Expression vectors with modified cole1 replication initiating site to control amount of plasmida copies

FIELD: biotechnology.

SUBSTANCE: disclosed is expression vector containing ColE1 replication system wherein homology of RNAI and RNAII of ColE1 replication initiating site with free tRNA is modified by one or more mutation in encoding region of RNAI or RNAII gene. Said mutations lead to substitution of one or more bases in loop 1, 2, 3 of RNAI or RNAII. Also described are bacterial cell transformed by said vector and method for production of desired protein from abovementioned bacterial cell. Claimed method includes cell culturing, protein isolation and purification thereof. Increased homology ratio of RNAI and RNAII with free tRNA causes increased amount of plasmida copies. Decreased homology ratio of RNAI and RNAII with free tRNA causes reduced metabolite load in cell that is highly for recombinant protein production.

EFFECT: improved method for controlling of plasmida copy amount.

12 cl, 1 dwg, 7 tbl, 2 ex

 

The present invention relates to improved expression vectors containing the site of replication initiation system ColE1, intended for the production of recombinant proteins and plasmid DNA.

To obtain on an industrial scale of interest recombinant protein or production of plasmid DNA have found an extensive use methods fermentation using genetically modified organisms (GMOs).

The main objective optimization of the fermentation process consists in obtaining cost-effective by maximum quantity of product with high quality. To achieve this goal it is necessary to optimize volumetric efficiency, which represents the number of units of the formed product per unit volume per unit time. Factors of importance for the optimization process are the amount of biomass per unit volume, i.e. the number of cells capable of producing the product, and the amount of protein that can produce every cell. To some extent producing capacity of the cell is proportional to the number of copies of the plasmid (PAC), i.e. the number of plasmids in the cell carrying the gene encoding the recombinant protein. In addition, the importance of the effectiveness of the system of transcription of recombin nnogo protein. While some promoters are weak and do not allow to fully realize the potential of metabolism, many promoters are too strong and leads to overexpression of recombinant protein. Because metabolic resources should be distributed between the expression of recombinant protein and the protein of the host body, the expression system, which has too much efficiency, quickly leads to the depletion of metabolic resources, which causes the death of cells.

In recent years, the use of plasmid DNA in the field of gene therapy was the basis for the development of a whole new industry. There is therefore a need to obtain sufficient amounts of plasmid DNA of high quality. In the process of obtaining the plasmid recombinant protein is not produced, instead of the cell "factory" is used for the production of plasmid DNA. To achieve this goal it is necessary to provide very high speed replication of plasmids, and a host cell must perform tasks other than the production of recombinant protein.

For fermentation processes using bacteria mainly used plasmids ColE1 due to the fact that when using this system it is possible to obtain a large number of copies of the plasmid.

Previously described plasmid ColE1 (Chan and others, 1985), conducted a detailed study of the mechanism of action site of initiation of replication of ColE1 (Cesareni and others, 1991). The process of replication of plasmid ColE1 begins with transcription of preprimer PHKII located at a distance of 555 base pairs against the course of transcription relative to the site of replication initiation, using RNA polymerase of the host (Tomizawa, 1985). // PHKII in the process of lengthening forms folds, with a specific structure, and after polymerization of about 550 nucleotides begins to form a hybrid with the DNA matrix. Transcription of preprimer ends heterogeneous and after the formation of the hybrid preprimer PHKII otscheplaut using RNase H, getting active primer with a free 3'-Oh-end that is available for DNA polymerase I (Tomizawa, 1990; Lin-Chao and Cohen, 1991; Merlin and Polisky, 1995).

Region ColE1 contains two promoters. PHKI is an antisense RNA molecule, consisting of 108 nucleotides, which is transcribed by a second promoter on the opposite chain and is complementary to the 5'-end PHKII. PHKI transcribed from the position located at a distance of 445 base pairs against the course of transcription relative to the site of replication initiation to approximate the locus where to start transcription PHKII (Merlin and Polisky, 1995; Tomizawa, 1990).

<> To view the regulation of the number of copies of the plasmid ColE1 characteristics kinetics play a more important role than the characteristics of the equilibrium. For example, some of the mutant strains with mutations in PHKII, which, although they do not affect the field of complementary PHKI, lead to reduced inhibition caused PHKI. This is likely due to effects on the half-life of the intermediate structures of RNA, reducing the time during which they are sensitive to the action of PHKI, which leads to an increase in the number of copies of the plasmid. This fact confirms the importance of intermediate structures PHKII and kinetics of formation of folded structures PHKII (Gultyaev and others, 1995).

It was found that the depletion of the (lack of) amino acids leads to the formation of large quantities of tRNA that are not associated with specific amino acids (below such tRNA labeled as "free tRNA"). This phenomenon can be compared with the above-described situation arising after the induction of expression of recombinant protein in the case when metabolic resources are exhausted.

Wróbel and Wegrzyn (1998) studied the strategy for selective induction of exhaustion five different amino acids. It was found that there is a positive correlation between homology loops of the tRNA anticodon corresponding to the depletion of certain aminokisloty certain loops in the structures PHKI and PHKII. It has been suggested that the translation mechanism captures a large part of the aminoacyl-tRNA, however, unlike the free tRNA have the opportunity to interact with other molecules, such as PHKI and PHKII. It is most likely that the interaction between tRNA and PHKI or PHKII can affect the interaction between PHKI and PHKII, leading to higher frequency of hybridization PHKII-DNA (assuming that the interaction of tRNA with PHKII does not change in any significant way the structure PHKII). The latter may lead to a higher frequency of replication and thereby increase the CCP.

Zavachev and Smith (1988) have compared the homology in total 21 tRNA and PHKI/PHKII. Among them, 11 were detected degree of homology with either PHKI or PHKII in excess of 40%. These tRNA were divided into three categories: (a) tRNA homologous PHKI: Arg, His, Leu, Lys, Phe and Thr, (b) tRNA homologous PHKII: f-Met (formylmethionine). Try and Gly, and C) tRNA homologous as PHKI and PHKII: Met and Val. All tRNAs have a loop anticodon, consisting of 7 nucleotides (Hjalt and Wagner, 1992). In the case of tRNA homologous PHKI, the highest degree of homology was observed in the area of the loop 2, while the lowest degree of homology was observed in the area of 5'-end PHKI.

Depletion of amino acids and cellular stress lead to increased amounts of free tRNA, which interact with item initiation of replication of plasmid ColE1. This interaction is due to the homology sequence of tRNA with three loop-like structures of RNA present in PHKI and PHKII site of replication initiation, and leads to interaction with regulatory CCP mechanism system. The result is a rapid increase in the CCP, which leads to disruption of the fermentation process.

To resolve these problems, WO 89/07141 was proposed expression vector having a replication ColE1 and having a mutation in the gene PHKII and/or rop gene, created to enhance expression. This was achieved without a significant increase in the number of copies of the plasmid.

Because the fermentation process using bacteria is effective only in the case when the system can be maintained for a long period of time, and because the increase in the number of copies of the plasmid is one of the main factors that lead to the collapse of the expression system, the object of the invention was to develop improved systems for the expression of differing prolonged life time (increased viability of the bacteria in the fermentation process.

In particular, the invention was the development of expression systems in which the number of copies of the plasmid after induction of the expression system limit compared to the uncontrolled amplification on the I, in order to maintain metabolic load below lethal levels.

The next task of the invention was to increase the speed of replication of plasmids and thereby release the plasmid DNA in the process of producing plasmids.

To solve these problems, in the claimed invention was used, the mechanism of replication ColE1-type. Specifically, this meant that by using the method of genetic engineering have changed, preferably reduced degree of homology of the site of initiation of replication of ColE1-free tRNA, or completely eliminated. Alternatively created a random library, designed for selection of plasmids with altered ability to replicate, for example, with a large number of copies of plasmids.

The present invention relates to an expression vector carrying the ColE1 replication system, in which the degree of homology PHKI and PHKII site of initiation of replication of ColE1-free modify tRNA with one or more mutations in the coding region of the gene PHKI and one or more corresponding mutations in the gene PHKII, and the specified(s) mutation(s) result in the replacement of one or more base pairs in the loop 1 or loop 2 or loop 3 PHKI and PHKII.

The term "mutation" includes mutations that increase and mutations that reduce the degree of homology PHKI and PHKII with free the mi tRNA.

For maximum preservation of secondary structure and melting point PHKI and PHKII in order not to degrade the ability to replicate, preferably using mutations, representing the replacement of complementary bases, i.e. mutation type And→T T→And C→G, G→C. May be present and the other(s) mutation(s), provided that it(I) does not affect the replication mechanism.

Unlike vector, ColE1, described in WO 89/07141, which has a mutation in the gene PHKII and therefore, thanks to its position relative to the promoter of the gene PHKI, vector according to the invention has a mutation in the coding region, more specifically in the areas of the loops as gene PHKI and gene PHKII that free homologous tRNA. Thus, the invention proposed a new strategy that aims to change the degree of homology and ultimately to regulate the speed of replication of plasmids.

In the context of the present description, the term "loop" refers preferably to unpaired loop structures PHKI or PHKII; however, this concept is not limited only by the scope of the loop and may also include nucleotides adjacent to the stem region, preferably no more than two nucleotides.

Mutation(s) may(may) be a substitution of one base in the loop 1, loop 2 or loop 3, or one or any number of substitutions of bases, including the I replace all of the bases, in loop 1 and/or loop 2 and loop 3.

Preferably, the mutation(s) present(s) in loop 2, which is an area having the highest degree of homology with the free tRNA.

The desired mutations in the loop(s) of the gene PHKI and PHKII you can create conventional methods mutagenesis and cloning.

According to one of embodiments they can be created as follows: using gene PHKI or gene PHKII or its fragment as a matrix, perform PCR using as primers the two oligodeoxyribonucleotides, one of which or both are desired(s) mutation(s). Preferably, PCR is a PCR, which is carried out in two stages. In the first stage amplified two overlapping segments, one of which has the desired mutation in the sequence of the primer between the restriction site, designed to connect the fragments, and binding site of the primer. Then amplificatoare fragments were cleaved with the appropriate restrictase, are ligated and used as a matrix in the next stage PCR amplification. At this stage, using the same primers as in the first stage, which does not contain the newly introduced restriction sites selected for binding against the course of transcription and downstream transcription with the nearest unique sites Rees is Ricchi specific plasmids.

Thanks to the complementarity of genes PHKI and PHKII both genes used to obtain the vector, or fragments thereof equally can be used as matrices when performing PCR amplification. In the presence of complementarity any(s) mutation(s) in one or more loops of the same gene lead to the creation of the corresponding mutations in other, complementary gene; in the above-described preferred method containing the mutation(s) primer serves not only to extend the use of the polymerase, but also as a matrix for DNA polymerase, thereby creating a complementary chain containing the mutation(s). Depending on which gene is used as a matrix: gene PHKI or gene PHKII, PHKII or PHKI will automatically carry complementary mutation.

Preferably as a matrix using a plasmid containing the full genes PHKI and PHKII. Alternatively, you can use a DNA molecule encoding a full gene PHKI or PHKII. Genes PHKI and PHKII described by Tomizawa and others, 1977.

In that case, if the matrix using a fragment of the gene PHKI or PHKII, the fragment must have a length sufficient to include all the necessary elements, i.e. the sequence that should be subjected to mutation, the binding site of primer and optionally one or more restriction sites. Preferably fragm the NT contains one or more loops (each of which consists of approximately 7 nucleotides) and the binding site of the primer (approximately 18 nucleotides), i.e. the minimum amount of suitable fragment of approximately 25-30 nucleotides.

According to a preferred variant of the invention, mutations with respect to their location and the number chosen in such a way as to provide a substantial change in the degree of homology with the maximum possible number of species of free tRNA.

So, in one of the embodiments of the invention the modification PHKI and PHKII exercise, starting with the modification of the loop 2, i.e. the loop with the highest degree of homology, and this modification is to exercise substitutions in the possible number of provisions. For example, as demonstrated in the experiment described in example 1, loop 2 can be modified by replacing six of the seven nucleotides, while maintaining without change of one base (position 693 in plasmid ColE1, Genbank GI 9507253), which thus may serve as part of a new restriction site, preferably Ncol site. Base replace the corresponding complementary to them by reason.

With this approach, if necessary, to completely eliminate homology with all the free tRNA. Therefore, this approach provides the maximum degree of freedom in the production of a wide variety of interest recombinant proteins n the matter what amino acid sequence they have, in particular, due to the regulation of replication of plasmids, ensuring the viability of cells by expression. In this case, the amplification of plasmids adjustable only for specific ColE1 replication mechanism and does not depend on fluctuations of metabolism in the cell-host; the number of copies of the plasmid remains almost constant during the fermentation process.

Unlike options, providing a complete elimination of homology between PHKI and PHKII and free tRNA specified homology can be modified, i.e. to increase or decrease only to a certain desired degree. For some applications, for example, in the case when the yield is insufficient due to the fact that the potential mechanism of expression is not implemented fully due to the decrease in the number of copies of plasmids and accordingly the presence of suboptimal amounts of plasmids ("gene dose"), it may be desirable to increase the speed of expression at the expense of a small increase in the number of copies of plasmids. This can be achieved by selective preservation of sequence homology with certain free tRNA, in particular, with rare tRNA. The literature describes a sequence homologous to certain free the RNA (Zavachev and Ivanov, 1988). This strategy can also be used to influence the ratio of secreted forms of the protein so that the protein can be present either in the form of intracellular Taurus or be in a soluble form. For example, when a certain degree of amplification can be formed predominantly intracellular calf, while a small decrease can stimulate the formation of soluble product.

In some cases it is preferable to increase the number of copies of plasmids by increasing the degree of homology sequences PHKI and PHKII free tRNA, in particular to obtain plasmid DNA. Mutations necessary to increase the degree of homology sequences are also described in the literature (Zavachev and Smith, 1988) and can be created based on the same principles as described for reducing the degree of homology of the sequence.

For a specific case and/or obtain product-specific method can be optimized by experimental studies of various mutations. Corresponding experiments can be performed as follows: intended(YM) for testing the plasmid-"candidate" or a set of plasmid-candidates carrying the mutation(s), transfection appropriate bacterial cell hosts, grown in appropriate laboratory the conditions, for example, in shake flasks, and observe the fermentation process, registering the parameters of interest, in particular, the growth, yield and its quality, the number of copies of plasmids.

According to another variant implementation, which is preferable for obtaining a wide variety of modifications of the sequences, perform a random mutation in one or several positions of the loop 1 and/or loop 2 and/or loop 3, thereby creating a library that can be used to construct expression vector intended for the implementation of any required property expression system. For example, plasmid-"candidate" can be selected with respect to the selection of the parameters that are most important for the production of recombinant proteins, such as growth rate, productivity and viability; however, the process is carried out using the above standard experimental method.

In addition, this approach allows to effectively regulate the rate of expression of the recombinant protein through manipulation of the CCP. In case of interest gene present in the vector, containing the library, selected plasmid will always be optimal from the point of view of the expression of the gene of interest.

what a time as in the ordinary course of fermentation after induction there is an almost tenfold increase in the number of copies of the plasmid (PAC), the use of the method according to the present invention can reduce or eliminate the homology between tRNA and PHKII. This leads to an increase in the share of neighborouing molecules PHKI, which do not interact with PHKII. Thus, the replication mechanism ceases to be associated with high levels of free tRNA, resulting in the excessive metabolism due to the expression of recombinant protein. The separation mechanism of replication of plasmids from metabolic stress caused by expression of a recombinant protein, allows to obtain a higher yield of recombinant protein.

Because replacement of the bases performed in accordance with the present invention, are present in PHKI and PHKII, which leads to a change of homology with all tRNA, the composition of free tRNA (dependent recombinant product) is not critical for this approach.

In addition to the modified system replication ColE1 expression vector according to the invention contains the elements necessary for expression of the protein, i.e. controlling the expression sequences that are functionally related to the cDNA sequence that encodes interest protein, including the promoter region is th initiation of translation, breeding markers (e.g. markers of resistance to antibiotics), restriction sites for embedding the DNA that encodes interest protein, etc.

Preferably the expression vector according to the invention is obtained on the basis of the following vectors:

pMBl (Bolivar and others, 1977);

pBR322 (Covarrubias and others, 1981; can be obtained from MBI Fermentas by catalog number No. SD0041; GenBank/EMBL, extension sequences J01749, K, L08654, M, M, M, M, M, M, M, M, V01119);

pUC18 (Yanisch-Perron and others, 1985; GenBank/EMBL, incremental sequence number L09137; can be obtained from MBI Fermentas by catalog number No. SD0061);

pUC19 (GenBank/EMBL, incremental sequence number L09136 can be obtained from MBI Fermentas by catalog number No. SD0051);

pTZ19R (GenBank/EMBL, incremental sequence number Y14835; can be obtained from MBI Fermentas by catalog number No. SD0141);

pTZ19U (can be obtained from MBI Fermentas by catalog number No. SD0161; GenBank/EMBL, incremental sequence number Y14835);

pBluescriptII KS (-) (Alting-Mees and others, 1989; GenBank/EMBL, incremental sequence number H);

pBluescriptII KS (+) (Alting-Mees and others, 1989; GenBank/EMBL, incremental sequence number H);

pBluescriptII SK (-) (Alting-Mees and others, 1989; GenBank/EMBL, incremental sequence number H;

pBluescriptII SK (+) (Alting-Mees and others, 1989; GenBank/EMBL, incremental sequence number H).

From the point of view of representing the th interest for protein sequence no restrictions, if the expression of the plasmid in E. coli results in having a functional activity of the protein.

In the experiments with the creation of the present invention, used a cDNA encoding a human Cu-Zn superoxide dismutase. Using a vector carrying this cDNA can be obtained viagrastoresyi dimeric protein having a molecular weight of 32 kDa, which consists of 153 amino acids and is released into the cytoplasm (Cserjan-Puschmann and others, 1999).

You can use any bacterial cell host that is compatible with plasmids ColE1 type, preferably strains of E. coli, in particular the strain HMS 174 (DE3) (Studier and Moffat, 1986) or Salmonella.

Another variant of implementation of the present invention relates to a cell host transformed by the expression vector carrying the modified site of initiation of replication of ColE1.

For transformation of strain-master can be applied by any conventional method, for example, electroporation or the deposition of calcium chloride or calcium.

The next object of the present invention is a method for representing the interest of the recombinant protein, which consists in the fact that the cell-master transform E. coli expression vector, which has a system of replication ColE1 containing mutations in one or more loops gene PHKI and gene PHKII grow to fit the existing conditions and allocate interest protein. The invention allows to speed up the process of producing recombinant protein, providing a compensation mechanism of interaction in the expression of recombinant protein and metabolism occurring in the cell host. The method according to the invention most preferably should be applied using periodic fermentation processes with water, i.e. processes in which the addition of nutrients associated with an increase in biomass. To fully realize the benefits of periodic processes with water, which can occur over long periods of time and thus allow to obtain higher yields of biomass with greater efficiency compared to conventional batch processes, it is necessary to have a stable and regulated expression system. This problem can be successfully solved by using the expression vector according to the invention.

In addition, since modifying the sequence of one or more loops PHKI and PHKI ColE1 can lead to a strong increase in the speed of replication of plasmids, vectors can be successfully used for production of plasmids, for example, for use in gene therapy. The advantages of the present invention lies in the possibility of reducing the stability of the so-called "kissing complex" ("kissin complex") PHKI/PHKII and thereby increase the speed of replication of plasmids.

Example 1

In our experiments we used a plasmid pET11a (derived plasmids pUC19 company Stratagene). This plasmid contains a gene for beta-lactamase, providing resistance to ampicillin. Expression of recombinant protein in this plasmid is controlled by the effective RNA polymerase of phage T7. The lac operator is located between the sequences of the promoter of phage T7 and site of translation initiation. This leads to the suppression of expression in the absence of inducer IPTG (isopropylthioxanthone). Plasmid pET11a-SOD carries the gene cDNA that encodes a recombinant human protein Cu-Zn-superoxide dismutase (csod), representing viagrastoresyi dimeric protein with a molecular mass of 32 kDa, consisting of 153 amino acids, which is not toxic to cells and is released into the cytoplasm (Cserjan-Puschmann and others, 1999).

For propagation of plasmids and expression of SOD used a strain of the bacterium Escherichia coli HMS174 (DE3) (Studier and Moffat, 1986). This strain contains the polymerase of phage integrated in the chromosomal DNA. Polymerase of phage T7 is important for the expression of recombinant protein. Transformants were selected on plates containing ampicillin (containing antibiotic Wednesday LB, 100 pkg/memecylon) (Maniatis, and others, 1982).

The transformation in the experiments conducted by the method of electroporation using the device is istwa type Bio-Rad Gene Pulser. The primers were obtained from Metabion company (Martinsried, Germany) in the form obtained by vacuum drying the powder, which was dissolved in water to obtain a mother solutions with a concentration of 100 pmoles/µl. PCR was carried out in thermoacetica (T-gradient, firm Biometra, Germany) using a coating of heat-resistant polymerase Dynazyme EXT at a concentration of 1 unit/μl (Finnzymes), 10-fold does not contain magnesium buffer with the addition of 10 mm MgCl (sold), 1 mm dNTP, DMSO and distilled water.

The primers used were pET11a-114back (SEQ ID NO:1), pEZ11a656for (SEQ ID NO:2), PHKI-Ncoback (SEQ ID NO:3) and PHKI-Ncofor (SEQ ID NO:4). Restrictase, lambda markers, the ligase of phage T4, phosphatase from calf intestine was obtained from the company MBI-Fermentas and used according to the recommendations of this company.

For fermentation used the fermenter with a capacity of 20 l firm MBR Bioreactor AG (Wetzikon, Switzerland) with attached controller type MBR IMCS-2000. The working volume of the fermenter was approximately 12 liters

Used culture medium: the amount of a nutrient medium, which is pumped into the system during the phase of the periodic recharge, continuously measured by weighing vessel. Pump for feeding regulated to provide a constant speed, growth μ=0,1. The addition of non started by the signal from the conductivity probe. The lack of contact with the environment p is solala to eliminate the risk of contamination of the fermenter. As a boot environment used semi-synthetic medium containing small amounts of tryptone and yeast extract to facilitate growth after the download begins. The components were mixed in a total volume of about 4 liters (4000 g). In order to avoid sediment, chemicals with the same number (see number below in table 1) first separately dissolved in distilled water. The glucose solution was added with distilled water to 300 g and autoclaved separately. Then all components except glucose solution were mixed with each other in the order specified and supplemented with distilled water to 3700,

Table 1:
The composition of the loaded environment (4000 g). The values given are in grams unless otherwise indicated.
No.Weight (g)Chemical
112Monopotassium phosphate potassium
124Trihydrate secondary hydrogen phosphate potassium
22Tripton (firm Oxoid Ltd., Hampshire, UK)
21Yeast extract
35The dihydrate triacrylate
32The heptahydrate of magnesium sulfate
40,2The dihydrate of calcium chloride
51000Trace element solution [ál]
680The chloride dihydrate, copper (II) [mg]
664Heptahydrate zinc sulfate [mg]
79Ammonium sulfate
77,4Ammonium chloride
866The glucose monohydrate


Table 2:
The composition of the recharge environment (6000 g). The values given are in grams unless otherwise indicated.
No.Weight (g)Chemical
1 18,00Monopotassium phosphate potassium
136,00Trihydrate secondary hydrogen phosphate potassium
251,57The dihydrate triacrylate
220,63The heptahydrate of magnesium sulfate
3to 2.06The dihydrate of calcium chloride
410313,71Trace element solution [ál]
5825,10The chloride dihydrate, copper (II) [mg]
5660,08Heptahydrate zinc sulfate [mg]
60,6The non
792,82Ammonium sulfate
876,32Ammonium chloride
9680,70The glucose monohydrate

To determine the percentage of bacterial cells carrying plasmids, and determine whether to grow cells carrying plasmid on the plates, containing the inductor IPTG that indicates whether load-bearing plasmid cells after induction to produce SOD in "normal" quantities, carried out the test of Koch.

The total amount of dry matter is determined by the dry weight of bacteria (SMBS).

For each sample glass was dried over night at 105°C, cooled in a desiccator and then weighed on an analytical balance.

MMR can be calculated based on the correlation of the size (number of base pairs) of genomic DNA and plasmid DNA.

To obtain plasmid DNA cellular debris derived from the drug sample, resuspendable 150 ál of solution I (50 mm glucose, 10 mm etc, 25 mm Tris-HCl, pH 8.0). Added 200 μl of LTOs (0.5%solution LTOs (firm ICN Biochemicals)was added 50 μl of lysozyme (firm Sigma), samples were mixed and incubated for 10 min at 37°C, the solution is homogenized by rotation. The samples were stored on ice until measurement using spectrofluorometer (type Hitachi F-2000).

To determine the amount of plasmid DNA was performed purification of DNA contained in the cell debris, using a set of type GFX (firm MBI, Fermentas) according to the supplier's instructions with the following modifications: after the stage of lysis was added a certain amount (˜2 μg) pUC19 as an internal standard. After elution of DNA in 50 μl of water was carried out is linearization the plasmid with Hind III for 1 h at 37° C. For the implementation of capillary electrophoresis sample containing cleaved by the restriction enzyme DNA was transferred into a sample vessel without causing air bubbles. The samples were loaded into the sampler. The calibration of the capillary was performed by washing buffer for 15-20 min the absorbance Measurement was carried out at 260 nm and 280 nm using a diode matrix. After analysis, the capillary was rinsed with buffer and kept at 4°C. the Amount of chromosomal DNA was calculated by subtracting the amount of plasmid DNA/mg SMB from the total amount of DNA/mg SMB. Because the amount of added internal standard is known, the PCC can be calculated according to the method described by Breuer and others (1998), by the following formulas:

To determine the amount of SOD having the ability to bind antibody (monoclonal antibody to SOD, clone 30F11, which was received from the company Novocastra Laboratories Ltd., UK) was diluted in the ratio 1:100 with buffer for sensitization of the surfaces (200 μg/ml). 100 μl of the diluted antibody solution was introduced into each well tiralongo microplate, incubated at 4°C overnight or at room temperature for at least 2 hours Tablet three times washed with buffer for washing, after which the buffer was removed by careful is trachymene tablet. The sample and standard in tablets for breeding bred consistently in the ratio of 1:2 using automatic device for pipetting. 50 μl of each individual cultivation was made (by automatic device) in sensitized antibody tablet, and incubated for 1 h at room temperature.

The tablet was rinsed with buffer for washing. Conjugated antibody was diluted in the ratio 1:500 buffer for cultivation (Porstmann and others, 1988).

Table 3 presents mutations at the site of replication initiation (table summarizes the provisions, which produced a replacement, the numbers correspond to the complete sequence ColE1 stored under the GI number="9507253 in Genbank):

Table 3:
692G→C
694T→And
695And→T
696C→G
697With→G
698And→T
699And→T
700C→G

The fermentation process with the use of plasmids pET11achSOD in the E. coli strain HMS174 (DE3) is illustrated in the drawing. Presents data on General and specific production of recombinant p is otein (SOD), and the speed of production, qp. Given the value of the total dry weight of bacteria (SMB) and the number of copies of the plasmid (PAC). In contrast to standard processes (for example, described in Cserjan-Puschmann, 1999) found that the CCP remains almost constant even after the induction carried out on the 45th hour fermentation.

The research results obtained using the test Koch, and the number of copies of the plasmid is presented in table 4

Example 2

In our experiments we used a plasmid pET11a-SOD described in example 1.

For propagation of plasmids and expression of SOD used a strain of the bacterium Escherichia coli HMS174 (DE3)as described in example 1. All manipulations with bacteria and plasmid DNA was carried out according to the method described in example 1. Oligonucleotides and enzymes were obtained from the same sources that described in example 1.

The primers used were pet11a-Sca-I-for (SEQ ID NO:5), pet11a-AlwN-I (SEQ ID NO:6), pet11a-Xba-I-back (SEQ ID NO:7) and PHK-I-randomXba-I-back (SEQ ID NO:8).

To select the most promising candidates from the pool of different clones used two approaches:

1. The selection of cells containing the plasmid with the highest number of copies.

Cells with a large CCP should have high resistance to ampicillin. Pool bacteria were sown on Petri dishes with LB-agar containing either 0,1 or 10 mg/ml ampicillin the and. Took 10 colonies from the plate with LB-medium containing 10 mg/ml of ampicillin, and carried out the sequence analysis. The survey revealed 7 clones (see table 5), the sequence of which differed from the sequences of the loop 2 PHKI and PHKII.

Table 5
PHKIIPHKI
ColE1Mut1ATCTACATGTAGAT
ColE1Mut9ATCTACATGTAGAT
ColE1Mut2TGGATACGTATCCA
ColE1Mut3TTCACCCGGGTGAA
ColE1Mut4CTGTATCGATACAG
ColE1Mut5AACATCCGGATGTT
ColE1Mut7AACATCCGGATGTT
ColE1Mut8AACATCCGGATGTT
ColE1Mut6GCTAGCGCGCTAGC
ColE1Mut10ACTGAAGCTTCAGT

The second selection criterion was the stability of plasmids. Bacteria containing a pool of plasmids were cultivated in shake flasks at 37°to achieve an optical density OD=2 using a synthetic medium containing ampicillin. After the implementation of the three subcultures (which corresponds with listello 20 generations) bacteria, received after the last subculture, were brought into the tablet with LB-medium containing ampicillin, to select for those bacteria that had contained the plasmid, with a single colony was selected for sequence analysis.

The results of this sampling are presented in table 6:

td align="center"> CTCGCCT
Table 6:
PHKIPHKIIPHKIPHKII
ColE1Mut11TTATGAGCTCATAAColE1Mut41CACCCAATTGGGTG
ColE1Mut12TTGCCACGTGGCAAColE1Mut42GCGGAAATTTCCGC
ColE1Mut13CTTACGATCGTAAGColE1Mut43GTGTCAATTGACAC
ColE1Mut14CATGCAATTGCATGColE1Mut44TCGCCNGCNGGCGA
ColE1Mut15GTGACAATTGTCACColE1Mut45TCGCCNGCNGGCGA
ColE1Mut16CCGACAATTGTCGGColE1Mut46TTTCCCGCGGGAAA
ColE1Mut17GGGGAAATTTCCCCColE1Mut47TACCCCGCGGGGTA
ColE1Mut18AGGCGAGColE1Mut48TCGCTAGCTAGCGA
ColE1Mut19AGGCCCTAGGGCCTColE1Mut49TCTTGCCGGCAAGA
ColE1Mut20TTGGTAGCTACCAAColE1Mut50TTGGTACGTACCAA
ColE1Mut21ATAGCAGCTGCTATColE1Mut51TCACCACGTGGTGA
ColE1Mut22TTGAGATATCTCAAColE1Mut52CCGCGAATTCGCGG
ColE1Mut23TTGGTAGCTACCAAColE1Mut53ACGCAAATTTGCGT
ColE1Mut24TTAGCGTACGCTAAColE1Mut54CTGAACTAGTTCAG
ColE1Mut25TTCTGCTAGCAGAAColE1Mut55CCCCCATATGGGGG
ColE1Mut26TTGCCATATGGCAAColE1Mut56CCCCCATATGGGGG
ColE1Mut27GATGGTTCTACCAAColE1Mut57TTTGCCGCGGCAAA
ColE1Mut28TTTTCGCGCGAAAAColE1Mut58TTCGCCGCGGCGAA
ColE1Mut29TACCCCCGGGGTA ColE1Mut59TTCGCCGCGGCGAA
ColE1Mut30CATTCGATCGAATGColE1Mut60GAGGTAGCTACCTC
ColE1Mut31GTTCCGATCGGAACColE1Mut61TGTCCAGCTGGACA
ColE1Mut32GTAGCCATGGCTACColE1Mut62CCTCTAATTAGAGG
ColE1Mut33ACTCTAATTAGAGTColE1Mut63ACGCAAATTTGCGT
ColE1Mut34CTTGGAATTCCAAGColE1Mut64TGGGTAGCTACCCA
ColE1Mut35CCCCCAATTGGGGGColE1Mut65TCTTCACGTGAAGA
ColE1Mut36TTGGTGTACACCAAColE1Mut66TTAGCACGTGCTAA
ColE1Mut37TTGCAATATTGCAAColE1Mut67TTGGTAGCTACCAA
ColE1Mut38TTGCGAGCTCGCAA
ColE1Mut39TGGTCAGCTGACCA
ColE1Mut40ATGTCAATTGACAT

When making this selection was applied the same fermenter and devices that were described in example 1. Used the same nutrient medium and the growth rate specified in example 1.

PAC defined and counted according to the method described in example 1, Characteristics of the clones-"candidates" from the point of view of influence on their CCPT was evaluated in experiments using periodic cultivation process with water. Results for prospective candidates is presented in table 7; the CCP different clones presents for reinducing and induced state.

Table 7:
Without inductionInduction after 3 hInduction after 5 h
ColE1WT54129298
ColE1Mut97502560600
ColE1Mut22136243380
ColE1Mut54346393

Clone ColE1Mut9 (and ColE1Mut1, which has the same sequence) turned out to be a very promising candidate in obtaining plasmids. The number of copies of plasmids equal to 750, the rough is about 14 times greater than the corresponding value characteristic of plasmid ColE1 wild-type.

PAC for clone ColE1Mut22 was also significantly higher (2.5 times).

Lower PAC for ColE1Mut54 can be favorable from the point of view of production of recombinant protein due to lower metabolic load.

1. The expression vector containing the ColE1 replication system, in which the homology PHKI or PHKII site of initiation of replication of ColE1-free modify tRNA with one or more mutations in the coding region of the gene PHKI or one or more of the relevant mutations in the gene PHKII, where the mutation(s) lead(s) to the replacement of one or more bases in the loop 1 or loop 2 or loop 3 PHKI or PHKII.

2. The expression vector according to claim 1, originating from the vector selected from a range that includes pMBI, pBR322, pUC 18/19, pTZ19R, pTZ19U, pBluescriptIIKS (+/-) and pBluescriptIIKS (+/-).

3. The expression vector of claim 1, wherein the mutation leads to a reduction in the degree of homology or elimination of homology PHKI/PHKII free tRNA.

4. The expression vector according to claim 3, where the mutation is present in the loop 2 PHKI and PHKII.

5. The expression vector according to claim 4, where the loop 2 PHKI and PHKII modify by mutations virtually throughout the entire sequence.

6. The expression vector according to claim 5, where six of the seven bases of the loops 2 are substituted for their corresponding complementary bases.

7. Express the ion vector according to claim 6, where loop 2 PHKI contains a sequence TGTAGAT instead of the sequence of wild-type and where the loop 2 PHKII contains a sequence of ATTESA instead of the sequence of the wild type.

8. The expression vector according to claim 6, where the loop 2 PHKI contains a sequence CTGAACT instead of the sequence of the wild type. TTGGTAG and where loop 2 PHKII contains a sequence AGTTGAG instead of the sequence of wild type STASIA.

9. Bacterial a host cell transformed by the vector according to any one of claims 1 to 8.

10. A host cell according to claim 9, which is a cell of E. coli.

11. A host cell of claim 10, which is a cell strain E. coli HMS 174.

12. The method of obtaining the interest of the protein, involving the transformation vector according to any one of claims 1 to 7, which contains the coding protein DNA, functionally associated with controlling the expression of sequences, the bacterial host cell that is compatible with ColE1 replication system, growing the host cell in suitable conditions and the isolation and purification of interest protein.



 

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FIELD: biotechnology, in particular production of glycopeptide-originated biologically active substances.

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3 cl, 1 tbl, 3 ex

FIELD: biochemistry, biotechnology, medicine, in particular bioactive peptides.

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FIELD: gene and protein- engineering, medicine, pharmaceutical industry.

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11 cl, 18 tbl, 27 ex

FIELD: biotechnology.

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38 cl, 4 dwg, 18 tbl, 39 ex

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EFFECT: valuable properties of plasmid.

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FIELD: biotechnology, gene engineering, medicine, pharmaceutical industry.

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EFFECT: effective agent for gene engineering, medicine, pharmaceutical industry, etc.

2 cl, 3 ex, 4 dwg

FIELD: molecular biology, biochemistry, medicine, oncology.

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EFFECT: valuable biological and medicinal properties of transcripts.

8 cl, 9 dwg, 1 tbl, 5 ex

FIELD: genetic engineering, biochemistry, virology.

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EFFECT: valuable biological and medicinal properties of plasmid and agents.

3 cl, 2 tbl, 6 dwg

FIELD: microbiology, molecular biology, genetic engineering.

SUBSTANCE: invention relates to designing recombinant strains of E. coli carrying the cloned sequences of genome of meliodosis pathogen, B. pseudomallei, and determining different spectra of the medicinal resistance. Method involves transformation of E. coli JM107 competent cells with Kpn I-fragments of chromosomal DNA of the strain B. pseudomallei 56770 SMR2 ligated with Kpn I-restricts of vector pUC19 followed by selection of clones showing the combined resistance to antibacterial preparations of different classes. Then method involves carrying out the plasmid screening of the recombinant clones and hybridization analysis for detection of chromosomal DNA fragment by using B. pseudomallei chromosome sequences as a probe. Then method involves assay of the presence of the expression product of chromosomal DNA cloned sequence by immunoblotting method with immunoglobulins of specific meliodosis antsera. The prepared recombinant strain ZV1 is deposited in the State Collection of pathogenic microorganisms "Mikrob" at number KM167 and it shows resistance to pefloxacin and streptomycin. Use of the invention provides carrying out investigations of molecular-genetic bases of the multiple medicinal resistance of B. pseudomallei and to study the functional role of separate proteins in formation of the polyresistance in the meliodosis pathogen.

EFFECT: valuable properties of strain.

1 dwg, 1 tbl, 2 ex

FIELD: biotechnology, molecular biology, microbiology, genetic engineering.

SUBSTANCE: invention relates to a method for preparing an immunogenic polypeptide inducing immune response that represents the protective response against infection with Bacillus anthracis. Proposed immunogenic polypeptide comprises from one to three domains of the full-scale Protective Antigen (PA) from B. anthracis or their variants and at least one of indicated domains represents domain 1 or domain 4 from PA or its variant. These variants of immunogenic polypeptide and full-scale PA are produced as result of expression in E. coli. Also, invention proposes a vector for expression in bacterial cells that comprises nucleic acid encoding abovementioned immunogenic polypeptide. Also, invention the developed method for prophylaxis of infection caused by B. anthracis based on administration of sufficient amount of immunogenic polypeptide. Also, invention proposes a vaccine for prophylaxis of infection caused by B. anthracis that comprises the effective amount of immunogenic polypeptide and a suitable carrier. Invention provides preparing the effective agent used for prophylaxis of infection caused by B. anthracis.

EFFECT: improved preparing method and valuable properties of polypeptide and vaccine.

22 cl, 5 dwg, 3 tbl, 6 ex

FIELD: biotechnology.

SUBSTANCE: claimed strain is isolated by exposition of strain TCH-OK with space irradiation. Purification ratio of surfaces contaminated with oil is 78.6 % for 30 days.

EFFECT: strain with accelerated utilization of crude oil and petroleum products.

2 ex, 4 tbl

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