Studies On Cloning Of HBikunin DomainⅡ And Expression Of Recombinant HBikunin DomainⅡ In Pichia Pastoris

Bikunin is a serine protease inhibitor that contains two Kunitz domains. DomainⅡis the C-terminal domain of Bikunin, which consists of 54 amino acid residues. The domainⅡis the active site for this glycoprotein. It has a serine protease inhibitory activity and has been suggested that this domain involved in the allergic and other inflammatory response. In addition, Bikunin is known to suppress the invasion and metastasis of cancer cells.1. Shake-flask expression of rhBikunin domainⅡand selection of high-level expression colonies1.1 Construction of expression vector pPICZαC- Bikunin domainⅡWith the recombinant plasmid of pPICZαC- bik as template, the sequence encoding hBikunin domainⅡwas obtained by PCR. The endonuclease sites of XhoI and SalI were added into the two ends of interested sequence. The results of sequencing demonstrated that the sequence was correct.1.2 Transformation of Pichia pastoris and selection of high-level expression coloniesThe linearized recombinant expression vectors were transformed into P. pastoris X-33 by electroporation using a Micropulser and screened on YPD plates containing 100μg/ml of zeocin. After the multicopy transformants appeared, ten colonies were cultured in shake flask and the genomic DNA was isolated from different clones to analyze rhBikunin domainⅡgene integrating into the P. pastoris genome by PCR. The result showed that Clone 6 expressed the maximum level of rhBikunin domainⅡ.The expression level of rhBikunin domainⅡwas evaluated by SDS-PAGE analysis of expression medium supernatant per day past 7 days induction by methanol at pH 3.0-6.0. Levels of rhBikunin domainⅡincreased gradually after induction and reached the peak at day 3, then began to decrease. The expression level reached a maximum when the pH value of the medium was 5.0.2. Large-scale expression and purification of rhBikunin domainⅡPichia pastoris has many advantages as a kind of expression host, and it's very suitable for large-scale expression of the extraneous proteins. We explored the large-scale fermentation process of rhBikunin domainⅡand found that the best pH value was 5.0, DO was between 25%~30% and the supply speed of methanol was 9-10 ml/h/L of initial fermentation volume. The concentration of rhBikunin domainⅡin the broth can reach to 50mg/L.The rhBikunin domainⅡsolution was purified with cation exchange chromatography (SP SepharoseXL) and reverse phase chromatography (Source 30).