弗氏链霉菌丝氨酸蛋白酶基因的克隆及表达

从一株具有极强的降解羽毛能力的弗氏链霉菌菌株（Streptomyces fradiae var.k11）中纯化得到了一种丝氨酸蛋白酶SFP2。经蛋白测序，得到部分氨基酸序列，设计简并引物，PCR扩增得到部分基因序列，通过构建基因文库，获得了包括信号肽序列在内的完整的基因sfp2(EMBL收录号AJ784940)，开放阅读框全长924bp，包括114bp的信号肽编码序列和810bp的酶原编码序列, 其中成熟蛋白编码基因长576bp，编码191个氨基酸，理论分子量为19.112kD。酶原编码基因和成熟蛋白编码基因均在大肠杆菌和枯草芽孢杆菌中得到了表达，酶原编码基因表达产物具有正常的生物学活性，证明了克隆基因的生物学功能。

Gene Cloning and Expression of Serine Protease SFP2 from Streptomyces fradiae var. k11

Extracellular serine protease SFP2 from Streptomyces fradiae var.k11 with high feather-degrading activity was purified. The partial amino acid sequences of internal peptide of purified SFP2 were determined, and the partial gene encoding SFP2 was cloned by PCR using the degenerate primers designed according to the amino acid sequences. Complete sfp2 gene was cloned by screening the genomic DNA library of Streptomyces fradiae var.k11. The Open Reading Frame of sfp2 including pre-pro-enzyme is 924bp long (EMBL Accession number: AJ784940). The signal peptide sequence is as long as 114bp, the precursor sequence is 810bp and the mature enzyme is 576bp long, encoding 191 amino acid resides with the putative molecular weight of 19.112kD. In E. coli and Bacillus subtilis, the two sequences encoding SFP2 pro-enzyme and mature enzyme were both expressed successfully. The pro-enzyme expressed had normal biological function and its mature product had normal enzymatic activity.