假单胞菌TS1138 L-半胱氨酸脱巯基酶基因的克隆与表达

Cloning and Expression of L-cysteine Desulfhydrase from Pseudomonas sp.TS1138

提取假单胞菌TS1138染色体基因,以自行设计的引物通过PCR方法扩增得到L-半胱氨酸脱巯基酶基因(cds),将其克隆至克隆载体pBluescript SKⅡ,并转化入大肠杆菌DH5α,测定了含有脱巯基酶基因(cds)的1．2 kb DNA片段的序列；同时,将其克隆至表达载体pETH,转化大肠杆菌BL21(DE3),IPTG诱导表达,用SDS-PAGE方法对表达产物进行分析,重组蛋白量占菌体总蛋白的36．8%,经Ni-NTA柱亲合层析后,重组蛋白纯化率达88%以上．用非变性PAGE方法对基因表达的脱巯基酶进行了鉴定．

The chromosomal gene of Pseudomona sp. TS1138 extracted and the L-Cysteine desulfhydrase gene （cds） was amplified by PCR with artificial primer. The amplified gene was recombined in cloning vector pBluescript SK Ⅱ and transformed into E. coll. DH5α. The 1.2 kb DNA fragment containing cds was detected. The cds was cloned into expression vector pETH, then transformed into E. coli BL21 （DE3） and afterward expressed by IPTG inducement. By SDS-PAGE analysisj, the expression protein amounted to 36.8 % of the total bacterial protein and the purification rate was above 88% after purified by Ni-NTA His-Bind Resin. The expression protein was identified by native-PAGE.