Synthesis of Biologically Active Polypeptides Mediated by Highly Specific Serratia 56K Protease
| Project/Area Number |
01550718
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
高分子合成
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| Research Institution | Kyushu Institute of Technology |
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Principal Investigator |
NISHINO Norikazu Kyushu Institute of Technology, Department of Applied Chemistry, Associate Professor, 工学部, 助教授 (40145165)
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| Co-Investigator(Kenkyū-buntansha) |
MIHARA Hisakazu Kyushu Institute of Technology, Department of Applied Chemistry, Assistant, 工学部, 助手 (30183966)
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| Project Period (FY) |
1989 – 1990
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| Project Status |
Completed (Fiscal Year 1990)
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| Budget Amount *help |
¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1990: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1989: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Keywords | Peptide Synthesis / Protease Catalysis / Serratia Protease / Trypsin / Biologically Active peptide / Growth Hormone Releasing Factor / 生理活性ポリペプチド / セラチア56Kプロテア-ゼ / ヒト成長ホルモン放出因子 |
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| Research Abstract |
Peptide synthesis mediated by proteases has an advantage that reactions proceed under mild condition without racemization and with few side products. Serratia Protease (SP) has high substrate specificity in hydrolysis of a peptide bond at the carboxyl side of Arg residue. We have found that SP catalyzes the formation of arginyl peptide bond with high specificity. In a model tetrapeptide synthesis, Abz-Gly-Phe-Arg-Xaa-Nba (Xaa : Leu, Ala, Gly, Abz : 2-aminobenzoyl, Nba : 4-nitrobenzylamine), SP catalyzed the arginyl bond formation in the following order, Leu >> Ala, Gly, indicating that SP has high sequence selectivity in the peptide bond formation. To apply the protease to the synthesis of large peptides, we attempted to synthesize a model peptide Bz-Gly-Arg-Gly-Phe-Arg-Leu-NH_2 which contains the specific Arg-Leu and non-specific Arg-Gly bonds. However, the product could not obtained due to hydrolysis of the Arg-Gly bond during formation of the Arg-Leu bond. This result indicated that
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hydrolysis of a peptide bond can not be avoided by use of any kind of specific protease.
To develop an efficient method to synthesize biologically active peptides by the use of proteases, it is necessary to find a solvent system which solubilizes substrates containing partially or fully protected peptides prepared by the convenient peptide-synthetic method and which keeps proteases active to give products in high yields. For this purpose, we examined a various mixtures of water-miscible organic solvents and water in the model peptide system, Abz-Gly-PheーArg-Leu-Nba, mediated by trypsin. In a solvent system of 4% H_2O/HFIP/DMF, the enzyme catalyzed the reaction in an 83% yield, while in a 30% yield in 50% H_2O/DMF which was frequently used by other researchers. It is noted that HFIP is a good solvent having high ability to solubilizes peptide-substrates and that the low-water-containing solvent shifts the reaction equilibrium to the peptide-bond formation to give a high yield.
We applied this solvent system to the synthesis of the active fragment of human growth hormone releasing factor (hGRF (1-29) -NH_2). The protected peptide fragments were synthesized by the solid-phase method using the oxime resin. These fragments were coupled by the solution method to give two large segments which were condensed by the aid of trypsin. The yield in this step was estimated as nearly 90%. The partially protected hGRF (1-29) -NH_2 was deprotected and purified by RP-HPLC to give hGRF (1-29) -NH_2, which was identified by RP-HPLC and peptide mapping of the tryptic hydrolyzates.
In conclusion, sequence-specific synthesis of peptides mediated by SP was not successful, but efficient method for synthesis of a biologically active peptide by the aid of trypsin has been developed. Less
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Report
(3 results)
Research Products
(7 results)
