JPH04502762A - somatotropin analog - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] somatotropin analog field of invention The present invention relates to analogs of animal somatotropin or growth hormone. More details In particular, the present invention specifically relates to novel bovine growth hormone analogs in which amino acid residue 99 has been changed. Regarding

Background of the invention Bovine somatotropin (BST) is a well-studied growth hormone (para. Dini A.C. (Pafad in i, A, C) et al., C.R.C. - Critical Reviews in Biochemistry (CRCCr1t) Rev. Biochem, ) 15:25-56 (1983)). somatotropy It was originally discovered in pituitary gland extracts of various animals. Generally, somatotropin is a conserved molecule, with similarities in amino acid sequence and structure between different animal species. You can see it.

matostatin has two intramolecular disulfide bonds, including bst. It is a globular protein consisting of a single chain of 00 amino acids. In detail, bst is 1 Single chain of 90-191 amino acids, spherical with two intramolecular disulfide bonds Structure and about 22. It has a molecular weight of OOO Daltons.

Natural BST extracted from the pituitary gland is heterogeneous. About the protein at least Six major forms have also been described. Its longest form has 191 amino acid residues and It has an ala-phe amino terminus.

The second form has 190 amino acid residues and a phe amino/terminus. third The form has 187 amino acid residues and a met amino terminus. remaining 3 of bst In this form, leucine is substituted with valine at position 127. Also, this heterogeneity In addition, unclear heterogeneity of bovine somatotropin has been described (Herbert et al. Hart, 1.C, et al., Biochemical Journal (Bi ochem J, ), 218:573-581 (1984), Wallace M ( Wa I 1ace M,) and Dixon H B-x7 (Di ckson, H.B.F.) Biochemical Journal (Biochem) , J.), 100: 593-600 (1965)).

When natural extracts are fractionated by anion exchange chromatography, it is clear that No heterogeneity in electrophoresis is seen. This means that the known form is It is shown that they have different relative efficacies in B. Also, bst's other obvious No species vary in a rat growth model when fractionated on an ion exchange column. (Hart et al. and Wallace and Dixon, supra).

Is the unclear heterogeneity that indicates biological diversity due to genetic variability? Is it due to post-translational modification in vivo or due to differences in phosphorylation? ・Liberti, J.P., et al., Biochemical and Biophys. Biochem, and Biop Hys, Res.

Comm,) 128 Nia 13-720.1985), produced artificially at the time of isolation It is unknown whether this is due to this.

Produced by recombinant microorganisms (rbSt) or extracted from pituitary tissue ruts Sisomatotropin is of commercial importance. It increases milk secretion in dairy cows and in beef cows. Increases size and meat production. To provide commercially acceptable production improvements It is estimated that up to 20 mg per animal per day is required. such administration The amount requires an efficient method of administration. The efficacy of BST described herein and stability are beneficial as the daily dose of drug administered to each animal can be reduced. It's a benefit.

Furthermore, one of the problems in the production of BST produced by recombinant methods is that it is acidic or Liquid processing of rbSt at alkaline pH and storage of asparagus at position 99 The gin residue is converted to isoaspartic acid. The obtained rbSt is This is called early eluating rbst. This is because it elutes earlier than native rbSt in reverse phase HPLC. Iso Aspartate is a product of ammonia formed by condensing the asparagine side chain with the peptide backbone. Formed when desorption occurs. In addition, during storage, the backbone of the peptide and rbSt Chain cleavage also occurs due to a condensation reaction with the asparagine residue at position 99 of the molecule.

The chain cleavage products are shared by a disulfide bond between cysteine residues 53 and 164. bindingly clustered and its relative to native or early eluting rbSt. From the relative elution position of "early-early elution rbSt" (early-early It is called eluting rbSt)j.

The instability caused by the modification of asparagine 99 inhibits its isolation. , amino acid substitution at position 99 because it disappears as a reconstitution product during disposal and storage. rbSt, which is more stable while retaining or enhancing its biological activity. It would be convenient to produce analogues.

Disclosure of information Analogues of rbSt are known (e.g. European patent applications 75°444 and 103,395 and Nucleic A c1d Res, ) 10(20):6487 (19B2)).

G Winter (G, Wjnter) and A.R. Fair Sud (A, R, Fersht), T.I.B. Niss (TIBS), 2 ．． p. 115 (1984), changes the amino acid composition at key sequence sites. The author comments on the effect of altering enzyme activity.

B Y Chick (P, Y, Chou) and G.D. Fazman (G , D, Fasman) (Ann, Rev, Bioc hem, ), pp. 47, 251-76 (197B)) uses amino acid sequences to It refers to predicting the secondary and tertiary structure of carbon/fine quality. Bee Y Chou (P, Y, Chou) and G.D. Fuzz 7:/ (G, D, Fasman) (Journal of Molecular 1 Biology (J, Mo 1. Bio 1. ), 115.135-75 (1977)) %X with reference to β-turns in protein. Sequence and X-ray structure are known From an analysis of 459 β-turn regions in 29 tannocorpores, they found that β -The two most frequently occurring amino acids at the third position of a turn are asparagine and asparagine. They were found to be gic acid and glycine. to all parts during the turn. The residues with the highest β-turn potential are proline and group. These are lysine, asparagine, aspartic acid and serine.

Summary of the invention The present invention replaces asparagine corresponding to residue 99 of natural bst with a different amino acid. biological activity of bSt and its analogs or during liquid storage by substituting stability or both.

Other living things, especially mammals including pigs, fish, sheep, horses, rats, monkeys and humans. Somatotropin from mammals can be similarly varied.

More particularly and preferably they are aspa located at amino acid residue 99. Lagin, especially glycine, serine, proline, aspartic acid, glutamic acid, with different amino acid residues including serine-serine or serine-aspartic acid. A substituted bst-like compound species.

More specifically, the animal somatotropin is found in cows, pigs, fish, sheep, horses, and rats. selected from the group consisting of human, monkey and human somatotropins.

Even more particularly, the animal somatotropin is bovine somatotropin.

Also, an effective amount of the somatotropin of the present invention is administered to an animal (particularly the animal is a cow). lysomatotropin is bst). Provides a method for promoting growth, especially in cattle.

Furthermore, an effective amount of animal somatotropin of the present invention may be administered to a female breeding animal (hereinafter referred to as The animal is a cow and the somatotropin is bst). Provided are methods for increasing milk production in breeding animals.

Detailed description Due to the heterogeneity of somatotropin molecules, the positions of amino acid residues in various somatotropins are The location numbers may differ. The term ``natural mammalian somatotropin'' refers to this It includes naturally occurring species. Chart 1 is the 99th place modified by the present invention. The specific sites of one species of bst are shown, corresponding to the groups. to other species or analogs Where relevant, numbering for other somatotropins may be different. Not possible. Using the bst asparagine 99 shown in Chart 1, one skilled in the art can somatotropins, e.g. mammalian somatotropins, e.g. mammalian somatotropins The corresponding amino acid in pin asparagine 99 or its analogs, The desired liquid storage stability, increased biological activity and uniformity of the present invention are You can get a uniform effect.

The invention encompasses both chemical and genetic modifications in the art.

Preferred genetic modifications include various amino acids replacing naturally occurring asparagine. by single site-directed mutagenesis to insert acid residues.

The term "animal somatotropin" refers to somatotropins derived from animals, e.g. mammals. lopins, derived from natural sources such as pituitary tissue or in naturally occurring forms Microorganisms transformed by recombinant genetics to produce somatotropin It includes somatotropin derived from Bovine somatotropin or soma of bovine origin When referring to a specific mammalian source, such as totropine, the somatotropin is a natural source. or derived from transformed microorganisms.

As used herein, the term "microorganism" refers to bacteria, yeast, actinomycetes, and cell culture. unicellular prokaryotes, such as single cells from higher plants and animals grown in Used to include both eukaryotes.

The term "natural" refers to naturally occurring forms of somatotropin, which include, for example: somatotropin from natural sources, such as in the glandular tissue, or in naturally occurring forms; recombinant genetics to produce somatotropin with the same amino acid sequence. It may also be derived from a microorganism that has been transformed.

Mammalian somatotropins are very similar in amino acid sequence and physical structure. are doing. Although the method described in the Examples is directed to bst, the method specifically If you are facing similar liquid processing and storage problems, have the necessary accessories available for replacement. Any animal somatotropin, such as a mammalian somatotropin, that has a sparagine residue Equally applicable to lopins.

The high relative potency of the bst analogs of the invention is readily demonstrated using hypophysectomized rats. can be determined (Evans.

H, M,) and Long J, A, Anatomica/ l/-L/:I-do (Anat, Rec,) 21:61.1921).

Pituitary bSt in the present invention, recombinant bSt (rbSt) and various bs Using the t analog, the relative increase in total body weight is recorded.

Site-directed mutagenesis: Site-directed mutagenesis to introduce specific changes to a DNA sequence. Various techniques of aberrant mutagenesis have been developed to produce the compounds of the present invention. (Kramer, W. et al., Nook) Leak Acids Research (Nucl, Ac1ds Res,), 12.94 pp. 41-56 (1984); d e c k i, W, ), Proceedings of National Academy of Sciences Demy of Sciences Op the United States of America Merica (Proc, Nat 1. Acad, Sci, USA), 83.7 pp. 177-81 (1986); Schiller, M. J. (Zoller).

M, J.) and Smith, M., Nu Creek Acids. ・Research (Nucl, Ac1ds Res,), 10.6487-6500 (19B2); Norrander, J. et al., Gene ( G e n e), 26.101-1o6 (1983); Kunkel T. Kunke I, T, A, Proceedings of National Academy of Sciences of the United States of America ・America (Proc, Nat 1. Acad, Sci, USA), 82 ．． 488-92 (1985); Schold, A. et al. D.N.A. (DNA), 3.469-77 (1984)).

The present inventors applied the primer characteristics of Sjöld et al. to 5 of the 7 analogs produced. Differential mutagenesis techniques were used. However, only one of the primers should be Used in the bridging reaction, and 18 μe of T4 gene 32 protein was added to the elongation reaction. added.

Colony filter hybridization: Filter hybridization The screening technique for Based on the ability to position and hybridize to form double-stranded segments (Ha Hanahan, D. and Meselman, M. e1s o n, M,), Methods in Zymology (Met h, Enz ymo 1. ), 100.333-42 (1983)). This conclusion The thermal stability of the combination depends on the number of pairs and unpairs contained within the double-stranded region.

The more unpaired it contains, the weaker the base-pair bond becomes, causing the DNA bond to cleave The temperature required for this is lower. colony filter hybridization (Bryan, R. et al., Miku Microbiology (1986)). Natural arrangement and projection By constructing mutant oligomers that maximize the temperature difference between sequences of natural mutations. and hybridize at lower temperatures to reduce the binding of the probes to the paired and most It can be a matched array. Perfect mating device for cleaning at elevated temperatures. The duplex remains bound, but the unpaired probe-DNA duplex remains bound. x becomes unstable and dissociates.

Therefore, the paired duplex has the darkest signal on the autoradiogram. This provides a method for detecting colonies with the desired sequence. Next, this DNA from the colonies is isolated and sequenced.

For filter preparation, place the nitrocellulose filter on a plate and moisten it. let Mark the filter and plate for orientation, then remove the filter. Carefully (lift the master filter plate off the plate. Bring the master filter plate to room temperature. Incubate overnight to repopulate the colony. The filter is 0.5M Place each piece on Whatman paper soaked in Na0HS1゜5M NaCl2 for 10 minutes. Denatured by placing in IM Tris, 1.5N NaCl, pH 7.4. Neutralize the soaked Whatman paper for 10 minutes each time, then replace the new paper with the soaked Whatman paper. Air dry on Toman paper for 30 minutes. Then, bake in a vacuum at 80℃ for 2 hours. .

A kit for radiolabeling mutant oligonucleotides used as probes. The enzyme reaction is as follows: 2μg oligo 2u 510X kinase buffer Q, y32-P ATP 100μCi% T4 bovinease 2μg and water 4μQ Mix and incubate for 1 hour at 37°C. 10m12 disposable column A 1 ml column was packed with DEAE Sephacel and 2-3 ml of salt buffer (1.5M NaC in TE, then low salt buffer) Equilibrate with 2-3 mQ of solution. Dilute the kinase reaction with 200ml of low salt buffer. and place directly into the column. Counts no longer elute from the column. Wash the column with 10 ml of low salt buffer until 100 ml. The probe has high salt concentration Elution was carried out in 4 mff of buffer.

For hybridization, place the filter in a crystallization dish and add 5X Denhard. Denhardts (1% BSA, 1% Ficoll and and 1% PVP), 5X SSC (0.75M NaCQ, 0.075M quench The batch was pre-incubated at 40 °C for 1 h in 0.1% SDS Hybridize.

The hybridization solution was replaced and the probe was added. Cover the vaginal opening and slowly Hybridization is carried out overnight with stirring. Then 5x ssc, o, t%SD Rinse the filter by changing S several times.

Water bath and cleaning solution (5X 5SC10, 1% 5DS) at cleaning temperature (46°C) Place the filter into the solution while heating to . one filter at a time Transfer to a new crystallization dish and wash 3 x 20 minutes, changing the dish after each wash.

They are then air-dried on Whatman paper, wrapped in Saran wrap and exposed if desired. Ru.

Preparation of vector DNA: DNA for sequencing was prepared using phenol/chloroform A single combined extraction was performed, and the DNA was passed through a Sephadex G-50 column. El Aji, except that it is spin-dialyzed. Erron (L, Age I 1on) and T Cheno (T, Chen) , Gene Analysis Techniques (Gene Ana 1. Techn,) , 3.86-89 (1986).

Sequencing = Double-stranded sequencing is performed according to the following method = 3μQ2NNaOH, 2m Add M EDTA to 12 μg of DNA and incubate for 15 minutes. Ru. 6μQ 3M Na0Ac, 1μg primer and 100μQ 95% ether Nol is added and the DNA is precipitated on dry ice for 20-30 minutes. The Pele Collect, wash and vacuum dry. Add it to 13 μm of water and 4 μm of RT buffer. (0,3M) Lis-hydrochloric acid, pH 8,3,0,375M NaCl, 37.5m Dissolved in M MgCQ=, 2.5mM DTT), y32P dCTP 2u Add Q and 1 μe of reverse transcriptase. 4 μe of the mixture was added to 1 μl of each 4 Eso Bends containing A mix, A mix, T mix or C mix Pipette into a test tube. Incubate the test at 42°C for 10 minutes. Ru. 1μQ chase mix (0.25mM dN) TPs) and incubate them for an additional 5 minutes. 10μC stop solution (80% formamide, 10mM NaOH, 1mM EDTA, 0.1% oxygen Rencia tool and 0.1% bromophenol blue) were added and the reaction was continued for 3 minutes. Boil and load 3 μQ of each onto a sequencing gel.

Induction method and 5DS-PAGE analysis: see PCT/US8810O328.

Example 1 Using site-directed mutagenesis techniques for double-stranded primer extension, rbSt cDNA Modified code for serine and proline amino acids at position 99 in m4 gene (filed on January 27, 1988, herein incorporated by reference) PCT patent application PCT/US8810032B). This method smell Then, clone the target sequence into a suitable plasmid and prepare plasmid DNA. Ru. NaOH creates a “nick” in the deoxyribose phosphate backbone of the DNA molecule The plasmid DNA is denatured by treatment with .

This relaxes the DNA and changes the oligomer containing the desired sequence. can hybridize to a plasmid sequence containing residue 99 of bst. I'm going to growl. The oligomer 〇3゛ end extends the primer and generates a mutagenic agent. Reverse transcriptase containing oligomers that synthesizes new DNA strands and replaces normal Aiura-like strands Create primers for DNA polymerase activity. The elongation reaction the oligomer-directed change ) increases the possibility of uptake. Transform the DNA into competent cells. , the obtained colonies were scanned by colony filter hybridization. Clean. Plasmid DNA is isolated and sequenced from positive candidates.

Used to construct serine and proline changes at position 99 in the rbSt m4 gene. The oligomers are produced by the previously described technology (PCT/US8810032B) . The oligonucleotide produced in this way called C3T-88 (Chart 3) According to the DNA sequence of Otido, asparagine AAC has changed to serine TCT. Another one, called C3T-89 (Chart 3), contains asparagine AAC. It has changed to proline CCG. Both of them are designed with Tm of 53℃, Therefore, as mentioned above, hybridization can be performed at 40°C, and stringency washing ( Stringent washing) is possible.

In parallel experiments, serine and proline oligomers were isolated from pBR322. Hybridize with Trp-BStm4 vector. This vector is Contains m4 cDNA of trp bromotor and rbSt (PCT/US 88100328). After primer extension, the DNA was used to incubate MC100O ( The Experiment with Jean Funi John Strain Kit , Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (the Experiments with Gene Fusi on 5train Kit, Col1d Spring Harbor Lab oratory, Colorado Spring Harbor, New York) Transform the competent cells (available from Biochemistry, Inc.). The transformed cells were plated. Grow to obtain 100-200 colonies on 10 plates. Then, the corresponding Transfer Ronnie onto nitrocellulose. The cells are lysed and their DNA is extracted from the fibre. Attach to router. A radiolabeled oligomer probe is labeled with the oligomer. prepared from C3T-88 and C3T-89 with Nasase and 32P-ATP.

Details of the probe operation for colony filter hybridization are described above. It is as follows.

Six candidates are highly visible on the autoradiogram for serine mutagenesis. It showed a strong positive signal. Plasmid DNA was isolated and sequenced for each candidate. Established. One of the candidates was found to have a serine change. This sudden change The different genes are called m4-99set. For proline mutagenesis, 6 2 positive candidates were analyzed by DNA sequencing and 2 were found to contain the desired change. It turned out that. These mutant genes are m499pr.

It is called.

The m4-99ser and m4-99pro genes were extracted from the parental vector with EcoRI - Excise it as a Hindllr fragment and use EcoRI of pURA-m4 vector. -Cloned into the Hindln restriction site (PCT/US88100328) , Chart 2 shows cloning of m4-99set gene into pURA-m4 vector. Indicates the Similar construction was performed for the m4-99pro gene. Kroni For sequence confirmation of the vector, pURA-99Se r and pURA-99 It's called Pro. These vectors are transformed into fermentation expression strain BST-IC (P CT/US8810032B).

Transformants from each cloning were induced and samples were separated on 5DS-PAGE. and evaluate the ability of the cells to express the modified rbSt gene under non-optimized conditions. . The results of the 5DS-PAGE analysis showed that pURA-99ser was isolated in each of the three inductions. and produced rbSt in 13.8%, 14.6% and 26.4% of total cellular protein. We showed that this occurs. The results of 5DS-PAGE analysis showed that in four different derivations , pURA-99Pro accounts for 28.2%, 29.6%, and 34.2% of total cellular protein. It was shown that rbSt was expressed in 0% and 41.9%.

Due to the low mutagenesis efficiency, the mutant oligomer has a low Tm and native sequence. shall be constructed so that there is a temperature difference of at least 5°C between the Tm of . Without this difference, effective screening of the candidates cannot occur.

Example 2 Add the desired amino acids (C-3T 90 and C-5T 91, Chart 3) to the food. Follow the procedure of Example 1, except for replacing with an appropriate oligomer at position 99. A bst analog with spartate and glutamate was also constructed.

Example 3 An analog with two amino acids substituted for asparagine at position 99 (Ser -3er and 5er-ASp) as described by Kramer et al. , Nucl Acid Research (Nucl.

Site-specific method of Ac1ds Res, ), 12.9441-58 (1984) In accordance with the law, Boehringer Mannheim Biochemicals r　Mannheim　Biochemica　s), P.O. Box 50816 No., Indianapolis, Indiana State 46250 (also Kramer W. ) and H.G.

(H, J, F r i t z), Me t h, E nz, ), 154.350-67 (1987)). The site-directed mutagenesis kit was constructed as described in the site-directed mutagenesis kit.

In this method, the DNA sequence to be modified is cloned into the M13mp9 vector. You need to This is vector pURA-m4 (PCT/US 881 0 O32B) was digested with restriction enzymes EcoRI and BamHI to obtain approximately 870 bp This is done by isolating a DNA fragment with a size of . This fragment was written by E. coli (E. , coli), tryptophan (trp) promoter, trpL ribosome Contains the binding site and the entire gene sequence of bst. This fragment is Attis et al., Molecular Cloning Laboratory Manual, Cold Spring Harbor Publishing, Cold Spring Barber, New: i-’) (Mol ecular Clo ning:A Laboratory Manual, Co1d Spring Harbor Publication, Co1d Spring Harb or, New Y.

rk); Jay Spring (J, Messing), Mess Enzym (Me M. Cloning into the E, coli and BamHI restriction sites of the 13mp9 vector. Ru. one using the induced vector M13mp9-m4 (Spring, supra). Strand DNA is isolated and used in mutagenesis methods as described above. these Cloning of the analog into the pURA expression vector was performed as in Example 1.

Here, the pBR322 vector was replaced with the double-stranded DNA of the M13 vector. Ru.

Example 4 Also, using the technique described in Example 1, replacing asparagine with glycine I built an analog that ranked 99th. The mutagenic oligonucleotide used was C -3T84 (Chart 3). It hoods the conversion to glycine.

Example 5 The activities of various analogs at position 99 were clarified. The measured parameters are based on the natural rbSt, Glyci 7-99 (GI Y-99) of Example 4 rbSt, Example Seri: y-99 (set-99) rbSt of Example 1 and aspartic acid of Example 2 3.5% fat correction mill of dairy cows intramuscularly injected with -99 (Asp-99) rbSt fat corrected m1lk yield (FCM) This is to evaluate the differences. Holstein dairy cows (45 head) were treated with rbSt injection. Based on the milk yield 3 days and 2 days before the start of ejaculation, Ranked from lowest to highest. Repeatedly randomize the cow based on milk yield 9 experimental groups (control without injection), Gly-99rbSt 5 mg and 20 m g daily, 5er-99rbSt 5mg and 20mg daily, Asp-99 rbst 5mg and 20mg daily and natural rbst 5mg and 2 0 mg daily). Injected into the semifemoralis muscle of dairy cows once daily for 21 days. Ta. daily for 3 days before the start of the injection, 21 days before the injection and 5 days after the last injection. Milk weight was recorded twice.

The concentration of rbSt was expected to be 10 mg/xQ.

The remaining post-injection rbSt solution, on average, contains native rbSt as determined by HPLC. 11.5 for St, 12.1 for Asp-99rbSt, Gly- 11.6 for 99rbSt and 11.6 for 5er-99rbSt. It was 6. Area percentages are Gly-99rbSt, 5er-99rbSt .

and Asp-99rbSt is larger than 99% of normal rbSt (“ Ordinary rbstJ refers to the amino acid sequence encoded by each individual gene, i.e. (meaning a non-degraded product), but for natural rbSt, the normal rbSt The average was 90.5. Natural rbSt has an average, early-e early-eluting) rbSt2%, early-elut ing) rbSt was 7.6%.

Statistical analysis of FCM within experimental groups used 3 days before the start of injection as a covariate and Based on average FCM for days 1 to 21 of radiation. Daily FCM (kg/day ) is the average of the rbSt morphology (P(.

12) or the administration X form of rbSt interaction (P<,90) There were no statistically significant differences. However, the statistical significance of the rbSt form Since P<,12 for the state, we have no information about the relative effect between the forms of rbst. Attempts were made to obtain further information. Based on the comparison between the four forms of rbSt, 5e FCM of dairy cows injected with r-99rbst is Gly-99rbSt or natural r Suggestions (P < , 05) were obtained. Compared to dairy cows injected with Asp-99rbSt, or G Dairy cows injected with ly-99rbst, Asp-99rbSt and native rbSt Differences in FCM between dairy cows injected with 5er-99rbSt No suggestions were obtained. The FCM of dairy cows administered 20 mg of rbSt was 5 m It was statistically significantly larger than that of dairy cows administered g.

Experimental group: Not compatible with injection mortar Change: Average FCM per day (kg/day) - cent change before interval after interval 1 after 5 Control 27.626.126.1 -1.5 -5.4 0 Natural 5ttrg 2 8.228.026.8 -0.2 -0.7 -4.3 Natural 2hg 26.6 30.226.0 3.6 13.5 ~ 13.5Asp-995mg 28. 129.229.2 1. I L9 0^5p-9920mg 24.529. 027.0 4.5 18.4-6.9Gly-995mg 29. :(29, 417,20,10,3-7,5Gly-9920ag 26.429.828 ．． 6 3.4 12.9, -4.2Set-995mg 28.831.03G , 4 2.2 7.6-1.9Set-9920mg 2g, 933.531. 6 4.6 15.9-5.7ml Injection on days 1-21 for pre-injection on days 1-3.

b Post-injections on days 1-5 for injections on days 1-21.

Example 6 In a separate experiment from that reported in Example 5, the activity of other analogs at position 99 was investigated. revealed. Again, the measured parameters have an asparagine at position 99 Natural rbSt (clinical rbSt), asparagine at position 99 is replaced with proline. rbSt (Pro-99rbSt) and asparagine at position 99 with glutamine. 3 of dairy cows intramuscularly injected with rbSt substituted with phosphoric acid (Glu-99rbSt) ．． To evaluate differences in 5% fat corrected milk yield.

Holstein dairy cows (35 cows) were harvested on the 3rd day before RBST injection and on the 2nd day of milk collection. Ranked from highest to lowest milk yield based on volume. milk yield The cows were randomly divided into 7 experimental groups (control without injection) in multiple series based on , Pro-99rbSt 5mg and 15mg daily, Glu-99rbSt 5mg and 15mg daily and natural rbSt 5mg and 15mg daily ). The drug was injected intramuscularly into the semi-dry muscle of dairy cows once daily for 7 days. injection Mil twice daily for 3 days before the start, 7 days after injection and 5 days after the last injection. The weight was recorded.

Dairy cows assigned 5 mg intake of Pro-99, Glu-99 and natural rbst The doses were on average 5.04 mg, 4.88 mg and 5.03 mg, respectively. It was g. Dairy cows assigned to receive 15 mg of rbSt received Pro-99, 15.12 mg, 14.64 mg and 14.64 mg of Glu-99 and native rbSt, respectively. and 15.10 mg. Early-early elution rbSt, early elution rbSt, oxidation The percentages of rbSt and post-oxidized rbSt are as follows: native rbst, Glu-99 The same appeared to be true for rbst and Pro-99rbSt. within the experimental group Statistical analysis of FCM used 3 days before the start of injection as a covariate and 1 day after injection. Based on average FCM of ~7 days. Natural rbSt, Pro-99rbSt and The FCM of cows in the and Glu-99rbSt groups was 28,7.28,8 and 28,8. (P=,95). Therefore, dairy cows fed natural rbst ranked 99th. Dairy cows that received rbSt in which asparagine was replaced with glutamic acid, and Differences in FCM of dairy cows fed rbSt in which sparagine was replaced with proline No suggestions could be obtained. For dairy cows in the rbSt 15 mg group, FC M was significantly larger than that of the rbSt5mg group. Injection of rbSt Upon interruption of the rbSt injection, the FCM of dairy cows previously injected with rbSt It decreased at the same rate for the next 5 days.

Example 7 For relative efficacy using the hypophysectomized rat growth bioassay, rbSt and and rbSt99 analog samples were examined.

Each experiment involved 200 pituitary removed females weighing between 75 and 150 grams. Sprague Dawley rats were used. that Purina Rat Chau (Purina Rat Chau) ow) ad libitum, and deionized and rechlorinated water.

Room conditions were 80°F and 50% relative humidity. Approximately 20 ventilation per hour with a photoperiod of 12 hours of light and 12 hours of darkness, and the photoperiod is approximately 6 a.m. It was started in 2000.

The rats were monitored for a 12 day preparatory period and allowed to adapt to the environment and food conditions. Weight is 1st Measurements were taken four times between offspring and day 12. 1 gram or less per day during the preliminary period Showing a weight loss of less than or a weight gain of 2 grams or less per day Rats that showed 10% were selected for injection with rbSt. Increase in average unit growth (ADG) The rats were ranked according to quantity and 25 rats were divided into 7 blocks. each Treatments within blocks were randomly assigned.

rbSt treatment continued for 10 days. During that time, inject the test compound twice daily for 9 days. A love pack (t) that measures the weight of the animal while taking into account its movements. Mettler model (Mettl) with ab-Pac programming e r Mode +) 3600 Top-o-ding O-balance (to Weight was tracked using ploading balance).

A stock solution of 2 mg/mQ rbSt was mixed with 0.03 M sodium bicarbonate and Prepared in 0.15M NaCl pH 9.5 buffer. Facilitates suspension of rbSt The lyophilized preparation was first dissolved in this buffer at pH 10.8 in order to The pH was then adjusted to 9.5 using 2N HCl2, and the pH was adjusted to 9.5. Make up to final volume with buffer solution and filter if necessary.

The stock solution was added to 37.5.75.1 using stock buffer (pH 9.5). Diluted to solutions of 50 and 300 mg protein/ml. Twice daily, each solution The rats were injected subcutaneously with 100 μQ of the buffer solution, and controls received 100 μQ of the buffer solution. . The experiment lasted 10 days with average daily weight gain tracked.

Statistical analysis of relative potency for various test samples of rbSt is as follows. = natural rbSt having asparagine at position 99 is used as an analytical standard, The relative efficacy value was determined to be 1.00. This sample contains bovine somatotropes derived from the pituitary gland. It had a potency of 1°15 against the same sample. 99th place asparagine Partic acid, glutamic acid, glycine, proline, serine and serine-serine The rbSt analogs substituted with rbSt control (respectively), 2°30.3. had a relative potency of 00.2.96.2, 50.3.06 and 3.03. . These values are significant at the 95% confidence level.

Example 8 r substituted with glycine, serine and aspartic acid in place of asparagine The aqueous stability of three of the bSt99 analogues was determined by two sets of incubations. It was compared with that of natural rbSt (asparagine at position 99) under these conditions. incu The first set of vations were subjected to conditions similar to those undergone during isolation and shaping of rbSt. To simulate the conditions, ( The protein concentration was 20 mg/ml. The second set of incubations is in Ringer's solution at pH 7.4 to simulate exposure to physiological conditions. , at 37°C (protein concentration 5 mg/mc). incubation sun All pulls were prepared aseptically.

Samples were removed from the 3 week incubation and subjected to isoelectric focusing, Stored at -20°C prior to analysis by 5DS-PAGE and reversed-phase HPLC. Ta.

Glycine-99 and serine-99rbSt analogs were However, natural asparagus has an IEF pattern that is virtually indistinguishable from rbSt. The rEF bar 9-7 of Ragin 99rbSt was created by introducing a negative charge at position 99. Shifted approximately 1.2 pH units lower. Natural rbSt and 99th analog ink The main difference observed between the IEF patterns of the incubated samples is that the more acidic The rate at which rbSt species were formed. The IEF pattern of natural rbSt is In both sets of kinivation conditions, the EF pattern of the 99th analog the more acidic rbst species at a faster rate. Natural rbSt and analogues One further difference between the IEF patterns of the incubated native rbSt In the sample, I The EF band is present. These bands are ranked 99th in analog IEF parameters. It was not observed during the turn.

When analyzed under non-reducing conditions, the incubated position 99 analog sample The 5DS-PAGE behavior was identical to that of native rbSt. however, When examined under reducing conditions, the formation of natural rbSt during the incubation The major rbSt fragment was found to be absent in the 99-position analog. However, Therefore, the asparagine residue at position 99 is replaced with glycine, serine, or aspartic acid. Substituting either of them avoids cleavage of the peptide bond between residues 99 and 100. It will be done. The two peptides formed as a result of this cleavage in native rbSt are typically 5 Covalent bond between cysteine residues at positions 3 and 164 remains intact and therefore the phase between reduced and non-reduced samples is It will be different.

Measure the amount of incubate formed and the amount of chain cleavage that occurs at position 99 of the rbSt molecule. Reversed phase HPLC was used for the determination. This is because in reverse-phase HPLC, these species This is because it elutes earlier than natural rbSt. Positions 99 and 100 of the rbSt molecule With rbst chain cleaved between positions 1) at the end of incubation in HIO Natural rbst was 8.5 area percent and incubation at pH 7.4. The explanation for the natural rbst being 11.1 area percent at the end of the arrived. By comparison, the amount of rbSt analog eluted at this position was - less than a cent. rb eluted at the position of isoaspartic acid 99rbSt St caused the native rbSt to be 35.8 at the end of the pH 10 incubation. area percent and native rbS at the end of incubation at pH 7.4. This explains why t is 49.9 area percent. 99th place rbSt Analog Among the groups, aspartate 99rbSt showed the maximum formation of rbSt; rbSt is 3.8 area percent at the end of incubation at pH 10 and 13.9 area percent at the end of incubation at pH 7.4. eluted at the position.

Therefore, the 99-position analog of the present invention is compared to natural asparagine 99rbSt. and exhibits excellent aqueous stability.

Example 9 With appropriate modifications and following the teachings of the previous examples, pigs, humans, sheep, and Similar analogs for somatotropin in horses, rats, monkeys, and birds also appear at position 99. It can be produced by substituting asparagine residues (Nis, Nis, Abdel-Meh). Guido (S, S., Abdel-Meguid) et al., Proceedings of National Academy of Sciences of the United States Tissot of America (Proc, Nat 1. Acad, Sci.

USA) 84.6434-37 (1987)). mammals in this region of the molecule. Because of the high sequence homology between mammalian somatotropins, asparagine 9 of bst The asparagine residue corresponding to It appears to be the third residue in the sequence, and therefore, as in the case of bst, leading to the formation of soaspartate and chain cleavage. For analysis of products such as those mentioned above. Therefore, if this is found to be true for other somatotropins, asparagine By replacing 99 with the appropriate amino acid, the formation of isoaspartate and Chain cleavage could be avoided.

Administration of bst analogs to dairy cows involves delivering the required dose to the animal's circulatory system. This is done according to known methods using any route available for this purpose. Administration method: oral, intramuscular Including intramuscular injections, subcutaneous injections and the use of timed-release implants. Like A new method of administration is by subcutaneous injection using a timed-release implant. be. Suitable vehicles for injection include sodium bicarbonate, sodium phosphate or or a physiologically compatible buffer such as ammonium phosphate solution.

Timed-release implants are known in the art (e.g., U.S. Pat. No. 333,919).

The effective dosage range is 1.0-200 mg per animal per day. give The greater the amount of bst, the greater the increase in growth, milk secretion or number of milk parenchyma cells that occurs. I hear it. Most preferably, the dosage range is 5-50 mg per day.

Mammalian growth hormones are very similar in terms of their amino acid sequences; Hormones from one animal source can enhance the growth of other, unrelated animal species. animal growth For the purpose of increasing speed, analogs of the invention can be used in cattle, sheep, rats, salmon and chickens. Like Watori, native bSt has been shown to have growth-related biological activities. It can be used for growth promotion in various species. Preferred animals are bulls. ), Hef 7-()16ifers) or Steer-(s　t　e　e　r　s) It is a cow used for beef cattle such as.

Beef cattle are slaughtered in layers just before they reach full maturity and size. bst a of the present invention By administering NALOG at any time during weaning up to layer killing, the growth rate of beef cattle can be improved. Can be used to increase the degree of Minimum 30 days depending on desired slaughter time Beef cattle are administered rbSt for up to 450 days. used for beer Animals are typically stratified at about 6 months of age and fed 10 to 30 m/day until stratification age. The bst analog of g is administered to obtain the desired increase in growth rate.

For the purpose of increasing milk secretion in cows, especially dairy cows, BBS is used between 30 and 90 days after giving birth. The t analog will be administered and continued for up to 300 days. Also, the bst analog is It also increases milk secretion in other commercial tag-producing animals such as sheep and sheep.

Chart 1. Amino acid sequence of bovine somatotropin alJLph* pro ala mat sar l@u sat gly Lau ph@ala a sn　al　va1Lau　arg　aha　gin　has　Lau　his gin leu ale ala asp ehr plw 1yspro se hr gly lyi asn glu ala gin gin Lys ar asp 1*u glu leu1@u arg ila s@r 1* u l@u lau ila gin s@t trp 1*u gly pr o 1 aul work u ala Lau m se arg glu l@u glu asp gly thr pro arg ala guygin il@l* u lys gin thr tyr asp lys phe asp eh r asn mat argsar asp asp ala lau l fist u lye asn tyr gly 1*u lau ier cys pha cys arg arg phe gly glu ala sir cys ala ph@chart 2. m4-99set gene to pURA vector cloning of fragment l Bgbst t, 4-99ser gene chart 3. Structure of rbSt analog Oligonucleotides used for construction C-5T 84 (gly) 5'-C: TkGCrTG to TCTTCACCc GTGC-5T 88 (ier) 5', - TC buy CACCTCrAll; GTGC-5T 119 (Pro) 5'-GTCTTckCCCC to CTr GACcTrGGTGC-5T 90 (asp) 5'-GAGTCTrCA CTGATAC(:T:rGGτGC-5T 91 (glu) 5'-GAG TCTTCACTGAAAGCTTG (23 to iGJ (sir-mar) 5 '-AGCAGAGTCTTCACCTCTTCCTCTrTGG'TGrTT GGCACCJ 57 (saw-tip) 5'-τCAGCAGAG'TC TTCACCTCTGACTCCTrGGTGITr(JCACCTCGf international search report international search report PCT/US 89105447 S^　33755

Claims (17)

[Claims] 1. Chart 1 shows the asparagine residues corresponding to the 99 residues of natural somatotropin. As shown in (a) Proline, (b) serine, (c) glycine, (d) serine-serine or (e) Serine-aspartic acid An animal species substituted with at least one different amino acid selected from the group consisting of Matotropin. 2. The mammalian somatotropy according to claim 1, wherein said asparagine is replaced with proline. hmm. 3. The mammalian somatotropin according to claim 1, wherein said asparagine is replaced with serine. . 4. The mammalian somatotropy according to claim 1, wherein said asparagine is replaced with glycine. hmm. 5. The mammalian somato according to claim 1, wherein said asparagine is substituted with serine-serine. Tropine. 6. The mammal according to claim 1, wherein the asparagine is substituted with serine-aspartic acid. Somatotropin. 7. Somatotropin in horses, pigs, fish, sheep, horses, rats, monkeys and humans The animal somatotropin according to claim 1, selected from the group consisting of: 8. The animal somatotropin according to claim 1, which is bovine somatotropin. 9. The animal somatotropin according to claim 1, which is bovine somatotropin. 10. The animal somatotropin according to claim 3, which is bovine somatotropin. 11. The animal somatotropin according to claim 4, which is bovine somatotropin. 12. The animal somatotropin according to claim 5, which is bovine somatotropin. 13. The animal somatotropin according to claim 6, which is bovine somatotropin. 14. The animal somatotropin according to claim 7, which is bovine somatotropin. 15. characterized in that an effective amount of somatotropin according to claim 1 is administered to an animal. How to promote the growth of animals 16. 16. The method of claim 15, wherein the animal is a cow. 17. A method characterized in that an effective amount of the animal somatotropin according to claim 1 is administered to a dairy cow. A method for increasing the milk cliff in dairy cows.