Li Jing, Chi Zhenming, Liu Zhiqiang, Yue Lixi, Peng Ying, Wang Lin
Unesco Chinese Center of Marine Biotechnology, Ocean University of China, Qingdao, China.
Appl Biochem Biotechnol. 2009 Oct;159(1):119-32. doi: 10.1007/s12010-008-8400-3. Epub 2008 Nov 5.
Metschnikowia reukaufii W6b isolated from marine environment was found to produce a cell-bound acid protease. The full-length cDNA (cDNASAP6 gene) of the acid protease (SAP6) from the marine-derived yeast M. reukaufii W6b was cloned. The insert was 1,755-bp long and contained an open reading frame of 1,527-bp encoding 508 amino acids. The deduced amino acid sequence included a signal peptide of 16 amino acids. The consensus motifs contained a VLLDTGSSDLRM active site and an ALLDSGTTITQF active site. The protein sequence deduced from the cDNASAP6 gene exhibited 12.9% overall identity with Cwp1 of Saccharomyces cerevisiae and a hydropathy profile characteristic of glycosylphosphatidylinositol cell-wall proteins. The cDNASAP6 gene without 48 bp encoding the signal peptide sequence was subcloned into an expression plasmid pET-24a (+) and fused with a 6-His Tag and transformed into Escherichia coli BL21 (DE3) for recombinant expression of the protease. The expressed fusion protein was found to have a unique band with molecular mass of about 54 kDa. The crude acid protease of the culture of the marine yeast strain W6b and the crude recombinant acid protease had milk clotting activity.
从海洋环境中分离出的鲁考夫梅奇酵母W6b被发现可产生一种细胞结合型酸性蛋白酶。克隆了源自海洋酵母鲁考夫梅奇酵母W6b的酸性蛋白酶（SAP6）的全长cDNA（cDNASAP6基因）。插入片段长1755bp，包含一个1527bp的开放阅读框，编码508个氨基酸。推导的氨基酸序列包括一个16个氨基酸的信号肽。共有基序包含一个VLLDTGSSDLRM活性位点和一个ALLDSGTTITQF活性位点。从cDNASAP6基因推导的蛋白质序列与酿酒酵母的Cwp1总体一致性为12.9%，具有糖基磷脂酰肌醇细胞壁蛋白的亲水性特征。将不含编码信号肽序列的48bp的cDNASAP6基因亚克隆到表达质粒pET-24a(+)中，与6-His标签融合，转化到大肠杆菌BL21(DE3)中以重组表达蛋白酶。发现表达的融合蛋白有一条分子量约为54kDa的独特条带。海洋酵母菌株W6b培养物的粗酸性蛋白酶和粗重组酸性蛋白酶具有凝乳活性。
