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庹晓晔, 柴家科, 蒋伟, 常东, 盛志勇
人β防御素3；,细菌膜穿透增加蛋白；,融合表达；,毕赤酵母,,人β防御素3；细菌膜穿透增加蛋白；融合表达；毕赤酵母,0引言,1材料和方法,2结果,3讨论,【参考文献】
Fusion expression of human βdefensin 3 and bactericidal/permeability increasing protein in P. pastoris
TUO XiaoYe, CHAI JiaKe, JIANG Wei, CHANG Dong, SHENG ZhiYong
1Department of Burns and Plastic Surgery
2Department of Clinical Laboratory, First Affiliated Hospital, Chinese PLA General Hospital, Beijing 100037, China
【Abstract】 AIM: To investigate the possibility of fusion expression of human βdefensin 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 pPICZαB followed by introduction into P. pastorisX33 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 ionexchange columns. Western Blot against hBD3 and BPI was performed respectively to identify the recombinant protein. RESULTS: Recombinant pICZαBhBD3BPI was proved correct by restriction analysis and sequencing. X33pICZαBhBD3BPI clone expressed the recombinant fusion protein in SDSPAGE electrophoresis after induction for 24 h. The protein product was both hBD3 and BPIpositive. A purity of 89% was reached after purification procedures. CONCLUSION: Its feasible to express hBD3 fused to BPI in P. pastoris yeast expression system. ......
TUO XiaoYe, CHAI JiaKe, JIANG Wei, CHANG Dong, SHENG ZhiYong
1Department of Burns and Plastic Surgery
2Department of Clinical Laboratory, First Affiliated Hospital, Chinese PLA General Hospital, Beijing 100037, China
【Abstract】 AIM: To investigate the possibility of fusion expression of human βdefensin 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 pPICZαB followed by introduction into P. pastorisX33 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 ionexchange columns. Western Blot against hBD3 and BPI was performed respectively to identify the recombinant protein. RESULTS: Recombinant pICZαBhBD3BPI was proved correct by restriction analysis and sequencing. X33pICZαBhBD3BPI clone expressed the recombinant fusion protein in SDSPAGE electrophoresis after induction for 24 h. The protein product was both hBD3 and BPIpositive. A purity of 89% was reached after purification procedures. CONCLUSION: Its feasible to express hBD3 fused to BPI in P. pastoris yeast expression system. ......