解脂亚罗酵母中HMGR基因的生物信息学分析及催化域部分表达

Bioinformatics analysis of HMGRgene and partial expression of catalytic domain in Yarrowia lipolytica

对解脂亚罗酵母中甲羟戊酸途径的限速酶HMGR进行了生物信息学分析,克隆其活性结构域(tHMG,第441~875位氨基酸),并与标记分子GFP共表达。生物信息学分析表明,HMGR基因可编码一个含有999个氨基酸的疏水性不稳定蛋白,该蛋白前500个氨基酸肽链含有较多的跨膜区,其理论分子量和等电点分别为254.93kDa和4.77;活性结构域包含506个氨基酸,理论分子量和等电点分别为127.74kDa和4.93,不含跨膜区。两个蛋白的二级结构主要为α-螺旋和无规则卷曲,三级结构均以同源四聚体的状态存在。在激发光(488nm)下观察融合表达tHMGGFP的酵母细胞,发现产生绿色荧光。经测定解脂亚罗酵母超表达催化域(tHMG)后,酶比活为1.14U/mg,比对照组提高了62.7%,此结果说明HMGR催化域部分可以独立表达,提高细胞内的HMGR酶活水平。

Bioinformatics analysis of the HMGR of rate-limiting enzyme in the mevalonate pathway from Yarrowia lipolytica was carried out. The activity domain （tHMG, amino acids 441; 875） was cloned and co-expressed with the labeled molecule GFP. Bioinformatics analysis showed that the HMGR gene can encode a hydrophobic labile protein containing 999 amino acids. The first 500 amino acid peptide chains contain more transmembrane region, the molecular weight was 254.93 kDa and theoretical pI was 4.77. The active domain contains 506 amino acids with a molecular weight of 127.74 kDa and a theoretical isoelectrie point of 4.93, which contains the non- transmembrane region. The secondary structures of the two proteins （HMGR and tHMG） are mainly α-helix and random coil, and their tertiary structures are in the state of homologous tetramer. The yeast cells expressing tHMG-GFP were observed at the excitation wavelength of 488 nm, and the green fluorescence was found. After overexpression of catalytic domain （tHMG）, the enzyme activity was 1.14 U/mg, which was 62.7% higher than that of the control. The results showed that the catalytic domain of HMGR could be expressed independently and it can increase the enzyme activity of HMGR in the cells.