人泛素偶联酶UBE2C/UbcH10在大肠埃希菌中的诱导表达与鉴定

Expression and identification of human ubiquitin-conjugating enzyme UBE2C/UbcH10 in E.coli

目的构建稳定表达人类泛素偶联酶E2C(UBE2C/UbcH10)蛋白的原核表达载体并在大肠埃希菌中诱导其表达,纯化并鉴定该重组蛋白。方法通过PCR扩增获得UbcH10基因,将其克隆至原核表达载体pET32a(+)中,构建重组表达载体,经EcoRI/XhoI双酶切及测序验证正确后,转化至大肠埃希菌BL21,经IPTG(终浓度1mmol/L)诱导表达4h。采用SDS-PAGE、Western blotting等方法鉴定表达产物,用Ni-Sepharose亲和层析和Sephadex G-50分子筛纯化UbcH10蛋白。结果成功构建UbcH10基因的原核表达载体,经IPTG诱导后在大肠埃希菌BL21中获得大量重组蛋白。该重组蛋白以可溶形式表达,表观分子量约为29kD,表达量约占菌体蛋白总量的40%。Western blotting检测在29kD附近出现预期条带,与目的蛋白的理论计算值相吻合。纯化后的UbcH10蛋白终浓度为8.82mg/ml,纯度达90%以上。结论成功表达并纯化了UbcH10重组蛋白,为进一步研究其结构、功能以及该蛋白在肿瘤发生发展过程中的作用奠定了基础。

Objective To construct prokaryotic expression vector of human ubiquitkrconjugating enzymes E2Cs （UBE2C/UbcH10） gene, and induce the expression of UbcH10 in E coli, and to purify and identify the obtained recombinant protein. Methods UbcH10 gene was amplified by PCR, and then was subcloned into prokaryotic expression vector pET32a （＋） to construct the recombinant vector pET32a （＋）/UbcH10. After being identified by enzyme digestion and DNA sequencing, the pET32a（＋）/UbcH10 was transformed into E.coli BI21, and the expression was induced with Immol/L IPTG for 4 hours. The expressed product was then analyzed by SDS-PAGE and Western blotting. The UbcH10 protein obtained was purified by Ni Sepharose affinity column and SephadexG-50 molecular sieve chromatography. Results Prokaryotic expression vector pET32a（＋）/UbcH10 was successfully constructed. A large amount of recombinant protein about 29kD was obtained in E. coli BL21 after IPTG induation, and it was soluble and accounted for about 40% of total bacterial protein. Western blotting showed that the molecular weight of recombinant protein was consistent with the theoretical value. The concen tration of purified protein was about 8. 82mg/ml, and the purity coefficient was up to 90%. Conchusions The successful expression and purification of recombinant protein UbcH10 will be valuable for the further study on the structure, function and its important role in tu morigenesis and tumor progression.