

2 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: mouse of line Balb/C is immunised with preparation of membrane proteins of cerebral endotheliocytes. B-lymphocytes of said mouse spleen are isolated and fused with cells of myeloma of line Sp2/0-Ag14 mouse, hybridomas are obtained, supernatants of obtained hybridomas are tested in immune-chemical way and hybridomas, producing monoclonal antibodies to abluminal membrane antigen of cerebral endotheliocytes, are separated. From the selected is hybridoma, characterised by the largest number of antibody-producing hybrid cells, synthesising antibodies belonging to class of G immunoglobulins preserving biological activity in blood serum for not less than 24 hours, monoclonal antibodies are separated from supernatant of selected hybridoma, in particular, by method of affine chromatography on protein-G-agarose.

EFFECT: method by invention ensures obtaining monoclonal antibodies to antigen of cerebral endotheliocytes with high immune-chemical activity, making it possible to visualise cerebral endotheliocytes both ex vivo, and in conditions in vitro and in vivo for characteristics of vascular system of gliomas.

2 cl, 9 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: gene therapy of malignant growths involves a recombinant genetically engineered construct containing an upstream promoter sequence of human gene NDUFV1 of the length of 3574 base pairs with a remote proximal sequence of the length of 91 base pairs and a controlled effector therapeutic cytotoxic gene, as well as the recombinant genetically engineered construct containing the upstream promoter sequence of human gene NDUFV1 of the length of 3574 base pairs with the remote proximal sequence of the length of 91 base pairs and a transposase gene under its transcription control in molar relation 100:1-1:100.

EFFECT: invention provides extended range of products for genetic therapy of various malignant growths caused by terminal cell growth.

3 dwg, 5 ex

FIELD: medicine.

SUBSTANCE: polypeptide, which is used in composition of pharmaceutical composition and in sets for screening of adhesion inhibitors of platelet adhesion or aggregation, is obtained in recombinant way applying matrix of cDNA of Anopheles stephensi salivary gland.

EFFECT: invention makes it possible to obtain polypeptide which possesses inhibiting activity with respect to platelet aggregation or inhibiting activity with respect to platelet adhesion.

10 cl, 4 dwg, 5 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: claimed invention relates to optimised fused protein for blocking BLyS or APRIL, which contains extracellular region of N-end of truncated TACI (transmembrane activator and CAML-partner) and Fc sequence IgG. TACI segment of fused protein contains sequence of amino-end region of extracellular region, starting with 13-th amino acid residue, complete sequence of stem area from TACI and is obtained from native sequence of TACI between 12-th and 120-th amino acids. Segment Fc of immunoglobulin IgG of fused protein contains hinge region, CH2 region and CH3 region, TACI segment and Fc segment are fused either directly or through linker sequence. In addition, claimed is DNA sequence which codes fused protein, expression vector, host-cell, pharmaceutical composition, containing fused protein, and application of fused protein for blocking BLyS or APRIL. Obtained fused protein does not degrade in process of expression, possesses high biological activity and high level of expression.

EFFECT: fused protein in accordance with claimed invention can be used in treatment of diseases, associated with abnormal immunologic functions and in treatment of diseases caused by abnormal proliferation of B-lymphocytes.

10 cl, 6 dwg, 8 ex

FIELD: medicine.

SUBSTANCE: there are offered: IL-6 receptor antibody, a coding gene, a vector and a host cell for producing the antibody, a method for producing the antibodies, and a pharmaceutical composition for treating IL-6-related diseases containing the antibody.

EFFECT: use of the invention provides new humanised IL-6 receptor antibodies that can find further application in therapy of the IL-6-mediated diseases.

8 cl, 22 dwg, 8 tbl, 9 ex

FIELD: medicine.

SUBSTANCE: protein is capable to inhibit thrombin activity specifically. Besides, what is offered is a nucleic acid coding said chimeric protein, a vector containing this nucleic acid and a host cell carrying the vector. The present invention also refers to a pharmaceutical composition containing the recombinant chimeric protein of neutrophils and girugen inhibition factor. An effect of the given composition consists in thrombocyte aggregation inhibition or peripheral leukocyte activation inhibition.

EFFECT: composition can be used for treating a cardio-cerebrovascular disease or preventing a cerebral ischemic injury or a cerebral hematoma.

13 cl, 11 dwg, 16 tbl, 17 ex

FIELD: medicine.

SUBSTANCE: invention refers to a method of producing a mutant lactobacillus Streptococcus thermophilus, to a milk ferment, a method of producing a fermented milk product and to the fermented milk product. The offered invention can be used for producing the fermented milk products with improved storage characteristics. A method of producing the mutant lactobacillus Streptococcus thermophilus characterised by weaker postoxidation, than a parent strain is implemented by introducing in DNA a genome of said parent strain of mutation codon 552 coding histidine, of teh domain HA of lactose permease. Said mutation induces replacement of said histidine by amino acid which is distinct from serine, tyrosine, histidine and threonine.

EFFECT: invention allows producing the mutant lactobacillus Streptococcus thermophilus characterised by weaker postoxidation and suitable particularly for producing the fermented milk products.

10 cl, 5 dwg, 3 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: there is offered a method of engineering yeast strains - stable human growth hormone (somatotropin) producers. Also, there are offered two strains Y-3506 of Russian National Collection of Industrial Microorganisms and Y-3507 of Russian National Collection of Industrial Microorganisms - stable human somatotropin producers. The producer strains have been prepared by sequential integration of expression plasmid into a recipient stain genome. Each plasmid carries in its structure a somatotropin gene (GH1); fused with a leader sequence controlled by the GAL1 promotor, as well as one of the genes URA3, LEU2, TRP1 and HIS3 complementing auxotrophic recipient strain mutations.

EFFECT: efficacy of the produced strains is 100-130 mg of somatotropin per 1 l of the medium.

3 cl, 7 dwg, 6 ex

FIELD: medicine.

SUBSTANCE: invention relates to field of medicine and deals with anti-sense oligonucleotides for treatment and/or prevention of, at least, one of the following diseases: asthma, hypereosinophilia. Essence of the invention includes oligonucleotides aimed against sequences of nucleic acids, coding receptor, selected from group, which consists of receptor CCR3 and common subunit of receptors IL-3, IL-5 and GM-CSF with sequences SEQ ID NO:1 and SEQ ID NO:14.

EFFECT: reduction of toxicity in comparison with hormonal medications.

39 cl, 8 ex, 11 tbl, 21 dwg

FIELD: medicine.

SUBSTANCE: offered is a microorganism producing homosuccinic acid, of Mannheimia, Actinobacillus or Anaerobiospirillum genus. Said microorganism has been produced by destructing a lactate dehydrogenase (ldhA) coding gene, a phosphotransacetylase (pta) coding gene and an acetate kinase (ackA) coding gene, without destructing a pyruvate format lyase (pfl) coding gene. Besides, offered is a method for producing said microorganism and a method for producing succinic acid with applying thereof. The offered mutant microorganism exhibits a property of high growth rate and efficacy of succinic acid in the absence or low production of other organic acids, as compared to the previous strains producing succinic acid.

EFFECT: said microorganism is available for producing succinic acid for industrial application.

13 cl, 7 dwg, 2 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: constructed are recombinant plasmid DNA pAUTL-GFP bearing a hybrid gene coding green fluorescent protein (GFP) and a signal sequence of autolysin from Chlamydomonas reinhardtii. The produced plasmid is used to transform an Agrobacterium strain. A suspension of the transformed agrobacterial cells in a logarithmic growth stage is incubated with cells of Chlorella microalgae for 14-16 hours A protein secretion level is evaluated by measuring fluorescence of a culture environment.

EFFECT: new compounds show effective biological properties.

2 cl, 1 dwg, 1 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: strain Pseudomonas species 181a recovered from Karelian breaking rock with using a selective minimal mineral medium containing insoluble calcium phosphate; it is deposited in the State Collection of Pathogenic Microorganisms and Cell Cultures GKPM-Obolensk (Obolensk settlement, Serpukhovsky area of Moscow region) No. B-6646. The strain shows high ability to release phosphates from insoluble mineral raw material; it is active against fungi Fusarium, stimulates plant growth, provides higher crop yield. The strain is non-toxic for plants and may be recommended for making a biophosphoric fertiliser with fungicidal properties on its basis.

EFFECT: invention provides higher efficiency of struggle with Fusarium blight control, and providing higher crop yield.

3 tbl, 6 ex

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