Protein factor, nucleotide sequence, the method of obtaining protein factor
(57) Abstract:Cloned and sequenced a fragment of genomic DNA from the sciatic nerve of the man, encoding the ciliary neurotrophic factor (SN-CNTF). The obtained DNA sequence suitable for expression of SN-CNTF person in E. coli and constructed recombinant expression vectors comprising these sequences. After transformation of these vectors suitable strain of E. coli, selection and cultivation of transformants from bacterial cells have identified the active form of recombinant SN-CNTF human mol. mass of 24 kDa and the N-terminal amino acid sequence matching the sequence of a native protein. 3 S. and 2 C.p. f-crystals, 1 table, 22 ill. The invention relates to neurotrophic factors and in particular to the ciliary neurotrophic factor (CNTF), caliraya his DNA and method for producing a recombinant CNTF.The strongest disorders, mental and physical, occur at the death of nerve or glial cells in the nervous system. The loss of nerve or glial cells can be called neurodegenerative diseases, such as senile sclerosis of the brain and multiple sclerosis, ischemia caused by stroke, travmaticheskoe promote the survival and functional activity of nerve cells and glial cells. There is evidence to suggest that neurotrophic factors prevent violations of the conditions listed above Appel, 1981, Ann. Neurology 10:499.Best characterized growth factor nerve (NGF).Experiments on animals show that NGF prevents death chainconstituting nerve cells diencephalon after injury and that NGF is able to prevent the loss of cognitive abilities, which happens with age. Hefti and Weiner, 1986, Ann. Neurology 20:275, Fisher et al, 1987, Nature, 329: 65. These results suggest the potential clinical utility of this factor for people in treating loss of cognitive abilities observed in disease, injury or aging.The complexity of the use of neurotrophic factors lies in their specificity only to those subpopulations of nerve cells that have the corresponding membrane receptors. Most nerve cells of the body NGF receptors are absent. Therefore, it was critical to identify new neurotrophic factors, which could ensure the survival of other types of nerve cells and glial cells.New neurotrophic factors have been found on their ability to maintain their survival in cstout survival of the ciliary ganglion motor neurons, which invert the skeletal and smooth muscles (ciliary ganglion nerve cells belong to the parasympathetic nervous system and their survival is not supported NGF).The presence of factors that facilitate the survival of ciliary ganglion cells marked for a variety of tissues and species. These ciliary ganglion neprotravnymi activity have the following similar chemical and biological properties: (1) this activity is present in high concentration in the sciatic nerves, (2) this activity can withstand the presence of the ionic detergent SDS and reducing agent beta-mercaptoethanol (BME) or dithiothreitol (DTT) during electrophoresis in polyacrylamide gel, and (3) on such gels this activity migrates with an apparent molecular weight between 24-28 kDa. Collins, 1985, Developmental, Bioligy, 109:255-258, Monthorpe et al/, 1986, Brain Pesearch, 367-282:286.Based on these similar properties suggested that the ciliary ganglion of neurotrophic activity responsible the same closely related molecules, commonly called "ciliary neurotrophic factor" or "CNTF".Without sufficient data, it cannot be argued that the proteins responsible for these activities are identical, so CNTF is RA - CNTF rabbit sciatic nerve (rabbit SN-CNTF)/
SN-CNTF, apparently, is in the peripheral nerves of higher concentrations. He is released from cells in the nerves in trauma. SN-SNTF supports the survival and growth of all studied nerve cells of the peripheral nervous system, including sensory, sympathetic and parasympathetic nerve cells. Thus, SN-CNTF exerts its effects on a wider range of nerve cells than NGF. As was recently shown, the rat SN-CMTF regulates the formation of specific types of glial cells in the Central nervous system (Hughes et al., 1988, Nature 335:70).It is assumed that the sciatic nerve CNTF - component response of the nervous system to injury. SN-CNTF released from cells into the damaged nerve, as you might expect, contributes to the survival and recovery of the damaged nerve cells and regulates the functional activity of glial cells necessary for regeneration. These considerations show that SN-CNTF may have therapeutic value in case of damage of the nervous system caused by illness or injury.Despite growing academic interest in SN-CNTF, the difficulty of allocating significant amounts of natural sources and osobnosti nerve cells. Previous attempts to purify rat SN-CNTF resulted in 800-fold enrichment compared with the crude extract of nerve cells in relation to the specific activity Manthorpe et al., 1986, Brain Research 367:282-286.However vosmisotletiya increase in specific activity was not enough to get a protein of the same species. The term "proteins of the same species" is used here and in the attached formula research to denote polypeptides with one with the same amino acid sequence in all active sites. In other words, if the operational part of the amino acid sequence is the same in two or more polypeptides, they are here, "proteins of the same species, even if there are minor heterogeneity in terms of length or charge.The purpose of the invention is obtaining SN-CNTF, purified to a much greater extent than ever previously achieved, so as to obtain a protein of the same species.Another objective of the invention is the creation of probes for screening cDNA and genomic libraries for cloning of animal and human genes encoding SN-CNTF.Another object of the invention is the obtaining of DNA sequences to animal and human CNTF.Another Ceti for human or animal CNTF could be expressed with obtaining human or animal CNTF.These and other objectives are achieved thanks to a method of cleaning SN-CNTF to such an extent that the specific activity increases more than 25000 times when compared to the extract and purified SN-CNTF, analysis of purified protein, getting on this basis for screening cDNA and genomic libraries SN-CNTF, cloning and determination of the nucleotide sequences of the rabbit and human CNTF and use them in a suitable recombinant expression system with obtaining biologically active SN-CNTF.It should be understood that both the foregoing General description and the subsequent detailed description are only exemplary and do not limit the invention. Accompanying drawings, which are incorporated and constitute a part of the specification, illustrate various embodiments of the invention and together with the description serve to explain the principles of the invention.In Fig. 1 shows exemplary results of chromatography on a column of Mono R; Fig. 2 - approximate distribution of neurotrophic activity in the eluate of each of the seven sections cut from the SDS-page gel after electrophoresis, and Fig. 3 - approximate results obraniakowi chromatography; Fig. 4 is an exemplary resultname fractions, it is shown in Fig. 3; Fig. 5 - profile of elution of peptides after treatment with endoprotease Asp-N and Lys-C, and Fig. 6 profile of elution of peptides after digestion by endoprotease Lys-C, and Fig. 7 is an exemplary results of the chromatography fractions of ammonium sulfate on a column of phenyl-Sepharose HIC; Fig. 8 is an exemplary results of chromatography on a Mono P chromatofocusing on column alkyl-Sepharose FPLC-HIC fractions; Fig. 9 is an exemplary results of chromatography preparative SDS-PEG fractions on C8 obraniakowi HPLC column ((A) illustrates the results of the initial cleaning procedures, (B) - procedure results consistent cleaning after adding two more HIC chromatography stages) of Fig. 10 is an exemplary results of electrophoresis SDS-page and Western analysis of purified obraniakowi chromatography SN-CNTF (lane 1 in each of the two parts shall be protein standard molecular weight (SDS SIGMA-7), track 2 - purified SN-CNTF, (A) illustrates the results of staining with silver, (B) illustrates the results of Western analysis with affinity-purified antipeptide-A - antibody); Fig. 11 is the nucleotide sequence encoding rabbit SN-CNTF (translation of this sequence gives the corresponding amino acid sequence that is printed is the selected, analysis of protein SN-CNTF), and Fig. 12 - nucleotide and corresponding amino acid sequence (three letter code) for the coding sequence of the human SN-CNTF. Sequences are between the lines. Where the nucleotide or amino acid sequence of rabbit differ from human, they're labeled above or below the line, respectively; Fig. 13 - the design of the expression vector CMVXVPL 2; Fig. 14 - the methods used for constructing CNTF-syn 1/3 for the expression of SN-CNTF (figure characteristic, but not at the scale, see example 7 for experimental details); Fig. 15 reflects the methods used to construct CNTF-syn 1/3 for the expression of CNTF (figure typical, but not to scale, see example 7 for experimental details); Fig. 16 is a synthetic nucleotides 1 through 4 used in the construction of CNTF-syn 1/3 and CNTF-syn 2/3 of Fig. 17 is a synthetic nucleotides 5 through 10, used in the construction CNTF-syn 2/3 of Fig. 18 - some features of the bacterial expression vector pT5T containing the insertion of DNA that provides the expression of CNTF (figure typical, but not to scale, see example 7 for experimental details); Fig. 19 reflects certain characteristics of bacamarte, see example 7 for experimental details); Fig. 20 - reducing SDS-polyacrylamide gel, in which were dispersed and painted Coomassie Brilliant Blue extracts of cells transformed by various expression constructs of Fig. 21 - reducing SDS-page, which were dispersed and detected by immunoblotting with affinity-purified anti-CNTF peptide And antibody extracts of cells transformed by various expression constructs of Fig. 22 reflected the bioanalysis serial dilutions of supernatants from bacterial cells expressing pT5T:CNTF-syn 1/3 or pT3x1-2: CNTF-syn 2/3 ( box reflects the bioanalysis extracted pieces from reducing SDS-PAA gel RTT:CNTF-syn 1/3 of the supernatant in the area of the gel directly above and below the 24 kDa).Although the invention concerns the allocation of CNTF from the sciatic nerve of rabbits, it is suitable for isolation and purification of this protein from other sources.Briefly, one preferred embodiment of the proposed method includes the use as raw material powdered material of the sciatic nerve of the rabbit. Crude extract of this material is centrifuged, the supernatant oxidized, and the resulting precipitate discarded. Then titrated supernatants a saturated solution of ammonium sulfate and removing the precipitate by centrifugation. Upon further addition of ammonium sulfate to the supernatant occurs the precipitation of the protein fraction containing the majority of the SN-CNTF activity.The above preparation is then applied to chromatofocusing FPLC column MonoP. Column fractions gather and analyze pH and CNTF activity. Fractions (indicated in Fig. 1 by an arrow with a vertical line) with the peak of SN-CNTF activity then further processed, as will be discussed in detail below.Focused fraction from multiple runs through the column MonoP applied to electrophoresis on SDS-polyacrylamide gel. The region of the gel corresponding to molecular weights from 22 to 27 kDa cut through the entire thickness of the gel on numerous strips and elute electrophoretic. Erwerbende proteins collected and the fraction with the highest activity in further purified using obratsova HPLC. This process is described in more detail in the examples that follow.It is advisable to conduct additional stages, which are included in the cleaning procedure in order to ensure easy handling of the material in the early stages. In the preferred embodiment between fractionation with ammonium sulfate and chromatofocusing chrome insert insert between chromatofocusing and preparative LDS-page (example 1).Developed treatment method resulted in the SN-CNTF in purified form (with more than 25,000-fold increase in specific activity compared to the crude extract).Increased degree of purification facilitates the determination of the amino acid sequence of SN-CNTF. In accordance with the invention defined amino acid sequence, the size of which was sufficient to generate oligonucleotide probes that facilitate screening for DNA and genomic libraries for cloning of animal and human genes encoding SN-CNTF.As will be discussed in more detail below, these genes are in turn made possible the production of large volumes of (1) animal SN-CNTF suitable for studying its ability to restore animal models of damage to the nervous system and human (3) SN-CNTF suitable for inclusion in pharmaceutical formulations useful in the treatment of lesions of the nervous system of man.Such peptides can be used to prepare antibodies. Antibodies to synthetic peptides derived and it is shown that they react with purified CNTF in Western blot analysis (Fig. 10).From the above work, the ultimate goal is to clone and sitechecker drugs for humans. As soon as genomic sequence is known, genes encoding SN-CNTF, can then be expressed in animal or bacterial cells. Because the sequence of the gene encoding CNTF rabbit and man, was determined, in the disclosed here of recombinant DNA technology for the production of CNTF could be used not only natural, but synthetic DNA sequence encoding SN-CNTF.Method of production of recombinant CNTF includes
a) preparation of a DNA sequence capable of providing in the host cell protein production, with CNTF-activity;
b) cloning the DNA sequence into a vector capable of introducing, replication and expression of DNA sequences in the host cell;
C) a transfer vector containing a foreign DNA sequence and the necessary elements into the host cell able to Express this DNA sequence encoding CNTF;
g) culturing the host cells under conditions suitable for amplification of the vector and expression of CNTF;
d) collection of CNTF and
e) creation of conditions for formation of the polypeptide tertiary structure, through which he has CNTF activity.In one embodiment the recombinant CNTF, may contain other nucleotides than the natural DNA sequence, this will still encode a polypeptide with the same primary structure as that of CNTF encoded CNTF genes in animals or humans.In another embodiment of the invention the natural DNA sequence encoding CNTF, is modified to enhance expression in the host organism or cell. Such modifications may include the following:
a) if the native DNA sequence is a genomic DNA, for subsequent expression in a bacterial system can be used to remove introns;
b) change in nucleotide sequence by introducing sequences recognized by various enzymes, for simplicity, the subsequent stages of ligation, cloning, and mutagenesis;
C) modifying the nucleic acid sequences by using codons preferred by the host organism used for the production of recombinant protein;
d) connection of the nucleotide sequences with the operational elements necessary to sustain and expression of DNA in a host organism or cell.Prepared in this way the sequence is inserted into exfactory E. coli
Vectors proposed for use in the invention include any vectors, which, as discussed above, the DNA sequence can be introduced together with any preferred or required operational elements, and the resulting vector can then be sequentially introduced into the host cell and to be replicated in a cage. Preferred vectors are those whose restriction sites are well known and which contain operational elements, preferred or required for transcription of the DNA sequence. However, certain applications of the invention relate and not fully understood at the present time vectors containing one or more of the described DNA sequences. In particular, it is preferable that all of these vectors had some or all of the following characteristics: (1) had a minimal number of sequences of the host body, (2) were stably maintained and bred in the desired host, (3) were able to be present in a large number of copies in the desired host, (4) had a regulatory promoter located so as to promote transcription of the gene of interest, (5) had at least one marker DNA sequence, AWANA DNA sequence, and (6) a DNA sequence capable of termination of transcription.In various preferred embodiments of the invention these cloning vectors that contain and are able to Express the DNA sequences of the invention contain various operational elements. These "operational elements", as discussed here, include at least one promoter, at least one sequence of the Shine-Delgarno and initiating codon and at least one termination codon. It is desirable that these "operational elements" also contained at least one of the following: at least one operator, at least one leader sequence for secretion of proteins, at least one gene for a regulatory protein and also other DNA sequences necessary or preferred for their inherent transcription and subsequent translation vector DNA.Certain of these operating elements can be represented in each of the preferred vectors of the invention. Any other additional operational elements that you may need, you can add these vectors, using methods known to anyone having ordinary knowledge in euut into suitable vectors, as described by Maniatis and other, Molecular Cloning, Cold Spring Harbor Laboratories (1984), cited here as reference.Vectors suitable for use in microorganisms other than E. coli, are also considered part of this invention. Such vectors are described in the table. In addition, certain preferred vectors are discussed below.b) Vectors Pseudomonas
Some vector plasmids are autonomously replicated in a wide range of Gram-negative bacteria, are preferred for use as cloning Shuttle vector into the hosts genome of Pseudomonas. Some of them are described Tait, R. C., Close, T. J. Lundguist, R. C., Hagiya, M. Rodriguez, R. L., and Kado, C. I. B. Biotechnology, May, 1983, pp. 269-275, Panopoulos, N. J. in Genetic Engineering in the Plant Sciences, Praeger Publishers, New York, pp. 163-185 (1981); and Sakagucki, K. in Current Toric in Microbiology and Immunology 96:31-45 (1982), each of which is specifically combined here in the links. One particularly preferred design usually contains plasmid RSF1010 and its derivatives, as described Bagdasarian, M., Bagdasarian, M. M. , Coleman, S., and Timmis, K. N. in Plasmids of Medical, Environmental and Commercial Importance, Timmis, K. N, and Puhler, A. eds, Elsevier/Noth Holland Biomedical Press (1979), specially made here in the links. The advantage of RSF 1010 in that it is a relatively small, vysokogorya, which is easily transtate Tac expression system, as described for Esherichia, since it appeared that the trp promoter E. coli readily recognizable PHK-polymerase Pseudomonas (Sakagucki, K. Curent Toris in Microbiology and Immunology 96:31-45 (1982) and Gray, G. L., Mckeown, K. A., Jones, A. J. S. Seeburg, P. H., and Heyneker, H. L. in Biotechnology, Feb. 1984, pp. 161 to 165).In cases where restriction minus strains of host species of Pseudomonas are not available, the transformation efficiency of plasmid constructs, isolated from E. coli, is low. Therefore, the desired passage cloning vector Pseudomonas before the transformation of the desired host through r-m+strain of other species (Bagdasarian, M., and other Plasmids of Medical, Environmental and Commercial Importance, pp. 411-422, Timmis and Puhler eds., Elsevier/North Holland Biomedical Press (1979).b) Vectors of Bacillus
The preferred expression system with the owner of the genus Bacillus includes the use of plasmid pUB-110 as a Shuttle cloning vector. As in other vector systems in Bacillus possible to Express CNTF invention intracellular or secretory proteins. The present application includes both systems. Shuttle vectors that replicate in Bacillus and E. coli, are available for constructing and testing different genes, as described by Dubnau, D., Gryczan, T., Contente, S., and Shivakumar, A. G., Genetic Engineering, Vol. 2, Setlow and Hollander eds. , occhialino attached to an area of the DNA codereuse protein, the signal sequence of alpha-amylase. For the synthesis of intracellular CNTF DNA sequence can be attached to the retaining frame to the binding site of the small ribosomal subunit alpha-amylase leader sequence.Transcription of any of these designs are preferably routed alpha-amylase promoter or its derivatives. This derivative contains a site of recognition RNA polymerase native alpha-amylase promoter, but also attaches and the area of the lac operator. Similar hybrid promoters, designed from the promoter of the gene penitsillinazy and lac operator, as was shown to operate in the host Bacillus in a regulatory manner, as determined Yansura, D. G. and Henner in Genetics and Biotechnology of Bacilli, Gansean A. T and Hoch, J. A., eds., Academic Press, pp. 249-263 (1984), specifically listed in the references. Gene lac I E. coli may also be included in a plasmid for introducing the regulation.g) the Vectors Clostridium
One of the preferred constructions for expression in Clostridium - plasmid pSU12 described Squires, C. H., and others in J. Bacteriol., 159: 465-471 (1984), transformed into C. pertringens method Heefner, D. L. al. as described in J. Bacteriol. 159: 460 to 464 (1984). Transcription is directed by the promoter of the gene of resistance to tetracycline. Translation is defined by the injured alien DNA introduced in yeast, can be accomplished in several ways, as described in Botstein, D. and Davis, R. W., in The Molecular Biology of the Yeast Saccharomyces, Cold Spring Harbor Laboratory, Strathern, Jones and Braach, eds, pp. 607-636 (1982), specially made here in the links. One preferred expression system for use in Saccharomyces as a host organism and CNTF gene 2 m plasmid. The benefits include a relatively high number of copies and stability with the introduction of cir' strains. These vectors can also include the beginning of replication and at least one marker of resistance to antibiotic from pBR322, to allow replication and selection in E. coli. Dvuhmetrovoy sequence and the yeast LEU2 gene provides the use in a defective LEU2 mutants of yeast.If it is expected that recombinant CNTF will eventually be expressed in yeast, it is preferable that the cloning vector was first introduced in Escherichia coli, where the vector would be replicated, then the vector would be transformed into the yeast for the final expression of CNTF.(e) cells of mammals
As the sequences for the expression of CNTF in mammalian cells serve as cDNA and genomic DNA encoding SN-CNTF.Must be available on the), specially made here in the links. Restriction DNA fragment carrying the DNA sequence encoding CNTF, you can insert in the expression vector, which already has a transcriptional promoter and enhancer, as described by Guarente, L. in Cell 52: 303-305 (1988) and Kadonada, J. T. et al. in Cell 51: 1079-1090 (1987). The promoter may be regulatory, as in the plasmid (Pharmacia Cat. N 27450601), if the constructive expression of inhibitor detrimental to cell growth. The vector should have full polyadenylation signal, as described in Ausubel, F. M., and others in Current Protocols in Molecular Biology, Wiley (1987) to mRNA transcribed from this vector, correctly processionals. In addition, the vector should have a beginning replication and at least one marker of resistance to antibiotic from pBR 322 to allow replication and selection in E. coli.In order discriminates stable cell line that produces SN-CNTF, the expression vector can carry the gene of breeding marker, such as marker of drug resistance, or to carry a complementary gene for the defective cell lines, for example, gene digidrofolyatreduktazy (dhfr) for the transformation of the dhfr - cell line, as described by Ausubel and others, supra. A plasmid carrying selectivity marker, can be totransform is written by way enter in a suitable cell host. These host cells can be cells of microorganisms or mammalian cells.(a) Microorganisms
It is considered that can be selected any microorganism having the ability to take exogenous DNA and to Express its genes and adjacent the operative elements. After the host organism is selected, the host organism is injected vector using conventional methods. Examples of such methods can be found in Advanced Bacterial Genetics by R. W. Davis et al., Cold Spring Harbor Press, Cold Spring Harbor, New York (1980). Preferably, the transformation took place at a low temperature, because temperature regulation is discussed as a means of controlling gene expression through the use of operational elements, as described above. In another embodiment, if the vector is introduced osmotic regulators, the change in pain concentrations during transformation requires to provide suitable control of alien genes.Preferably, the microorganism-host was a facultative aerobe or gone anaerobic. Among the yeast is advisable to use Saccharomyces and especially Saccharomyces cerevisiae. Among the bacteria are preferably genera Bacillus, Escherichia and Pseudomonas, especially Bacillus subtilis and Escherichia who ü introduced into cultured mammalian cells by several technologies, such as calcifolic: DNA-coprecipitation, electroporation, or the fusion of protoplasts. The preferred method is coprecipitate with calcium phosphate as described by Ausubel and others, supra.There are many stable cell types, which are easily transformed and capable of transcribing, processional and translate the DNA sequence and to produce protein SN-CNTP. However, cell types can be variable with respect to glycosylation of proteins and post-translational modifications of amino acid residues (or any other). Thus, the ideal cell types - those that produce recombinant CNTP, identical to the natural molecule.The host cell is cultivated under conditions suitable for the expression of CNTF. These conditions are mainly specific to the host cell and can be easily determined by the average it professional qualification in this area in light of the published literature concerning the conditions for the growth of such cells and recommendations contained herein. For example, Bergey''s Manual of Determinative Bacteriology 8th Ed. , Williams & Wilkins Company, Baltimore, Maryland contain information about the conditions of cultivation of bacteria. Such information is necessary for the cultivation of yeast and mammalian cells can be obtained and the m of the embodiments, the cells are grown to high density in the presence of appropriate regulatory conditions, which inhibit the expression of the DNA sequence. When reached optimal cell density, environmental conditions change to suitable for expression of the DNA sequence.Recombinant SN-CNTF can be purified after expression in a host cell or organism.In the preferred embodiment of the invention CNTF is present in its biologically active state when removing the bacterial cultures. In an alternative preferred embodiment to create the conditions in which downregulation of CNTF taking its active structure (at an appropriate stage of the cleaning process).For purification of recombinant protein using some combination of the following steps: anion exchange chromatography (Mono-Q, Mono-S and/or DEAE-Sepharose), helpanimals chromatography (Sepharose), chromatofocusing (Mono-P) and chromatography with hydrophobic interaction (OCTI - and/or phenyl-Sepharose).The following examples are provided to illustrate certain preferred embodiments of the invention and not limit it. All references in these examples, specially made here.Example 1. Preparation of protein.Materials
Sedale the n from Schwartz /Mann Biotech. Cleveland ohio. Phenylmethylsulfonyl (PMSF) Epsilon-aminocaproic acid, benzamidine, pepstatin, dithiothreitol (DTT), poly - L-ornithine (R) and 3/4,5-dimethylthiazol-2 yl) 2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma Chemical Co., St. Louis, Missouri. _ MonoP chromatofocusing FPLC column obtained from Pharmacia, Inc., Pisataway, New Jersey. C8 obraniakowi HPLC column obtained from Synchrom, Inc. Lafayette, Indiana. Acetonitrile was purchased from J. T. Baker Chemicak Co., Phillipsburg, New Jersey. Triperoxonane acid obtained from Pierce Chemicals, Rockford, Illinoice. Endoprotease Asp-N and Lys-c obtained from Boehringer Mannerheim Biochemicals, Indianapolis, Indiana. Fetal calf serum was purchased from Hyclone Laboratories, Logan, Utah. Cultural environment and saline solutions obtained from Irvine Scientific, Santa Ana, California. Cultural cups obtained from Costar, Cambridge, Massachusetts. Pathogen-free fertile embryonic chicken eggs for practical use is obtained from Spafas, Roanoke, Illinois.b) Analysis of SN-CNTF
Culture main chick embryo ciliary ganglia were prepared as described previously (Collins, 1978, Develop. Biol. 65:50; Manthorpe et al., 1986, Develop. Brain Res. 25:191. Ciliary ganglia were removed from fertile, pathogen-free chicken eggs, which were incubated for 9 to 10 days at the 38oC in a humid atmosphere. Ganglia were subjected to chemical rozdelenim HEPES buffer, pH 7.2 for 10 min at 37oC, and then treated with a solution of 0.125% bactericide 1:250 (Difco, Detroit, Michigan) in a balanced salt solution Hanks, modified as described above, 12 min at 37oC. Trypsinization was stopped by the addition of fetal calf serum to a final concentration of 10%.After this processing ganglia was transferred into a solution consisting of the modified Dulbecco eagle medium without bicarbonate containing 10% fetal calf serum and 10 mm HEPES, pH 7.2 and mechanically separated by passing 10 times through a Pasteur pipette, the end of which was burned smoothly and stretches to a diameter that allows for 2 to fill the eyedropper.Separated ganglia were placed in a Cup (for tissue cultures with a diameter of 100 mm) to the culture medium (see above), supplemented with 10% fetal calf serum, 4 mm glutamine, 60 mg/l penicillin-G, 25 mm HEPES, pH 7,2 (40 divided ganglia per Cup) at 3 o'clock This procedure was carried out in order to divide menariknya cells, which were stuck to the Cup, and nerve cells, which are not sticking. After three hours unattached nerve cells were collected by centrifugation, resuspendable in culture medium and were sown at 50 μl per well in floors the notches per well).Microtiter wells pre-treated with a solution of polymethine (1 mg/ml) in 10 mm sodium borate, pH 8.4 V overnight at 4oC, washed with distilled water and dried in air.10 μl of serial dilutions of sample were added to each well and the plates were incubated 20 h at 37oC in a humid atmosphere containing 7.5% of CO2for the purpose of analysis of neurotrophic activity. After 18 h, each well was added 15 μm solution of tetrazolium dye MTT (1.5 mcg/ml) in the environment of Dulbecco without bicarbonate containing 10 mm HEPES, pH of 7.2, and the culture was placed back into the incubator (37oC) for 4 h and Then was added 75 μl of a solution of 6.7 ml of 12M HCl to 1 liter of isopropanol) and the contents of each well homogenized 30 times to destroy the cells and suspending the dye. Then determined the absorption at 570 nm compared to 690 nm by correction for each well using an automatic reader microtiter plates (Dynatech, Chantilly, Virginia). The absorption of the contents of the wells that did not contain any of neurotrophic agent (negative control), subtracted from the absorption of the sample-containing wells. Final absorption is proportional to the number of living cells in each well, which tract is as an equivalent division, which gives 50% maximal survival of nerve cells. The trophic activity in units per 1 ml is obtained by dividing the total number of activity units on volume analysis (60 μl).The specific activity was determined by dividing the total activity trophic units on the amount of protein present in the sample.C) Cleaning of SN-CNTF
At the end of each of the following stages of the product was either used immediately or were left at -70oC not more than one week before use.Stage 1. Cooking coarse extract.100 g (wet weight) of the rabbit sciatic nerve (nerves about 300) were thawed and triturated using polychrony rotary homogenizer (Kinematica, Switzerland) for 1 min in 10 volumes (W/V) water, containing 10 mm EDTA, 1 mm Epsilon, aminocaproic acid, 1 mm benzamidine and 0.1 mm PMSF, and centrifuged at 140000 g for 30 min at 4oC. the Supernatant was filtered through a glass filter to remove floating lipids.Stage 2. Treatment with acid and ammonium
Under centrifugation, referenced below, were performed at 17000 g for 20 min and all operations were performed at 4oC, unless otherwise noted. Crude extract was centrifuged. and to pH 6.3 1 N. NaOH and the resulting precipitate was again removed by centrifugation. To the above supernatant solution was added ammonium sulfate to 30% saturation and the precipitate was removed by centrifugation. Further addition of ammonium sulfate to the supernatant to achieve a 60% saturation resulted in precipitation of the protein fraction containing the greater part of SN-CNTF activity. The precipitate was dissolved in 20 mm sodium chloride, pH of 6.7, containing 1 mm EDTA, 0.1 mm PMSF and 0.1 μm pepstatin to bring the concentration up to 8 to 13 mg/mlStage 3. Chromatofocusing
The above preparation were dialyzed overnight against 500 times larger volume of 10 mm sodium phosphate, pH of 6.7 with one change of buffer and centrifuged at 1400000 g 30 minutes the Supernatant was passed through 0.22 μm (pore size) nylon filter and put in 3 doses (2 ml each time) on Mopar chromatofocusing FPLC column (4 ml), equilibrated 25 mm Tris-HCl buffer, pH 5.8. The column was washed with the same buffer until the return of absorption of the eluate at 280 nm to the baseline. The sample was then chromatographically with polybuffer, pH 4,0 (1 to 10 dilution RV from Pharmacia).Column fractions were collected and analyzed pH and CNTF activity. Fig. 1 shows the results of the chromate is given in each fraction. Faction is marked by an arrow with a vertical line (with the peak of SN-CNTF activity around pH 5) was collected and treated with solid ammonium sulfate to reach 95% saturation, and the precipitate was collected by centrifugation, resuspendable in saturated solution of ammonium sulfate and centrifuged to remove polybuffer. The precipitate was dissolved in sufficient quantity of 10 mm sodium phosphate buffer, pH of 6.7 to achieve a protein concentration of 3 to 5 mg/l (referred to as "the collected fraction"). Typically 1 l initial coarse extract requires 8 separate runs on the column.Stage 4. Preparative electrophoresis in the gel with sodium dodecyl sulfate (SDS)
The collected fractions from multiple runs through MonoP column were combined and were dialyzed against 100-fold volume of 10 mm sodium phosphate buffer, pH of 6.7 for 8 h with one change of buffer, then ran a 15% reduction DDS polyacrylamide gel in accordance with the method of Lemli, 1970. Each allows the gel has a size of 0.3 cm in width, 14 cm in height, 11.5 cm in width. 5,5 mg protein were applied to each gel. Electrophoresis was carried out at 15oC and 40 mA/gel until such time as the standard molecular weight (painted) 20 kDa has not reached the end allowing grivaud sheet of nitrocellulose (0.45 μm pore size in the form of a roll, obtained from Millipore Corporation, Beadford, Massachusetts), pre-soaked in water, 2-soaked sheets and 2 sheets of dry chromatography paper (3 MM Chr, obtained from Whatman, Hillsboro, Oregon), a glass Cup and 500 ml glass flask weight. After 30 - 45 min outlines of the gel was observed on the nitrocellulose paper using water-insoluble marker. The paper was washed 3 times with 10 mm Tris-HCl buffer, pH 8.0, containing 0.15 to NaCl and 0.3% NP - 40 detergent, and then stained for 15 - 30 min 1: 1000 diluted Kohinuor Rapidograph Ink (available in stores stationery) (in the above buffer).The initial gel was placed on a glass Cup and leveled his footprints painted on nitrocellulose paper, under glass. The region of the gel corresponding to molecular weights between 22 - 27 localized mark, in accordance with stained molecular weight standards (BRL, Bethesda, MD), banished in the narrow lanes on both ends of each gel. This area is cut across the entire width of the gel in the form of seven 2.5 mm parallel slices, using the curvature of the bands detected painted nitrocellulose paper. Each individual piece of the gel was cut into smaller pieces (2,52 mm) and proteins were suirable electrophoretic within 6 h in 1:1 dilution in the morning,2 ml. Fig.2 represents a plan of allocation of neurotrophic activity in the eluate from each of the seven pieces (marked in order of decreasing molecular weight). The fraction with the highest activity (strip (d) further purified using back-phase HPLC.Stage 5. Back-phase HPLC.Dithiothreitol (DTT) and 10% triperoxonane acid (TFA) were added to the eluates gel to achieve a final concentration of 2 and 0.3%, respectively. The sample was filtered through 0.22 μm nylon filter, downloaded C8 obratsova HPLC column and was suirable H2O/0.1% of TFA:acetonitrile/0.1% of TFA gradient. Fractions were collected in siliconized tubes containing 5 μl of 0.4% Tween-20. Analyzed the neurotrophic activity of aliquot of each fraction. Protein concentration was determined by absorption at 215 nm and combined with the distribution of neurotrophic activity. Fractions with peak SN-CNTF activity (fraction 37-40, Fig.3) were collected for sequencing as described in example 2. In a separate drug factions, including neighboring peak CNTF activity, equivalent fractions 36-44 in Fig.3, also analyzed on a painted silver reducing DDS-page (Fig.4).Performed additional chromatograph chromatographic stage based on the principle of chromatography on hydrophobic interaction (HIC). The first HIC stage is a conventional chromatographic procedure, inserted after stage 2.Dissolved material after ammonium sulfate precipitation further diluted with 10 mm sodium phosphate buffer, pH of 6.7 (Buffer a) And ionic strength (measured with conductivity), equal ionic strength buffer containing 0.3 M ammonium sulfate and 5% isopropanol (Buffer A). Then add isopropanol to dilute the sample to a final concentration of 5% and the mixture is applied on a column of phenyl-Sepharose (Pharmaccia, Inc., Piscatanay, New Jersey), equilibrated with buffer A. Not more than 3 mg of protein sample is applied on a 1 ml volume of the column. 1 l coarse extract the sciatic nerve gives 50 ml again dissolved sediment, precipitated with ammonium sulfate, which is then diluted up to 70-100 ml, as above, and put in 110 ml of phenyl-separato column. With the column elute sequentially, starting with 3 volumes of buffer B, then, using 2 volumes of buffer containing 50% ethylene glycol (Buffer), washed with 5 volumes of water 18 ml fractions.Fig. 7 shows the results of each chromatographic run. Profile buervenich proteins was measured continuously at 280 nm (thick line). O. D. - circuit combined profile elyuirovaniya bioactivity SN-CNTF in catit from the column during the elution buffer C (indicated by an arrow with a vertical line in Fig.7). Column fractions containing the major part of bioactivity, were collected and concentrated, dialigue under pressure using a membrane Amicon JM-10 (Amicon Division, W. R. Grace & Co., Danvers, MA) to approximately 1/10 of the initial volume, which usually led to a final protein concentration of 2.5-3.0 mg/ml Concentrate were dialyzed for 6 hours against 3 shifts 55-fold volume of Century Cialisovernight material was passed through 0.2 μm (pore size) filter Acrodise (Gelman Scinces, Inc., Ann Arbor, MI) and were downloaded multiple application (2 ml) at chromatofocusing column as described above.Without this HIC column stage 1 l coarse extract the sciatic nerve required 8 separate runs through MonoP chromatofocusing column, and with the addition of HIC column stage 1 l coarse extract could be processed in one chromatofocusing run.The second HIC stage is entered after the original stage 3: chromatofocusing on MonoP. To each 1 ml chromatofocusing material (3-5 mg/ml protein) was added to 2 ml of 50 mm phosphate buffer, pH of 6.7, containing 15 M ammonium sulfate (buffer D). The mixture is then passed through 0.2 μm (pore size) filter Acrodise and loaded (multiple application of 2 ml each) alkyl-Separato column HR 10/10 (FPLC(Pha baseline. Then the column was treated for elution 60 ml linear gradient of buffer D buffer E (50 mm phosphate buffer, pH 6,7) and collecting 1 ml fractions.Fig. 8 illustrates the results of one such FPLC-HIC column run. The continuous line represents the profile lirovannomu protein (O. D. 280). Combined with it line - plot of SN-CNTF activity in each gradient fraction. The fractions containing the biological activity (indicated by an arrow with a vertical line in Fig.8), were collected and concentrated in Centricon-10 conc. (Amicon) to 0.5 ml of Sample was diluted by adding 2 ml of buffer to the upper limit and again concentrated by centrifugation to a final volume of 0.5 ml Dilution and re-concentration was repeated 2 more times and finally concentrated sample were driven in reducing SDS-15% polyacrylamide preparative gel, as described above, except that prior to dialysis is not required.Fig. 9 compares the final purification step on obraniakowi HPLC in the initial cleaning procedure (upper part) and in the purification procedure after adding the two stages HIC (bottom). Each part shows the profile buervenich proteins (thick line O. D. 280) and the combined SN-NTF activity (the local protein. Prepared original procedure described above, the protein is purified to homogeneity. The advantage of the new purification procedure that 8 l of the initial material can be processed as well as 1 l, using the original method.Example 2. Sequencing of purified neurotrophic factor.Fractions with peak SN-CNTF activity (37-40, Fig.3) were collected and concentrated to 50 µl under a vacuum evaporator centrifuge. The concentrated sample consisted of 0.14% tween-20. It was diluted in 1% ammonium bicarbonate to a final volume of 350 μl and were treated by endoprotease Asp-N or endoproteases Zys-C overnight at 37oC. the Mixture was concentrated to approximately 50-100 µl in a vacuum evaporator centrifuge and loaded on a 1 ml sample loop on a narrow Aquapore RP-300 C8 obratsova HPLS column (Broronbee Labs), 2,1220 mm, suirable H2O/of 0.1% TFA acetonitrile/0.1% of TFA gradient. Peptideatlas fractions were collected manually in appendrows tubes, based on the absorption at 215 nm. Fig.5 shows the profile buervenich peptides after digestion by endoprotease Asp-N (absorption at 215 nm). Fig.6 shows the profile buervenich peptides after digestion by endoprotease Lys-C after reducing the gas protein sequencing machine.Additional amino acid sequence was obtained with the digestive enzymes trypsin and endoprotease Glu-C(Boehringer Mannheim Biochemicals, Indianapolis, Inc.). This additional protein sequence has allowed some of the amino acid sequences defined above, to combine into large peptides (overlapping amino acids). New amino acid sequence and those that merged with the previous sequences are given below:
Example 3. Preparation of antibodies to neurotrophic factor.Antibodies that react with purified rabbit SN-CNTF, useful for screening libraries to obtain the gene that encodes rabbit SN-CNTF. In addition, antibodies that neutralize its biological activity, are used in intact animals, in order to define the biological role of this neurotrophic factor.In order to prepare such antibodies, synthesize relevant areas of the sequence SN-CNTF using the Applied Biosystems automated protein sintezator. Such synthetic peptides covalently Swaziland, large doses after 3 and 6 weeks with incomplete adjuvant. Serum samples taken from each pig and use in Western band against purified SN-CNTF in order to determine responding antibody in serum with purified protein. Positive in the Western analysis of the samples subsequently tested for their ability to neutralize neurotrophic activity in bioanalysis used for cleaning. Seropositive Western and neutralization analysis further purified as follows: (1) serum adsorb protein carrier in order to remove antibodies raised against this protein, then serum re-test the above analysis, (2) the IgG fraction antibody purified from serum by standard methods and re-test the above analysis. Both these stages will provide a polyclonal antibody, the purity of which is sufficient to be used for screening expression libraries with cloning of the mRNA and the gene SN-CNTF.Antibodies were producirovanie in rabbits to a synthetic peptide "A" corresponding to part of the amino acid sequence of rabbit SN-CNTF given in example 2 ( E-S-J-V-K-H-Q-G-L-N-K-N). Below in this example, the given methods. Affinity-purified the antibodies is different rabbits through the affinity column, bearing covalently bound synthetic peptide and then suirable bound peroxidase antibodies. Nefrackzionirovannam immune antisyware gave the title Ca105in ELISA (using peptide And for covering holes and serum in a final dilution of 1:50 for Western blot analysis). Affinity-purified antipeptide And antibody prepared as described above was used in Western blot analysis at a final concentration of 80 mg/mlAs antipeptide And - anticavity and affinity-purified antibodies that interact with purified rabbit SN-CNTF. Preimmune serum do not interact with the SN-CNTF under these conditions. Aliquots of the fractions of the peak CNTF at the final stage of purification obraniakowi HPLC (Fraction # 46, Fig.9, part b) were driven on two separate tracks, followed the tracks containing protein molecular weight markers. The gel was cut into two parts, each of which contained one track purified CNTF and neighboring protein markers. One of these slices were stained with silver to localize proteins Bio-Rad Laboratories, Richmond, CA), and the other explored the Western blot analysis (Towbin and others, 1979, Proc. Natl. Acad. Sci., USA. 76: 4350) on proteins that react with affinity-purified antipeptide And antibodies.Leted And antibodies. This specific recognition, as an unrelated marker proteins on the left track to the right is not recognized antipeptide And antibodies, although present in high concentrations, as shown on the left is painted silver tracks (Fig. 10). Preimmune serum from the same rabbit also does not recognize two lanes cleared NTF. These results show that there are at least two different CNTF, which differ by Ca 5000 Da molecular weight on reducing SDS-page.To prepare antipeptide And antibodies, synthetic peptides And conjugatively with hemocyanin (KLH), to increase its antigenicity. For conjugation of 1 mg of peptide a and 1 mg of KLH (Calbiochem, La jolla, CA) in 50% glycerol was dissolved in 0.5 ml of PBS (20 mm sodium phosphate buffer, pH of 7.4, containing 0.15 M NaCl). 10% glutaraldehyde was added dropwise with stirring to a final concentration of 1% and left the reaction to stand at room temperature over night under stirring, then diluted to 5 ml of PBS. The conjugation mixture was emulsiable with complete adjuvant Friend 1:2 and were injected with under the cuticle many dorsal location (subcutaneously in the back) two new Zealand white rabbits (100 µg peptide And the rabbit. Three weeks later, each krolikowski supporting the injection was carried out with 2-week intervals until while anticavity did not give a titer of at least 100,000 in ELISA (Tainer and others, 1984, Nature 312. 127) when using coated with peptides holes. Serum was prepared from blood taken from the ear vein 5 weeks after the first injection, and every two weeks after. Serum was stored at -70oC.To prepare the peptide-affinity column, the peptide And covalently attached to a chromatographic column matrix as follows: 3 mg of peptide And dissolved in 0.4 ml of BBS containing 4 M guanidine hydrochloride was added to 4.5 ml of 0.1 M NaHCO3, pH 8.0 and 0.5 M NaCl. One gram of freeze-dried activated SN Sepharose 4B (Pharmacia) was washed, and it is swollen in 200 ml of 1 mm HCl and directly transferred into a solution of peptide A. the Mixture was rocked overnight at 4oC. Then the gel was besieged in a centrifuge and the supernatant was retained to determine the number of peptide a, which remained associated with the matrix. 15 ml of 0.1 M Tris buffer, pH 8.0, was added to the precipitate gel and incubated at room temperature for 2 h to block unreacted mating group matrix gel. Then the gel was placed in a column (3 ml) and washed three times by the following sequence of buffers: (1) 10 volumes of 0.1 M acetate buffer, pH 4,0, sod what they 0,02% sodium azide. The difference in concentration of free amino groups was determined in the solution of the initial peptide and in the supernatant after conjugation, using fluorescamine (Chen et al, 1978, Arch. Biochem. Biophys, 189-241; Nowicki, 1979. Anal. Letters 12: 1019). This analysis showed that 92-95% of the peptide conjugial with matrix Safronova gel.Before affinity purification of antipeptide And antibodies 8 ml serum of immunized rabbit were dialyzed overnight against 2 liters of PBS. Peptide A-separato column was washed sequentially with 10 volumes each of the following solutions: 0.1 M glycine-HCl, pH 2.5, the PBS 0.1 M triethylamine, pH 11,5; PBS. Cialisbuynow serum is passed through the column three times to achieve complete bonding antipeptide And antibodies. The column was washed with 20 volumes of PBS, and then elute sequentially 4 volumes of each of the following solutions: 0.1 M glycine-HCl, pH 2,5; PBS; 0.1 M triethylamine, pH 11,5; then PBS, and 1 ml fractions collected. Eluate of glycine and triethylamine of prolivov neutralized immediately 1 M Tris, pH 9 and 7, respectively, and aliquots analyzed by antipeptide And-antibody ELISA analysis using coated with peptide A-holes. Fractions with the highest titer (usually within 3 volumes beginning of glycine and triethylamine of ELSI) were collected and were dialyzed CNY supernatant maintained at -70oC.Example 4. Cloning of the gene
Given the possibility that rabbit and human sequences SN-CNTF not identical, it is reasonable to first obtain clones of the gene rabbit through hybridization with synthetic oligonucleotides based on the protein sequence, and then use the clone rabbit as hybridization clones in gene screening of man.A. SN-NTF gene
To obtain the genomic sequence of the rabbit encoding SN-NTF genomic library rabbit (Clontech) were transferred into a bacterial strain of E. coli 58 nm and detected with approximately 1000000 recombinant clones. Regions of the protein sequence of the rabbit SN-NTF, which can be represented by the small number of codons, back translate and synthesize the corresponding degenerate oligonucleotide samples. Rabbit oligonucleotides mark kinases in accordance with a standard Protocol (Maniatis and others (1982, Molecular Cloning, A Laboratory manual, cold Spring Harbor Laboratory). DNA kinase obtained from US Biochemical Corp. and gamma-labeled ATP obtained from ICN. Oligonucleotides have been labelled with a specific activity of at least 1000000 pages on picomol.When screening the genomic library of p. the t in accordance with the methods Manuatis and other (1982, ibid) and hybridizing night with radioactively labeled oligonucleotide breakdown. Hybridization mixture includes 6 SSC, 2x Denhard, 0.05% sodium pyrophosphate, 1 mm EDTA, 0,1% SDS, 100 μg yeast tRNA (Sigma), pH 8.0. The hybridization temperature should be a few degrees below the measured (calculated) Tm of the oligonucleotide. Clones that hybridize with multiple samples based on different regions of the protein sequence, diffuse to the individual colonies, and zones of hybridization is sequenced by the method of dideoxy-cliff. (Sanger and others, 1977, Proc. Natl. Acad Sci 74: 5463), using sequenase (US Biochmicals Corp.) to identify clones that encode SN-CNTF protein sequence.B. SN-CNTF mRNA sequence
Total cellular RNA derived from rabbit and human sciatic nerves. The tissue is homogenized in guanidine thiocyanat/beta mercaptoethanol solution and RNA purified by sedimentation in a gradient of cesium chloride (Glison and others, 1974, Biochemistry 13: 2633).Polyadenylated RNA selected by chromatography on oligo (dt) cellulose (Avid and Leder, 1972, Proc. Natl. Acad. Sci 69: 408). Quantitative RNA blot hybridization analysis can be performed with "antimuslim" oligonucleotide probes to assess prevalere what's for screening (at least 99% probability) of the resulting clones CNTF. A sufficient amount of double-stranded complementary DNA can be synthesized as described after Gubler and Hoffman, 1983, Gene 25-263 and insert in the vector lambda gem (Promega Biontech) in accordance with Palazzolo and Meyerowitz, 1987, Gene 52: 197.Clones encoding rabbit SN-CNTF, identify the hybridization of recombinant phage plaques as described above. The identification of these clones was checked by determination of the nucleotide sequences in order to establish compliance with completely known protein sequence. Screening with cDNA probes of the human sciatic nerve (Feinberg and vogelstein, 1983, Anal Biochem 132: 6), which is a more reliable procedure for the detection of cross-hybridization than the use of smaller oligonucleotides used for screening the rabbit cDNA libraries.Polymer chain reaction (PCR) (Saiki and others, 1988 Science 239: 487) was used for amplification of DNA fragments corresponding to the amino acid sequence of rabbit CNTF. Such DNA fragments amplified from human and rabbit genomic DNA and cDNA of the sciatic nerve of a rabbit and sympathetic ganglia. Amplificatoare DNA fragments were subclinically and sequenced using standard techniques (Maniatis and others, Iringa cDNA library of the sciatic nerve of a rabbit and a genomic DNA library of a man. Positive clones cDNA-rabbit and positive genomic clones person was isolated and partially sequenced. The open frame sequence corresponding to the coding (equivalent mRNA) sequences for CNTF rabbit or human, confirmed the sequence of the DNA fragments coding region obtained using PCR. The resulting coding sequence for SN-CNTF rabbit and man are given in Fig. 11 and 12, respectively.Part of the amino acid sequences obtained from rabbit SN-CNTF, aired back in degenerate oligonucleotides #'S 1, 13, 7 and 12 and complementary #'S 3, 14, 8 and 17. Amino acid sequence (top) and location and numbering of the respective sense and antisense degenerate oligonucleotides (bottom) is given below
(5') ****1****** ****13***** *****7*****(3')
(3') ****3****** ****14***** *****8*****(5')
The nucleotide sequence of the semantic versions of each of the degenerate oligonucleotides is given below (where N represents any nucleotide)
#1 5'-TA(T/C) GTN AA (A/G) CA(T/C) CA(A/G) GG-3'
#13 5'-AA(T/C) AA(A/G) AA(T/C) AT(A/T/C) AA (T/C) (C/T)T-3'
#7 5'-GA(T/C) GGN GTN CCN ATG GC-3'
#12A 5'-AA(A/C) TT (the global polymerase chain reaction was performed using either human, or rabbit genomic DNA as a sample and oligonucleotides # 's 1 and 8 or #'S1 and 17 as primers in order to amplify the corresponding regions of genes CNTF human and rabbit. Southern blots of these reaction products (processed by the oligonucleotide # 13) discovered the existence of labeled bands size 66 mo (#'s 1 and 8) and the size of 366 mo (#S1 and 17).The same PCR reaction described above was performed using cDNA prepared from either mRNA rabbit sciatic nerve, or mRNA rabbit sympathetic ganglion. RNA was prepared from rabbit sciatic nerves or the sympathetic ganglia and was passed through an oligo-dt column to collect the messenger RNA (mRNA), as described above. Complementary DNA (cDNA) was prepared using reverse transcriptase, using mRNA as a sample and oligo-dt as primer. When the PCR reaction was used as a primer, either cDNA or oligonucleotides # 's 1 and 8 or #'S1 and 17 and amplified those fragments, which had the same sequence that amplificatoare with genomic DNA of the rabbit (Fig. 11). This indicates that in the encoding region of the gene CMTF between oligonucleotides #'S1 and 17 there are no introns.To get more coding sequences (equivalent which uses mRNA rabbit sciatic nerve, as the sample and oligo-dt Not I linker adaptor as a primer. Subsequently added EcoR I/XmnI linker adaptor (5'-ATTACHAUTH-3') to the 5'-end of the double-stranded cDNA by legirovaniem on blunt ends (Maniatis, et al., ibid). Polymerase chain reaction was performed using this cDNA as a sample and the oligonucleotide #8 and EcoR I/XmnI linker-adapter, as primers. Southern blot-reaction products (processed labeled oligonucleotide #13) revealed the existence of a labeled band of approximately 200 mo.To obtain cDNA clones rabbit CNTF, a cDNA library was prepared from poly(A)+mRNA sciatic nerve of rabbits by the method described above, except that used a lambda gt-vector (Stratigene) lambda-gem 2. Approximately 4 to 106plaques of the library was skanirovali using a sample prepared with a random phase subclone M13 PCR fragment was obtained using cDNA rabbit sympathetic ganglion in the sample and the oligonucleotide #8 and EcoR I/XmnI - linker adaptor as primers (see above). Separate primary positive was considered to be plaque purified tertiary screening. During digestion, EcoR I DNA of this clone gave three fragments in addition to the frag is issued CNTF - specific oligonucleotides and fragments mentioned above. The DNA sequence 1,5 TPN and cDNA fragment showed that it corresponds to the full coding sequence of the rabbit SN-CNTF (Fig. 11).To obtain clones of genomic DNA for human CNTF was skanirovali about 3 to 106plaques genomic DNA library of a man in the vector lambda EMBL 3, using a sample prepared with a random tagging of M13 subclone PCR fragment obtained from human genomic DNA as template and oligonucleotides #1 and #17 as primers (see above). Six positive plaques cleaned by sequential screening, hybridisable with additional CNTF-specific oligonucleotides and PCR-fragments, 0,6 TPN Bam HI restriction fragment of one of these subclones, hybridizers with oligom #13, was subcloned into the Bam cut H13 HI mp 19 and sequenced.Sequence DNA fragments obtained by PCR from rabbit cDNA clone were combined, based on areas of overlapping sequence to obtain the coding sequence (equivalent to mRNA) for rabbit, shown in Fig. 11. Sequence DNA fragments obtained by PCR sequence to obtain the coding sequence for human SN-CNTF shown in Fig. 12. Nucleotide sequence of the rabbit CNTF and humans are approximately 89% identical (Fig. 12), indicating that the sequence of rabbit and man come from homologous genes encoding CNTF. As shown in Fig. 11, part of the nucleotide sequence for rabbit CNTF confirmed by amino acid sequences obtained from purified SN-CNTF, and referred to in the previous examples.Polymerase chain reaction was performed using matrix and the primers described above. The program of the reactions was as follows: denaturing cycle, 1 min at 95oC, the cycle of annealing, and 1.5 min at 40oC and the cycle extension circuit, 4 min at 72oC. the Reaction was carried out for 30 cycles. The reaction products were applied on 2% agarose gels and transferred to membranes Leta-Bind (Bio-Rad. Richmond, CA) for southern analysis. In order to identify amplificatoare portion of the coding sequence CNTF, southern blots were processed labeled oligonucleotide #13, about which we know that it lies between the oligonucleotides used for the priming reaction in the sequence CNTF protein. Labeled on the of the ends of the fragment maple DNA polymerase (New England Biolabs, Beverly, MA) in the presence of all four dNTP and processing of DNA polynucleotides T4 (US Biochemical Corp., Cleveland, OH) and ATP. Suitable pieces of DNA was then subcloned into M13mp10 vector, cut with SmaI (dephosphorylating, commercial product from Amersham Corp. , Arlington Heights, IL). Recombinant phage containing the desired fragment, identified by the screening procedure Benton and Davis (1977, Science 196: 180), using labeled oligonucleotide #13 as the sample. These recombinant clones were grown to obtain sufficient for sequencing the number of single-stranded DNA and then sequenced by the method of dideoxy-chain termination (Sanger and others, ibid).When hybridization was performed long used labeled DNA samples included 5 SSCP, 2x denhardt's solution, 2 mm EDTA, 0.05% sodium pyrophosphate, 0.1% dodecyl sodium sulfate (SDS), 250 µg/ml sperm DNA herring (nonspecific competitor for binding) pH 8.0. Hybridization was carried out at 65oC and the blots or the filters were washed at 65oC in 0.1 SSCP and 0.1% SDS. At short hybridization oligonucleotide samples consisted of 6 SSCP 2x denhardt's solution, 2 mm EDTA, 0.05% sodium pyrophosphate, 0.1% of SDS, 100 μg/ml yeast tRNA (nonspecific competitor for binding), pH 8.0, the temperature of hybridization and the conditions which otide (Maniatis and others, ibid).Example 5. The expression of the genes encoding SN-CNTF in animal cells.The expression of SN-CNTF in cultured animal cells requires the following steps:
a) Construction of expression vector;
b) line Selection of host cells;
C) Introduction of the expression vector in the host cell;
d) manipulation of the recombinant cell host, to increase the levels of expression of SN-CNTF.a) SN-CNTF expression vectors designed for use in animal cells, can be of several types, including strictly constitutive expression design, inducible gene construct, as well as to target specific cell types. In all cases, the promoters and other regulatory regions of the gene, such as enhancers (inducible or not) and polyadenylation signals, are placed in a suitable position in relation to sequence cDNA in the gas plasmid vectors. Two examples of such structures follow. (1) Design, using the district strictly constitutive promoter, doing, using signals of genetic control of SV40 in this arrangement, as in the plasmid pSV2CAT, as described Gormah and others, Mol. Cel. Biol. 2: 1044-1051, 19 the thread coding sequence chlorophenyltrichlorosilane (SAT) SN-CNTF cDNA, using standard molecular biological techniques (Maniatis and others , supra). (2) the Inducible genetic design make using plasmid RMK, which contains the promoter region of metallothionein mouse (MT-1) (Brinster and other Cell 27: 228 - 231, 1981).This plasmid can be used as a starting material and it should be manipulated to obtain metal-induced gene construct.b) you Can use different cell lines to Express the SN-CNTF, working with vectors described above for producing an active protein. Two potential cell lines, which are well characterized for their ability to enhance the expression of foreign gene - mouse line Ztk-and cell line Chinese hamster ovary (CHO) dhfr-however , the expression of SN-CNTF is not limited by this choice.Line of animal cells, which can also be used for expression, which includes cells monkey kidney COS-7, which are useful for transient expression, and human embryonic kidney cells 293.in) Vector DNA should be entered in this cell line, using any of the known technologies. The method used here, includes technology Calcifer the s SN-CNTF, seacadets with a second expression vector coding for breeding marker. If Ztk - cell transfection as a breeding marker gene is timedancing and selection were carried out as described in Wigler, etc. in Cell 16; L 777-785, 1979, in the case CHOdhfr-cells of breeding marker-digidrofolatreduktaza (DHFR) with the selection, as described Ringold and others in J. Mol. Appl. Genet. 1: 165-175, 1981.g) Cells, which Express a gene construct, grown under conditions that will increase the levels of production SN-CNTF. Cells bearing structures with metallothionein promoter, can be grown in the presence of heavy metals such as cadmium, which will lead to a 5-fold increase in the use of MT-1 promoter (Mayo and others, Cell 29:99-108) and, respectively, comparable to the increase in the levels of protein products SN-CNTF. Cells containing SN-CNTF expression vectors (either SV40 or MT-1-derivative), together with DHFR-gene-expression vector can be subjected to gene amplification Protocol described by Ringold and others in J. Mol. Appl. Genet. 1. 165-175, 1981, using methotrexate, a competitive antagonist of DHFR. This leads to a greater number of copies of the DHFR genes present in these cells, and simultaneously increased gene copies SN-CNTF that of the expression vector pCMVXVPL2 used to Express the coding sequence of the rabbit CNTF for a short time in COS-7 cells. This plasmid vector contains the early promoter and enhancer, the cytomegalovirus (CMV) (Boshart and other CM 41:521-530, 1985).This plasmid can be constructed, as shown in Fig. 13. The polyadenylation signal is supplied SV40 sequence and map coordinates 2589-2452, see Reddy and others, Science 200: 494-502, 1978. Begin replication of SV40 included in this plasmid to facilitate its use in COS-cells for rapid gene-expression analyses.Rabbit SN-CNTF quickly expressively in COS-7 cells as follows: 1,5 TPN EcoRI fragment of cDNA clone rabbit sciatic nerve, containing the complete coding region for the rabbit SN-CNTF (example 4), was subcloned into restrictively EcoRI vector pCMVXPL2. Selected a single clone, which after digestion with SacI and Bam HI restriction gives fragments corresponding in size predicted for insertion into the vector fragment of 1.5 TPN in the correct orientation. Plasmid DNA from this construct was prepared by the method of alkaline lysis followed by centrifugation in a CsCl density gradient (Maniatis and others, ibid). This DNA was introduced into COS-7 cells by the method of Sompayrac and Dama (Proc. Natl. Acad. Sci USA 78: 7 is rtsii.48 h after transfection Wednesday night and the precipitated cells were collected. The precipitated cells were extracted by the rapid destruction of ultrasound in ice in 20 mm sodium phosphate, pH 6,7, containing 1 mm EDTA, 0.1 mm PMSF and 0.1 μm pepstatin. A series of dilutions as cell extract, and the night environment from each culture were analyzed on the activity in the survival analysis of the ciliary ganglia.Cell extracts from cultures transfected with a vector containing a cDNA fragment CNTF, had a titer of Ca 15000 Tu/ml in bioanalysis and approximately 50 ng/ml CNTF, as determined by Western blot analysis. No cell extracts from cultures transfected with a single vector or the environment from any of the cultures did not show any detektiruya bioactivity CNTF protein or its presence in Western blot analysis. This result clearly demonstrated that CNTF cDNA we cloned that encodes a protein with the biological activity of a genuine SN-CNTF.Example 6. Selection SN-CNTF from recombinant animal cells.Since SN-CNTF is synthesized by host cells in a way similar to the natural material, the methods described above for purification of natural protein, applicable for purification and characterization of recombinant forms. Skilled in atolia depending on the situation.Example 7. Production of recombinant human CNTF.In one of the embodiments of the invention for the production of recombinant human CNTF was used bacteria Escherichia coli. If this were applied two alternative methods for constructing DNA, which must be Express, and two different expression vector. All these variant expression systems produce soluble biologically active CNTF protein with high yield in a bacterial cell extract. Methods of obtaining these producing systems described below. Therefore, the position of characteristic features of some of the figures given in parentheses, for example 233, denotes the number of pairs in the coding sequence of human CNTF (Fig. 12) at which to begin any feature or trait, in the course of transcription from A (1) initiation codon ATG.1. Preparation of DNA for the expression of CNTF.The strategy I to construct the 5' end (Fig. 14):
Clone genomic DNA of a person for CNTF in phage lambda EMBL3 of example 4 was digested with restriction enzymes Sal I and Hind III and 4.3 TPN fragment was purified by electrophoresis in a gel. It contains the coding sequence of CNTF above Hind III site (233). This 4,3-TPN fragment also ressio in bacterial cells, this intron was deleted site-directed in vitro mutagenesis using a synthetic oligonucleotide as described G. A. Mc Clary, F. Whitney, G. Geiselsoder (1989) Biotechnigues 7:282-289.A. Deletion of the intron site-directed mutagenesis using fahmideh vector and genetic selection.Site-directed mutagenesis for deletions 1,3-TPN-intron was carried out in a 4.3-TPN Sal I/Hind III DNA fragment, subcloned in formigny vector, Bluescript SK M 13 (-) (Strategene). This vector was chosen because it can take Sal I/Hind III insertions, large size (4,3 TPN). Famiglie vectors plasmid vectors containing the intergenic region, bacteriophage f1, which allows you to replicate as single-stranded DNA. In addition famiglie vectors have several advantages over single-stranded vectors bacteriophage M13. As family less than half the size of M13 vectors, which prefer insertions of size less than 2.3 TPN, large insertions can more easily subconious in family, and the possibility of spontaneous deletions decreases. Another advantage fahmid is that insertions can be sekvestirovatj directly from double-stranded superspiritual DNA, which facilitates their characteristics.Mutagenesis was carried out using the Muta-thi, relA1, pCS 105 [Cap] CS236 bears F'factor, electroysis on chloramphenicol and allows you to "save" single-stranded hamidou DNA using a suitable helpername phage R 408 used in the present work. "Saved" single-stranded fahmida DNA undergoes partial substitution for uracil, due to the dut (dutpase) and Ung (uracil-glycosylase)-mutations in CS 236. DNA priming with replaced uracile used for mutagenesis, selectively destroyed by the transformation in cells of the hosts that contain ung locus of the wild type, in this case DH5Lthus allowing the preferred replication, newly synthesized mutant DNA.To perform mutagenesis cleared gel 4,3 TPN-Sal I/Hind III fragment ligated into the digested and purified in the gel Sal I/Hind III SKM 13 (-). Legirovannoi DNA was introduced into cells of strain CS 236, made competent by the method of Hanahan, as described in J. Mol. Biol., 166:557 (1983). Transformants selectively on plates containing 50 μg/ml ampicillin (to select famid) and 30 μg/ml chloramphenicol (to select for the preservation of the F'factor). The transformants were checked for the presence of the correct insertion restriction analysis of DNA. The transformant carrying the correct insertion (pSHM-D19), used in Polak helper. Strain CS 236 containing pSHM-D19, were grown in broth of Buria containing ampicillin (50 μg/ml) and chloramphenicol (30 µg/ml) to A600approximately 0.3. Cells infected R 408 helper phage at a multiplicity of infection of 20, then shaken at 37oC 8 to 14 hours Of "saved" fahmid were extracted single-stranded sample.Site-directed (intron deleterule) mutagenesis was performed using 72-mo oligonucleotide (oligonucleotide 1 in Fig. 16). An oligonucleotide probe 1 has a base 1 - 30, complementary to the coding circuit immediately following /3'/ and the base 31 - 71, complementary to the coding circuit immediately above /5'/ intron, which should be deleteroute of CNTF genomic DNA. For use in the reactions mutagenesis oligonucleotide was fosforilirovanii using polynucleotides T4. The reaction mutagenesis using Rio Rad Muta Genekit, performed in accordance with the manufacturer's specifications, except that DNA after reaction mutagenesis was used to transform E. coli strain DH5L. Deletion mutants were characterized by restriction mapping of DNA and DNA-sequence double-stranded DNA from mutants with suitable restriction maps. Correctly deleteriously devoid of intron mutant, in Bluescript marked pMCN-21.Restriction analysis Delany with Nhe I protruding end; this allowed them to be combined with the remaining 3'-fragment of the gene after digestion with Nhe I.C. Attaching oligonucleotides 2 and 3 to the coding sequences with the remote intron.Oligonucleotides 2 and 3, containing added N-ends of the CNTF gene, were annealed to each other and ligated with the cut Nhe pMCN-2a. Legirovannoi DNA was then digested Bam H I and Hind III to release the DNA fragment, designated CNTF-Syn I, which contains the DNA sequence suitable for expression in E. coli and coding for human CNTF above Hind III site (233).Strategy 2 to construct a 5'-end (Fig.15):
Implemented an alternative strategy, in which introns are removed site-directed mutagenesis, but prepared more fully synthetic DNA sequence encoding CNTF above Hind III site (233), but without the intron. To make this synthetic construct DNA, were synthesized oligonucleotides from 5 to 10, Fig.17. These oligonucleotides are designed to form a partially overlapping double-stranded pairs: 5, 6, 7 and 8, 9 and 10. Each pair consists of double-stranded single-stranded protruding ends, intended for correct ligating them together in the order (5 and 6)-(7 and 8)-(9 and 10). OId, called CNTF-Syn 2. This synthetic DNA also contained: (1) the modified codons, which take into account the preferred use of codons in E. coli (see Fig. 12 and 17), (2) 5' translational linker used above (Fig. 16 and 17), and (3) 5' Bam H I speaker and 3' Hind III protruding ends to facilitate ligation and cloning (Fig.17).Preparation of 3'-end for the expression.Design (Fig. 14 or 15) clone human genomic DNA CNTF in phage lambda EMBL3 of example 4 was cut by the restriction enzyme Hind III, and 2, 1 TPN-fragment containing the coding sequence Hild III site was cleared in the gel. This 2,1 TPN fragment cloned in cut Hild III plasmid pFMBL8 (Donti and others, 1983, Nucleic Acidc Res 11: 1645). Spe I site (613) inserted from 2.1 TPN-the insertion of DNA oligonucleotide-directed mutagenesis 13 nucleotides following the stop codon, the final CNTF sequence using synthetic oligonucleotide 4 (Fig.16). The mutant plasmid was cut by Hind III and Spl I to release the underlying encoding fragment was gel purified and called CNTF - Syn 3 (containing coding sequences for human CNTF below Hind III site (233)).Preparation of full expression construct.CNT-Syn I ligated ternative DNA fragment, each of which encodes human CNTF and is suitable modifications of the DNA sequence to enhance the efficient expression in E. coli. These DNA fragments are then expressed in E. coli after sublimirovanny in: (A) bacterial expression vector PT5T-based promoter of phage 17 or (B) a bacterial expression vector, pT3xI-2, based on the hybrid lactose promoter of tryptophan operon ('Tac').2. Expression of CNTF using expression vector system based on "T7 promoter", the characteristics of this vector is shown in Fig. 17.A. Description pT5T.Based on the T7 promoter expression vector pT5T is largely the same as pJU 1003 described Squires, et.al., J. Biol. Chem. (1988) 263: 16297-16302, except that it is shorter than the size of the DNA between the unique Bgl III site 5' T7 promoter and the Cla I site in the gene for resistance to tetracycline. The sequence of this DNA is as follows:
ATCGATGATA AGCTGCAAA CATGAGAATT GAGCTCCCCG GAGATCCTTA
B. Design full expression vector.Cleared gel vector was linearizable Bam H I and Spe I restriction enzymes. CNTF - Syn 1/3 mixed with linsidomine vecto the first design).C. Expression of recombinant human CNTF in E. coli: pT5T: CNTF - Syn 1/3 transformed into E. coli strain BL21 (DE3) for expression. This strain, described by Studier and Moffat, J. Mol. Biol. (1986) 189: 113 - 130, contains the gene for RNA polymerase under the control of the T7 induced I PTG lac promoter on neveretheless lithogenes the bacteriophage lambda. 10 skanirovaniya transformed two glona how it was discovered, Express IPTG-induced protein migrating with a molecular weight corresponding to the largest CNTF ( 24 kDa). These two clones designated as pT5T: CNTF - Syn1/3-5a and 5c. DNA sequencing pT5T : CNTF - Syn 1/3 - 5a and 5c confirmed that the data sequence of correct recombinants.High level expression of recombinant CNTF was achieved by growing cells in Luria broth with 15 μg/ml tetracycline to a cell density corresponding to A6000.5 to 0.8. Cells were grown for 1.5-4 h or without IpTG ("neindutsirovannom") or IPTG was added to final concentration of 1.0 mm ("induced").G. analysis of the expressed protein by electrophoresis in SDS polyacrylamide gel followed by staining dye Coomassie or Western blot turns.The cells were collected by rapid centrifugation and dissolved SDS, 0,0625 M HCl, pH 6,8 (and boiled 2 min) (Fig. 20 and 21). In cells transformed pT5T : CNTF - Syn 1,3-5a and induced by IPTG for 2 h (lane 5, Fig.20) observed intensely colored dye Coomassie strip extending in the position expected for SN-CNTF (24 kDa). If the cells were grown without IPTG (lane 4, Fig. 20) observed a significantly lower intensity of this band, as expected for a protein, the expression of which is under the control of the lac operon. Cells transformed by the vector pT5T without insertions CNTF did not show such a band no induced (lane 3, Fig.20), nor in neindutsirovannom (lane 2, Fig.20) variant. Track 2 includes the molecular weight standards.Identical SDS - polyacrylamide gel were transferred to nitrocellulose or did immunoblotting with affinity-purified antibodies to CNTF peptide A (E-S-Y-V-K-H-Q-G-L-N-K-N). In cells transformed pT5T : CNTF - Syh 1,3-5a and induced by IPTG for 2 h (lane 5, Fig. 21), there is a tight band, recognizable afinno-purified antibodies to CNTF peptide A and going at the position expected for CNTF (34 kDa). If the cells were grown without IPTG (lane 4, Fig.21) observed much less intense band at this position. Cells transformed by the vector pT5T without inserti.20).Lane 1 contains molecular weight standards.In addition, cells transformed pT5T : CNTF - Syn 1, 3-5 and induced by IPTG for 2 h, was destroyed by passage three times through the cell compressor jacket. An aliquot coarse cell lysate was divided into fractions of supernatant and a precipitate by centrifugation at 20000 timing in JA-20 rotor (Beckman) for 15 minutes Aliquots rough lysate, supernatant and precipitate, representing the same number of initial cell suspension, also ran on the same SDS-polyacrylamide gel, transferred to nitrocellulose and did immunoblotting with afinno-cleaned antipeptide A antibodies. The supernatant of the lysate (lane 8, Fig. 21) contained much more immunoreactive CNTF than the precipitated lysate (lane 9, Fig.21). The level of supernatant CNTF was comparable to nefrackzionirovannam lysate (lane 7, Fig.21).D. the Bioactivity expressed CNTF.Cells collected by rapid centrifugation, resuspendable in 20 mm Tris-HCl, pH 8.2 1/35 initial volume of cell suspension, was destroyed by passirovannym through cellular compressor jacket three times and crude cell lysate was divided into fractions of supernatant and a precipitate by centrifugation at 20000 rpm in SA-20 rotor (Beckman) for 15 minutes Poemate ciliary nerve ganglion cells (as described in example 1). Supernatant showed significant biological activity at a dilution of 1:1,000,000 (Fig. 22). The specific activity of the recombinant human SN-CNTF, as was estimated on the basis of bioactivity and the number of CNTF protein, calculated from immunoblots approximately 275 TU/ng. This specific activity is more than twice the specific activity of the purified CNTF protein, indicating that recombinant CNTF biologically active in bacterial cell extrate. Lysates of cells transformed pT5T, without the insertion of CNTF, did not show detectable bioactivity.The supernatant also chased by electrophoresis in 15% polyacrylamide reducing SDS-gel and cut into 1 mm slices of width), which were extracted overnight in cell culture medium at 4oC with shaking, and analyzed for activity as described in example 1. The insert of Fig. 22 illustrates that the peak of bioactivity detected in the fractions corresponding to the 24 kDa (as expected for CNTF).E. Amino acid sequence expressed by CNTF.The area around 24 kDa (similar to Fig. 20) cut out from the SDS polyacrylamide gel, but not stained, Kumasi. This mater what s the following amino acid sequence: AFTEHSPLTPHRRDL[? ]S... A question mark icon corresponds to C in the human sequence (Fig. 12), which cannot be determined by this method. This sequence corresponds to the expected human CNTF (Fig. 12) and provides additional evidence that is properly expressed. It also indicates that aminoterminal methionine removed during expression, leaving alanine, as aminoterminal amino acids in the expressed protein.The above results demonstrate that immunologically cross-reactive CNTF expressively at a high level in a biologically active form, much of which is soluble after lysis of bacterial cells.3. Expression of CNTF using the expression vector, based on the hybrid promoter 'TAC' (see Fig. 19 characteristics of the vector).A. Description pT3XI-2 (modification of pKK223-3).The starting plasmid for this design served as a plasmid pKK 223-3 purchased from Pharmacia. Plasmid pKK223-3 is part of a gene tetracyclic sustainability. This non-functional gene was replaced with the complete genome of resistance to tetracycline pBR 322. Plasmid pKK 223-3 digested completely Sph I and partially Bam H I, 4,4 TA 5' GATCTAGAATTGTCATGTTTGACAGCTTATCAT 3'
3' ATCTTAACAGTACAAACTGTCGAATAGTAGC 5'
and 539 mo a DNA fragment from Cla I / Sph I restricciones gene, tetracycline resistance pBR (PL Biochemicals, 27-4891-01). The resulting plasmid was designated pCJ1.Then XhoI linker purchased from New England Biolals, insertional in the Pvu II site of plasmid pCS1, to obtain plasmid pCSX-1. This insertion violated the rop gene, which controls chopinot plasmids. EcoR I Fragment containing the lac I gene, was purified from the plasmid pMC9(Colos and others, Proc. Natl. Acad. Sci USA (1983), 80:3015-19), and then was inserted into the Xho I site with Xho I EcoR I adapters having the sequence
5' TCGAGTCTAGA 3'
3' CAGATCTTTAA 5'
The sequence of polylinker between the EcoR I and Pst I sites in the plasmid pKK223-3 was then replaced polyglycerol the sequence given below
5' AATTCCCGGG TACCAGATCT GAGCTCACTA GTCTGCA 3'
3' GGGCCC ATGGTCTAGA CTCGAGTGAT CAG 5'
The obtained plasmid vector outlined pCSXI-1.In the end, the gene for tetracycline resistance was replaced with a similar genome, which had sites for the restriction enzymes Hind III, Bam H I and Sal I, disturbed by bisulfite mutagenesis. The following procedure was used to get to mutate the gene for tetracycline resistance pBR322. Plasmid pBR322 was cut by Hind III and then was subjected to bisulfite mutagenesis nutritinal Hind III, to linearize any plasmid that has escaped mutagenesis. E. coli SM 109 (Janisch-Perron and others, Gene (1985) 33: 103-119) was transformed with this plasmid and then sown on the selective medium. Isolated plasmids from the tetracycline-resistant colonies were checked for the absence of a Hind III site in the gene for resistance to tetracycline. Successfully mutated plasmid was designated pT1. A similar procedure was used to introduce mutations into the BamH1 site pT1 and obtain plasmid pT2. Plasmid pT2 in turn was subjected to mutagenesis to remove the Sal I site, and received plasmid pT3Cla I/Bsm I fragment pT3 carrying the mutant gene for tetracycline resistance, allocated and used to replace the homologous fragment pCJXI - 1 to create pT3XI - 2. A mutant gene for tetracycline resistance is still encodes functional protein.B. Creating pT3XI - 2 - 10TC3FGF syn (preparation of the tac - promoter vector for CNTF).Was originally synthesized gene basic fibroblast growth factor (FGF). This gene encodes the sequence described by Sommer and others, (1987, Biochem. Biophys. Res. Commun. 141.67), but uses codons that are preferred in the genes with high expression level in E. coli. The structure of this gene is such that (see Squires and others, 1988, ibid) EF provides the AMI and sequenced. The structure of this gene following completely.Key features of the gene identified.Then he was isolated by restriction BamHI and Hind III and insertion into Bam H I / Hind III cut pSU 1003/ Squires and others, 1988, ibid; with pSH 1001003-Syn FGF. This plasmid was digested xbaI and Hind III and xba I/Hind III fragment carrying the FGF gene, was identified. This fragment is ligated in RTG-2, split EcoR I and Hind III, using EcOR I-xba I linker
pATT TCC ACA ACG GTT TCC, CT3';
3'GG TGC CAA AGG GAG ATCp5'.The new plasmid was designated pT3XI - 2 - 10TC3 FGF syn.By the Insertion of CNTF expressionid structures in Tac promoter vector.pT3XI - 2 - 10TC3 FGF syn uncoupled BamH I and Spl which I held to the linearization of 7.4 TPN expression vector and the release of approximately 0.5 TPN FGF DNA fragment. In separate reactions CNTFsyn 1/3 and CNTF syn 2/3 ligated with the purified gel Bam H I/Sp lI cleaved DNA fragment of the vector to form plasmid pT3XI - 2: CNTF - syn 1/3 and pT3XI - 2 : syn 2/3, respectively.D. Expression in E. coli pT3XI - 2 : CNTF - syn 1/3 transformed into a phage-resistant strain of E. coli SM 07, 14 transformants were grown and analyzed for the expression of CNTF electrophoresis in SDS - polyacrylamide body staining with Coomassie Brilliant Blue 4 transformant showed intensely colored stripes is in Luria broth with 15 μg/ml tetracycline until the cell density, appropriate A6000,8. Added IpTG to a final concentration of 1.0 mm and the cells were allowed to grow for 2 hours All four transformant showed almost the same density CNTF as Kumasi-painted and gel immunoblotting. Apparent level of CNTF expression in these transformants (according to gels) was about 1/4 compared to demonstrate pT5T:CNTF - syn 4/3 - 5a, growing, like the previous one, with tetracycline. 7 ml of this cell suspension was added to 50 ml of Luria broth in large flasks and were grown at 27oC with shaking up A600approximately 0.5. Then IPTG was added at a final concentration of 0.5 mm to induce the expression of CNTF and continued to grow until reaching A600approximately 1.2, which usually takes 4 - 6 hours the Cells were collected by centrifugation at SA-20 rotor at 7000 rpm for 5 min at 4oC and washed again by centrifugation in 50 mm phosphate buffer, pH 8.0 (Buffer A) at 4oC. the Supernatant was removed and the precipitated cells were frozen at -20oC as pasta.The cell paste is suspended in buffer B (buffer a containing 1 mm of EGTA and 1 mm EDTA) (0.5 g of paste per 1 ml buffer) at 4oC and was performed three times through the cell compressor of the French to destroy the bacteria is dalali by centrifugation for 15 min at the highest speed in Mirofuge. This stage usually leads to a reduction in the content of nucleic acids, as measured by the ratio of A260/A280from 25 to less than 5%.The sample is then applied to a column of Q-Sepharose (Pharmacia), equilibrated with buffer B. CNTF is a major component of the lysate of damaged cells, so the chromatography may be accompanied by analysis of the column fractions on SDS - polyacrylamide gel. Main, Kumasi-colored stripe SN-CNTF, approximately 24 kDa. Using this analysis, CNTF wash off with Q-Sepharose column Buffer B.The fractions containing the bulk of the CNTF protein, collect, dialist against Buffer C (5 mm phosphate buffer, pH 8.0, containing 1 mm of EGTA and 1 mm EDTA) and applied to a column of Q - Sepharose, balanced with Buffer C. the Column was washed with Buffer C until until A280not returned to baseline, indicating that the unbound proteins are washed from the column. In Buffer C CNTF binds to the Q - Sepharose column, and then elute 0 - 0.1 M NaCl gradient in Buffer C. CNTF emerges from the column at approximately 40 mm NaCl and with more than 90% purity, judging by colored Kumasi-SDS-polyacrylamide gel fraction of the peak CNTF.The sequence of the gene in FGF listed at the end of the text.Vasser, M. Proc. Natl. Acad. Sci. USA 80, 21-25 (1983).3. Shimatake, H. and Rosenberg, M. Nature 292, 128-132 (1981).4. Derom, C., Gheysen, D. and Fiers, W. Gene 17, 45-54 (1982).5. Hallewell, R. A. and Emtage, S., Gene 9, 27-47 (1980).6. Brosius, J., Dull, T. J., Sleeter, D. D. and Noller, H. F. J. Mol. Biol. 148 107-127 (1981).7. Normanly, J., Ogden, R. C., Horvath, S. J. and Abelson, J. Nature 321, 213-219 (1986).8. Belasco, J. G., Nilsson, G., von Gabain, A. and Cohen, S. N. Cell 46, 245-251 (1986).9. Schmeissner, U., McKenney, K., Rosenberg, M. and Court, D. J. Mol. Biol. 176. 39-53 (1984).10. Mott, J. E. , Galloway, J. L. and Platt, T. EMBO J. 4, 1887-1891 (1985).11. Koshland, and D. Botstein, D. Cell 20. 749-760 (1980).12. Morva, N. R., Kakamura, K. and Inouye, M. J. Mol. Biol. 143, 317-328 (1980).13. Surin, B. P. , Jans, D. A., Fimmel, A. L., Shaw, D. C., Cox, G. B. and Rosenberg, H. J. Bacteriol. 157, 772-778 (1984).14. Sutcliffe, J. G. Proc. Natl. Acad. Sci. USA 75, 3737-3741 (1978).15. Peden, K. W. C. Gene 22, 277-280 (1983).16. Alton, N. K. and Vapnek, D. Nature 282, 864-869 (1979).17. Yang, M., Galizzi, A., and Henner, D. Nuc. Acids Res. 11(2), 237-248 (1983).18. Wong, S.-L., Price, C. W., Goldfarb, D. S., and Doi, R. H. Proc. Natl. Acad. Sci. USA 81, 1184-1188 (1984).19. Wang, P.-Z., and Doi, R. H. J. Biol. Chem. 251, 8619-8625, (1984).20. Lin, C.-K. , Quinn, L. A. Rodriguez, R. L. J. Cell Biochem. Suppi. (9B), p. 198 (1985).21. Vasantha, N., Thompson, L. D., Rhodes, C., Banner, C., Nagle, J., and Filpula, D. J. Bact. 159(3), 881-819 (1984).22. Palva, l., Sarvas, M., Lehtovaara, P., Sibazkov, M., and Kaariainen, L. Proc. Natl. Acad. Sci. USA 79, 5582-5586 (1982).23. Wong. S.-L., Pricee, C. W., Goldfarb, D. S Thompson, L. D., Rhodes, C., Banner, C. Nagle, J., and Filula, D. J. Bact. 159(3), 811-819 (1984).26. Yansura, D. G. and Henner, D. J. PNAS 81, 439-443 (1984).27. Gray, G. L., McKeown, K. A., Jones, A. J. S., Seeburg, P. 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The DNA fragment encoding ciliary neurotrophic factor sciatic nerve (SN-CNTF) a person having the nucleotide sequence represented in the North, or equivalent sequence, as defined by the degeneracy of the genetic code.2. Recombinant SN-CNTF person obtained in E. coli cells, transformed with the recombinant vector containing the DNA fragment on the e, the ability to increase the survival of ciliary ganglion nerve cells.3. Recombinant SN-CNTE man p. 2, characterized in that characterized the derived amino acid sequence represented in C.4. A method of obtaining a recombinant SN-CNTF man, which lies in the cultivation of E. coli cells, transformed with the recombinant vector containing the DNA fragment under item 1, encoding SN-CNTF person, and the subsequent selection of a product from bacterial cells.5. The method according to p. 4, characterized in that the cultivated strain of E. coli BL21 (DES), transformed with the recombinant vector pT5T : CNTF-S UE 1/3.
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