摘要
【目的】获取人蛋白酪氨酸磷酸酯酶4A2(PTP4A2)基因并高效表达纯化,对于产物的体外活性进行测定。【方法】用RT-PCR技术,从肝癌细胞系HepG2的总RNA中,获得编码PTP4A2基因功能片段的DNA。测序后,通过酶切亚克隆至表达载体pGEX-4T-2,构建重组表达载体,并导入大肠杆菌E.coliBL21中,IPTG诱导表达重组的GST融合蛋白。对重组融合蛋白过谷胱甘肽琼脂糖柱进行纯化,western印记进行鉴定,质谱检测蛋白分子量。通过其对底物磷酸多肽的去磷酸化反应,鉴定其活性。【结果】获得编码肝癌细胞PTP4A2(全长氨基酸)的基因序列,测序结果与已发表的基因序列相一致。重组GST融合蛋白经western分析,在相对分子质量Mr=45000处,有特异的蛋白条带。重组蛋白经谷胱甘肽琼脂糖柱进行纯化后,得到了高纯度的融合蛋白,质谱检测其分子量为45470.6。底物多肽去磷酸化反应表明纯化产物具有较强活性。【结论】成功克隆人PTP4A2基因,并在E.coliBL21中高效表达,亲和层析后获得高纯度GST融合蛋白,质谱检测分子量与预期结果一致,纯化产物具有蛋白磷酸酯酶活性。
[Objective] To obtain human protein tyrosine phosphatase type 4A2 （PTP4A2） gene expressed in E. coil and purify the target proteins.[Methods] PTP4A2 genes were amplified by RT-PCR from total RNA of HepG2 human hepatoma cell line, then were inserted into pGEM-Teasy plasmid. After auto-sequencing, the target gene fragment was cloned into prokaryotic expression vector pGEX-2T in fusion form and transformed into E.coli BL21. Recombinant GST fusion proteins were expressed via the induction of IPTG and purified through glutathione agarose column. The molecular weight of fusion protein was detected by SELDI mass spectrometry. In vitro PTPase assays were used to determine the activity of fusion protein. [Resuhs] The sequence of cloned PTP4A2 gene was identical with those early reported. GST fusion protein was separated by SDS-PAGE, One protein band of Mr= 45 000 appeared on SDS-PAGE gel. The expressed fusion proteins were highly purified and its molecular weight was 45470.6 detected by mass spectrometry. [Conclusions] The human PTP4A2 gene had been successfuUy cloned and efficiently expressed in E.coli, and the GST fused target protein had been highly purified with high activity in vitro.
出处
《中山大学学报（医学科学版）》
CAS
CSCD
北大核心
2007年第2期137-141,共5页
Journal of Sun Yat-Sen University:Medical Sciences
基金
国家自然基金资助项目(No.30100218)
广东省科技厅资助项目(No.2004B30301010)
关键词
蛋白酪氨酸磷酸酯酶
基因克隆
表达
纯化
质谱
protein tyrosine phosphatase
clone
express
purify
mass spectrometry
