Cloning and Expression of Recombinant Human Matrix Metalloproteinases-1 from Pichia Pastoris Expression System

• Main Authors: Po-Wen Tseng, 曾柏文
• Other Authors: Shann-Tzong Jiang
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/40115699422714569810

碩士 === 靜宜大學 === 食品營養研究所 === 98 === Matrix metalloproteinases are zinc-binding endopeptidases that collectively degrade the constituent macromolecules of the extracellular matrix. MMP-1, can degrade collagen, is thought to participate in skin wrinkle and has been implicated in playing important roles during metastasis of malignant tumors. In the study, one polynucleotide (1158 bp) encoding mature human MMP-1 was designed, based on the amino acid sequence of the protein and the genetic codon usage frequency of Pichia pastoris, and constructed by assembly PCR, cloned into pGAPZαC and pPICZαC expression vectors, respectively, and finally transformed into Pichia pastoris X-33 expression host. After 3 days shaking cultivation and under control of GAP promoter and AOX1 promoter, respectively, high level of active form of recombinant human MMP-1 was expressed secretively byα-signal sequence. According to the results of SDS-PAGE, periodic acid schiff staining and N-terminal sequence analysis, there are two recombinant human MMP-1 proteins were expressed and could be purified by nickel- trap chromatography. The larger one (about 60 kDa) is a active form glycosylated rhMMP-1 but the smaller one (about 45kDa) is not glycosylated. In the future, the recombinant human MMP 1 could be used in food industry applications as meat tenderizer and used in searching MMP-1 inhibitors as anti-wrinkle reagents in cosmetic applications or as anticancer reagents in medical application.