Fusion expression of human ¦Âª²defensin 3 and bactericidal/permeability increasing protein in P. pastoris

    TUO Xiaoª²Ye, CHAI Jiaª²Ke, JIANG Wei, CHANG Dong, SHENG Zhiª²Yong

    1Department of Burns and Plastic Surgery

    2Department of Clinical Laboratory, First Affiliated Hospital, Chinese PLA General Hospital, Beijing 100037, China

    ¡¾Abstract¡¿ AIM: To investigate the possibility of fusion expression of human ¦Âª²defensin 3 (hBD3) and bactericidal/permeability increasing protein (BPI) in P. pastoris. METHODS: DNA fragments encoding hBD3 mature peptide and BPI were linked via a Linker sequence and cloned into yeast expression vector pPICZ¦ÁB followed by introduction into P. pastorisª²Xª²33 by electrical transduction. Positive clones identified by genomic PCR and phenotype analysis were induced by methanol for protein expression. In supernatants, recombinant protein was purified by phenyl sepharose high performance and source 30Q ionª²exchange columns. Western Blot against hBD3 and BPI was performed respectively to identify the recombinant protein. RESULTS: Recombinant pICZ¦ÁBª²hBD3ª²BPI was proved correct by restriction analysis and sequencing. Xª²33ª²pICZ¦ÁBª²hBD3ª²BPI clone expressed the recombinant fusion protein in SDSª²PAGE electrophoresis after induction for 24 h. The protein product was both hBD3ª² and BPIª²positive. A purity of 89% was reached after purification procedures. CONCLUSION: It¬ðs feasible to express hBD3 fused to BPI in P. pastoris yeast expression system. ......