Expression and activity of human serum albumin-recombinant human disintegrin domain of ADAM15 fusion protein

Objective To investigate the expression of recombinant human disintegrin domain of ADAM15(a disintegrin and metalloproteinase 15)and human serum albumin(HSA)fusion protein(HSA-rhddADAM15-His)in Pichia pastori and determine the activity of expressed product. Methods The rddADAM15-His gene was amplified by PCR using rhddADAM15gene as a template,and cloned into plasmid pBluescript-HSA. The constructed recombinant plasmid was digested with SnaBⅠand NotⅠ,and the recovered HSA-AD-His fragment was inserted into vector pPIC9k. The constructed recombinant plasmid pPIC9k-HSA-rhddADAM15-His was transformed to P. pastoris GS115 for expression under induction of methanol. The expressed product was analyzed by SDS-PAGE,purified by Blue Sepharose,nickel ion chelate and DEAE anion exchange chromatography,then identified by SDS-PAGE and Western blot,and determined for activity by wound healing test and SRB assay. Results Restriction analysis and sequencing proved that recombinant plasmid pPIC9k-HSArhddADAM15-His was constructed correctly. The expressed fusion protein HSA-rhddADAM15-His, with a relative molecular mass of about 76 000,existed in a soluble form,reached a purity of about 75% after purification,and showed specific reaction with rabbit antibody against rhddADAM15. The fusion protein showed significantly inhibitory effect on the migration,while showed no significantly inhibitory effect on the proliferation,of mouse melanoma B16 cells.Conclusion HSA-rhddADAM15-His fusion protein was successfully expressed in P. pastoris and purified,which laid a foundation of study on its action mechanism.