人腺苷甲硫氨酸脱羧酶的原核表达及纯化

Prokaryotic expression and purification of human S-adenosylmethionine decarboxylase

目的克隆人腺苷甲硫氨酸脱羧酶(S-adenosylmethionine decarboxylase,AdoMetDC)cDNA,原核表达并纯化重组AdoMetDC蛋白。方法采用巢式RT-PCR法从人宫颈癌HeLa细胞株总RNA中克隆人AdoMetDC cDNA,插入载体pET-15b中,构建原核表达质粒pET-15b/AdoMetDC,转化大肠杆菌Rossetta(DE3),IPTG诱导表达。表达的重组蛋白在尿素变性条件下经Ni-NTA树脂亲和层析纯化,SDS-PAGE和Western blot法鉴定纯化的重组AdoMetDC蛋白。结果克隆出编码全长人AdoMetDC的cDNA序列,并构建了重组原核表达质粒pET-15b/AdoMetDC;表达的重组AdoMetDC蛋白经SDS-PAGE分析,分别可见相对分子质量约35 000、30 000、14 000的前酶、自催化分裂后的α亚单位和β亚单位;前酶和β亚单位可被Ni-NTA树脂有效纯化;纯化的重组蛋白可被抗His抗体和抗AdoMetDC抗体特异性识别。结论原核表达并纯化了人AdoMetDC,为其功能研究和临床应用奠定了基础。

Objective To clone the eDNA of human S-adenosylmethionine decarboxylase（AdoMetDC ）, express in prokaryotic cells and purify the expressed protein. Methods Human AdoMetDC eDNA was cloned from the total RNA of HeLa ceils by nested RT-PCR and inserted into vector pET-15b. The constructed recombinant plasmid pET-15b/AdoMetDC was transformed to competent E. coli Rossetta （DE3） and induced with IPTG. The expressed recombinant protein was purified by Ni-NTA chromatography at condition of denaturation with urea, and identified by SDS-PAGE and Western blot. Results The full-length eDNA sequence of human AdoMetDC was cloned, and recombinant plasmid pET-15b/AdoMetDC was constructed. The expressed recombinant AdoMetDC protein showed bands of proenzyme as well as subunits α and 13 obtained by autocatalytic cleavage, with relative molecular masses of about 35 000, 30 000 and 14 000 respectively, on SDS-PAGE profile. Proenzyme and subunit 13 were effectively purified by Ni-NTA chromatography. The purified recombinant protein was specifically recognized by the antibodies against His and AdoMetDC. Conclusion Human AdoMetDC was expressed in prokaryotic ceils and purified, which laid a foundation of study on its function and clinical application.