Sugiura Tatsuki, Yamagishi Kenji, Kimura Toshiyuki, Nishida Tomoaki, Kawagishi Hirokazu, Hirai Hirofumi
Department of Bioscience, Graduate School of Science and Technology, Shizuoka University, Japan.
Biosci Biotechnol Biochem. 2009 Aug;73(8):1793-8. doi: 10.1271/bbb.90152. Epub 2009 Aug 7.
Two genes, encoding YK-LiP1 and YK-LiP2, were cloned from the white-rot fungus Phanerochaete sordida YK-624, and a homologous expression system for the gene was constructed. Two full-length cDNAs (ylpA and ylpB) were isolated by degenerate RT-PCR and RACE-PCR. The results of N-terminal amino acid sequence analysis of native YK-LiP1 and YK-LiP2 showed that ylpA and ylpB coded for YK-LiP2 and YK-LiP1 respectively. The promoter of glyceraldehyde-3-phosphate dehydrogenase cloned from P. sordida YK-624 (PsGPD) was used to drive the expression of ylpA. Expression vector pGPD-g-ylpA was transformed into a P. sordida YK-624 uracil auxotrophic mutant, UV-64. The YlpA protein was secreted in active form by the transformants after 4 d of growth in a medium containing an excessive nitrogen source, whereas endogenous YK-LiP1 and YK-LiP2 were not produced. The physical and catalytic properties of the purified YlpA protein were very similar to those of YK-LiP2. These results suggest that homologous expression of recombinant YK-LiP2 was successful.
从白腐真菌黄孢原毛平革菌（Phanerochaete sordida）YK-624中克隆了编码YK-LiP1和YK-LiP2的两个基因，并构建了该基因的同源表达系统。通过简并RT-PCR和RACE-PCR分离出两个全长cDNA（ylpA和ylpB）。对天然YK-LiP1和YK-LiP2的N端氨基酸序列分析结果表明，ylpA和ylpB分别编码YK-LiP2和YK-LiP1。从黄孢原毛平革菌YK-624中克隆的甘油醛-3-磷酸脱氢酶启动子（PsGPD）用于驱动ylpA的表达。将表达载体pGPD-g-ylpA转化到黄孢原毛平革菌YK-624尿嘧啶营养缺陷型突变体UV-64中。在含有过量氮源的培养基中生长4天后，转化体以活性形式分泌YlpA蛋白，而不产生内源性YK-LiP1和YK-LiP2。纯化的YlpA蛋白的物理性质和催化性质与YK-LiP2非常相似。这些结果表明重组YK-LiP2的同源表达是成功的。
