1. Cloning And Expression Of The Gene Coding For The Glucagon Derivates In Streptomyces Lividand TK24 And E.coli BL21 And Purification Of The Recombinant Protein 2. The Studies Of Cloning And Expression Of Rat α-Amidase In E.coli BL21

Glucagon, secreted by theα-cells of the pancreatic island, is a peptide consisting of 29 amino acids. It coordinated with insulin in the metabolic process of sugar, protein and fat ,which balances the function of insulin by increasing the blood sugar level, promoting the decompose of protein and fat to decrease the synthesis of protein and fatty acid. Glucagon has been used in clinical for treatment the serious syndromes in low blood sugar level for the patients suffering diabetes caused by insulin and the infants .It also can be used for inhibition wriggling of the stomach intestine temporarily in the radiation therapy.The studies indicated that the c-terminal amidating of hormone peptides is the important process in the post-transcription. The c-terminal amidating is essential for the biological activity of numerous peptides, some of their activities even have 10000 times difference whether or not amidating. The amidating peptides may have the longer half-time and the more compacted avidity with their receptors. Natural glucagon is not presented with c- terminal amidating, but the studies of its structure and function showed that the glucagon with c-terminal amidating may be more compacted binding with glucagon receptor.In our studies, the gene of glucagon-gly was amplified by PCR and inserted into the plasmid pMSA with replacing the gene of sCT to form the plasmid pMGA. After transformation, the recombinant strain Streptomyces lividans TK24 [pMGA] was constructed. The protein produced by the strain was purified with 93.4% purity. The results of sequencing analysis for the 14 residues of amino acid at N- teminal of the protein showed that it was the same as the natural glucagon. The molecular mass of the protein is 3494 determined by MALDI TOF ,which is identical with the molecular mass of the glucagon with c-terminal amidating. According to the results mentioned above, the glucagon with c-terminal amidating has been produced by the strain Streptomyces lividans TK24 [pMGA]. The gene encoding glucagon-gly was amplified by PCR and inserted into the plasmid pET30a. The strain E.coli B121 [pET-G] was constructed after transformation the recombinant plasmid pET-G.. Analysis with SDS- Tricine -PAGE ,Western blot and ELISA , the results indicated that the molecular mass of the fusion protein with biological activity producing by the strain is 9500. With the Ni²⁺ affinity column chromatography, the fusion protein was purified and digested by the thrombin . With the HPLC, the protein was purified to about 88.3% purity and its molecular mass was 3531 determined by MALDI TOF , which accorded with the molecular mass of glucagon-gly.With the plasmid pMSA as the template, the gene encoding theα-amidase was amplified and cloned into the plasmid pET30a .After transforming the recombinant plasmid pET-A into E.coli B121, the strain of E.coli B121[pET-A] was obtained .The molecular mass of the recombinant protein produced by the strain is about 80,000 determined by SDS-PAGE and Western blot , which is similar with the molecular mass of theα-amidase with His-tag. With the Ni²⁺ affinity column chromatography, the protein was purified to the purity with 86.6% ,which has the biological activity amidating the peptide Tyr-Val-Gly at the c-terminal to be the peptide Tyr-Val-NH₂.Such results identified that the protein isα-amidase indeed.In this thesis, the glucagons with c-terminal amidating was produced by the recombinant strain Streptomyces lividans TK24 [pMGA], which has never been reported before as our knowledge. The gene encoding glucagon-gly and the gene encoding theα-amidase were expressed separately in E.coli B121, which lied the ground works for producing the glucagon with c-terminal amidating in vitro. This glucagon derivate may have longer half time in vitro with potential prospect. Such results will be helpful for the studies preparing various peptides amidating at c-terminal.