硫酸化胆汁酸水解酶基因BSSH1克隆、表达及活性

Cloning,expression and biological activity of the bile acid sulfate sulfatase gene BSS H1

目的从铜绿假单胞菌克隆出硫酸化胆汁酸水解酶(BSS)基因,并表达蛋白产物、研究其活性。方法通过序列比对确定铜绿假单胞菌中的目的序列BSS H1;通过PCR扩增获得BSS H1基因全序列,将其插入pET30b(+)载体,并将重组表达载体转化到大肠杆菌BL21(DE3)中诱导表达。通过优化诱导表达条件,实现重组蛋白BSS H1的高效表达;通过BSS-HSD酶联法初步研究其酶活性质。结果工程菌扩大培养3 h、以终浓度为1 mmol/L IPTG诱导5 h时蛋白表达量最高;研究发现BSS H1能催化底物生成3α和3β两种构型的羟基胆汁酸,在pH 8.5时,其活性分别是(632±65)U/L和(52±4)U/L。结论成功构建BSS H1高效表达菌株。BSS H1能催化生成两种立体异构体的特性在其他BSS中未见报道,其机制尚不明确,值得深入研究。

Objective To clone a gene encoding BSS HI from Pseudomonas aeruginosa and to express the product. To study its enzymatic activities. Methods A target sequence named BSS Hlwas identified in Pseudomonas aerugi- nosa genome through sequence alignment. The complete sequence of BSS HI was amplified by PCR. The obtained segment was inserted into pET30b（ ＋ ） vector. The recombinant expression vector was transformed into Escherichia Coli BL21 （DE3） and was induced to express the protein. A high expression yield was achieved by optimizing the conditions of induction. The BSS-HSD double enzyme-linked method was employed in the study of the enzymatic characters. Results The highest level expression of the enzyme was achieved when the engineered E. coli had grown for 3 hrs and was induced by IPTG with a final concentration of 1 mmol/L for 5hrs. The hydrolyzates of bile acid sulfate included both 3α- and 3β-hydroxy bile acid. At pH 8.5 the activity of the cloned enzyme was （632 ±65 ） U/L for 3α-catalyzing and （52± 4） U/L for 3β-catalyzing respectively. Conclusions An engineered strain of high BSS HI expression was obtained. The unique characteristic of BSS H1 that it can transform the substrate into two types of stereoisomers has not been reported in the study of other BSS. The mechanism is unknown.