药物中间体S-苯基-L-半胱氨酸的酶法合成

Enzymatic synthesis of pharmaceutical intermediates S-phenyl-L-cysteine

关键词:  S-苯基-L-半胱氨酸  酶法合成  O-乙酰丝氨酸(硫醇)裂解酶  非蛋白质氨基酸

Key words:  S-phenyl-L-cysteine  enzymatic synthesis  O-acetylserine sulfhydrylase  unnatural amino acids

摘要:

目的　利用在大肠杆菌中高效重组表达的O-乙酰丝氨酸（硫醇）裂解酶A(OASS-A)合成药物中间体S-苯基-L-半胱氨酸，并对合成产物进行纯度及结构鉴定。　方法　通过PCR扩增目的基因 Cys K，并连接到pET-22b(+)载体上构建原核表达质粒，重组体转到感受态大肠杆菌BL21（DE3）中进行蛋白诱导表达，重组蛋白采用Ni 2+-树脂柱亲和层析纯化，利用SDS-PAGE鉴定表达纯化蛋白，通过HPLC对合成产物纯度进行检测， 应用 1H NMR技术对化合物进行结构鉴定。　结果　成功构建了OASS-A原核表达载体，在IPTG诱导下获得了高效表达。重组酶高效合成非蛋白质氨基酸S-苯基-L-半胱氨酸。合成的化合物以晶体形式析出，采用HPLC和 1H NMR技术对其结构进行了鉴定，合成产物的纯度为100%。　结论　利用大肠杆菌中重组表达的OASS-A能够高效合成药物中间体S-苯基-L-半胱氨酸。

Abstract:

Objective　 To construct expression vector of O-acetylserine sulfhydrylase A (OASS-A), purify and determine its chemical structure.　Methods　Full-length Cys K gene was amplified by polymerase chain reaction (PCR) and ligated with pET-22b(+) expression vector to construct prokaryotic expression plasmid. The recombinant was transformed into competent E.coli BL21(DE3) for protein expression with IPTG induction. Recombinant protein was purified with Ni2+-resin affinity chromatography and identified by SDS-PAGE. The purity and chemical structure of the synthesized compound were determined using HPLC and 1H NMR techniques.　Results　The prokaryotic expression vector for OASS-A was successfully constructed and fusion protein was expressed at a high level with IPTG induction. The unnatural amino acids S-phenyl-L-cysteine was effectively synthesized by recombinant OASS-A.　Conclusion　The pharmaceutical intermediate S-phenyl-L-cysteine was effectively synthesized by recombinant OASS-A from E.coli.