Studies On Expression Of Recombinant Human Bikunin Domain Ⅰ In Pichia Pastoris

Bikunin is a serine protease inhibitor, it contains two Kunitz domains. Bikunin domainⅠis the N-terminal domain of Bikunin, which consists of 54 amino acid residues. The N-terminus of Bikunin domainⅠmay play a role through a cooperative interaction to form the appropriate conformation for binding to the cells,The Bikunin domainⅠis the active site for this glycoprotein.It has been shown that Bikunin domainⅠinhibit cathepsin G and granulocyte elastase.1 Construction of recombinant plasmid pPICZαC-hBikunin domainⅠWith the recombinant plasmid of pPICZαC- hBikunin as template, the sequence encoding hBikunin domainⅠwas obtained by PCR. The restriction sites of XhoI,Kex2 (5' end) and EcoRI (3' end) were added into the two ends of interested sequence. The PCR product was digested with XhoI and EcoRI and then ligated into the linearized vector pPICZαC. The results of DNA sequence analysis demonstrated that the DNA encoding hBikunin domainⅠw as corrected inserted into pPICZαC vector and the sequence was consistent with we designed.The selected plasmids were linearized with Sac I and transformed into competent P. pastoris strain X-33 by electroporation using a Micropulser (Bio-Rad, USA). Electrocompetent cells of P. pastoris were prepared according to the instruction of the manufacturer. Transformants were selected on YPD plates.Colonies appeared after 72 h of incubation at 30°C, 9 single clones were selected to culture.The isolated genomic DNA from the transformants was used as template for the PCR with the primers mentioned above.The positively transformed yeast cells harboring different expression plasmids were cultured for about 168 h in a shaking flask. For the secreted high expression hBikunin domainⅠ, the culture filtrates of the transformants were run on an 18% (w/v) SDS-PAGE gel and stained with Coomassie blue R250.The result demonstrated that the expression medium supernatant per day over 168 h induction by methanol .Levels of hBikunin domainⅠincreased gradually after induction and reached maximum at 96 h, then at 120 h began to decrease. At the first day of culture,isolated the genomic DNA of the transformed yeasts to perform PCR using the expression primers. Clone 4 expressed the maximum level of hBikunin domainⅠ. 2 Optimization of expressional conditionsThe expression level of hBikunin domainⅠwas appraised by SDS-PAGE and TIA analysis of expression medium supernatant per 24 h past 192 h induction by methanol at pH3.0, pH3.5, pH4.0, pH4.5, pH5.0, pH5.5, pH6.0, pH6.5. Levels of hBikunin domainⅠand reached the maximum at 96 and levels of express production reached the peak yalue when the pH value of the medium was 4.0 after induction.3 Study on purification of rhBikunin domainⅠThe high expression strain was plated on YPD plates containing Zeocin ,then wasinoculated in 10 ml BMGY, after 24 h cultured in 200ml BMGY medium , finally, after 36 h inoculated in 2 L BMGY of flask . waiting for glycerin was exhausted , inducing the expression of rhBikunin domainⅠwith methanol of 10 ml in pH 4.0 for 72 h. This situation show that the level of expression reach the maximum at 72 h by SDS-PAGE and TIA analysis of expression medium supernatant per 24 h past 192 h. The wet weight and OD600 were monitored per 12 h.Apply AKTA Explorer 100 chromatography system, the optimal concentration of NaCl for elution was 0.5 M and the optimal pH for blinding was 3.0. When the level of rhBikunin domainⅠreached the peak, we centrifuge the culture. The rhBikunin domainⅠsolution was purified with cation exchange chromatography (SP Sepharose XL) reverse phase chromatography (Source 30). Following these processes, we could get a high purity rhBikunin domainⅠin high concentrations of methanol. In order to remove the methanol, the solution must be futher purified by vacuum distillation. Then we could get a more than 90% purity protein of rhBikunin domainⅠ.As mentioned above, the expression plasmid pPICZα-hBikunin domainⅠand Pichia Pastoris high efficient expression system for rhBikunin domainⅠwas constructed successfully. It established a significant foundation for the deep researching and can provide basis methods for the developing research of fermentation and purification.