人基质金属蛋白酶-9在酵母Pichia pastoris中的表达

基质金属蛋白酶 - 9( MMP- 9)可促进恶性肿瘤的侵袭、转移 ,并在组织重建、胚胎发育以及伤口愈合等生理过程中发挥重要作用 .为研究这一蛋白的性质 ,并以之为靶标筛选抗肿瘤转移药物 ,在酵母 Pichia pastoris中实现了重组人 MMP- 9蛋白的高效、高活性、分泌表达 .首先用 PCR扩增了 MMP- 9基因编码区 (不含信号肽序列 ) ,经测序证实后 ,将其插入 p PIC9质粒中 ,构建表达载体 .用 Li C1 - PEG法转化酵母后 ,采用明胶 -酶谱法筛选获得 5株高效分泌表达 MMP- 9的克隆 ,经PCR证实 MMP- 9基因整合在阳性克隆的染色体中 .重组蛋白分子量为 93k D,表达量为 1 0 mg/L.重组蛋白可水解明胶及 型胶原 ,并可经有机汞 APMA诱导发生自剪切 ,转换成 85k D的激活形式 ,表明重组蛋白具有与天然人 MMP- 9蛋白相似的底物水解活性和自剪切激活特性 .

Expression of Human Matrix Metalloproteinase-9 in Yeast Pichia pastoris

Matrix metalloproteinase 9(MMP 9) plasys a pivotal role not only in the physiological processes such as tissue remodeling,embryo development and skin damage repairing,but also in the pathological processes such as cancer invasion and metastasis.To investigate the function and targeting at this protein for screening anti metastasis drugs,human MMP 9 protein was expressed and secreted in yeast Pichia pastoris. As the first step of expression strategy,MMP 9 gene coding region was amplified by using PCR with a deletion of the signal peptide sequence.Following sequencing,the DNA fragment was inserted into pPIC9 vector to construct the expression plasmid,followed by transformation of the yeast GS115 strain with LiCl PEG method.After gelatin zymography analysis of the culture supernatants,5 recombinant clones were found to express and secrete MMP 9 protein,while the MMP 9 gene fragment could be PCR amplified out from chromosomal DNA of these clones.The recombinant protein was found with a molecular mass of 93 kD,and its expression level could be as high as 10 mg/L.The recombinant protein could degrade both gelatin and type Ⅳ collagen.It was also found to be induced to autocleavage to an activated 85 kD protein by organomercurial APMA.These results suggest that the recombinant protein is similar to the native human MMP 9 not only in the substrate specificity but also in other biochemical characteristics such as APMA induced auto cleavage.