PTD-SARA融合蛋白的表达、纯化及鉴定

Expression, Purification and Identification of PTD-SARA Fusion Protein

目的在大肠杆菌中表达PTD-SARA融合蛋白,并对其进行纯化和鉴定。方法使用大肠杆菌偏爱密码子合成PTD-SARA全长基因,将其克隆入载体pQE-30中,构建重组表达质粒pQE-30-PTD-SARA,转化感受态大肠杆菌M15,IPTG诱导表达。表达产物经Ni2+-NTA亲和层析纯化后,进行Western blot鉴定及N-端测序。结果重组表达质粒pQE-30-PTD-SARA经双酶切及测序鉴定证明构建正确。1.0mmol/LIPTG诱导4h,目的蛋白的表达量最高,约占菌体总蛋白的25%,主要以包涵体形式存在。纯化的融合蛋白纯度为94%,浓度为0.2mg/ml,且具有良好的反应原性,N-端有14个氨基酸。结论已成功表达并纯化了PTD-SARA融合蛋白,为其功能的研究奠定了基础,也为临床防治肾脏纤维化提供了一个新的策略。

Objective To express FFD-SARA fusion protein in E. coli and purify and identify the expressed product. Methods The full-length PTD-SARA gene was synthesized by using E. coil-preferred codon and cloned into vector pQE-30. The constructed recombinant plasmid pQE-30-PTD-SARA was transformed to competent E. coli MI5 for expression under induction of IFFG. The expressed protein was purified by Ni^2＋ -NTA affinity chromatography, identified by Western blot, and its N-terminus was sequenced. Results Both restriction analysis and sequencing proved that recombinant plasmid pQE-30-PTD-SARA was constructed correctly. After induction with 1.0 mmol / L IPTG for 4 h, the expression level of target protein reached a peak value of about 25% of total somatic protein. The expressed product mainly existed in a form of inclusion body and reached a purity of 94% and a concentration of 0.2 mg/ml after purification. The fusion protein with 14 amino acids at N-terminus showed good reactogenicity. Conclusion PTD-SARA fusion protein was successfully expressed and purified, which laid a foundation of study on its function and provided a new strategy for prevention and treatment of kidney fibrosis.