HIV-1型整合酶蛋白的表达、纯化及复性研究

Expression,purification and refolding research of HIV-1 integrase

目的对HIV-1整合酶蛋白进行原核表达、纯化以及复性研究。方法从HIV-1 HXB2中PCR扩增出全长的整合酶基因,连入原核表达载体pET-30a中,得到pET-30a整合酶表达质粒。再将质粒转入大肠杆菌BL21中诱导表达,经镍柱纯化后得到整合酶蛋白。纯化后蛋白在FoldIt复性液中进行稀释复性确定最佳复性液,然后蛋白在此条件下复性并用反相柱回收,最后通过对复性蛋白抽干及再溶分析复性蛋白的物理稳定性。结果HIV-1整合酶蛋白主要以包涵体形式表达,表达量占菌体总量的10%。经镍柱纯化后蛋白的总浓度为0.9 mg/ml。蛋白的最佳复性液是:Tris-Cl 55 mmol/L, NaCl 264 mmol/L,KCl 11 mmol/L,Gu-HCl 550 mmol/L,EDTA 1.1 mmol/L,以及附加成分中的GSH、GSSG。复性后蛋白抽干后再溶,溶液清亮透明,SDS-PAGE可见有寡聚体状态的蛋白。结论成功构建了pET-30a整合酶表达质粒。纯化后蛋白的浓度较高。确定了最佳的蛋白复性液。并且初步检测了复性蛋白的物理稳定性。这为蛋白体外活性研究和AIDS药物研究打下了基础。

Objective To expression, purification and refolding of HIV- 1 integrase （ IN ） . Methods The full length IN gene fragment of HIV-1 was amplified by PCR from HXB2 and inserted into pET-30a vector. The expression of IN gene was induced by transforming pET-30a-IN into E. coli （ BL21 ）. The IN protein was purified by Ni affinity chromatography. The best refolding buffer of purified IN protein was confirmed by using Foldh Screen, which is a protein folding screen and can evaluate 12 factors in 16 unique solutions in vitro. Applying the best buffer condition, the purified IN protein was refolded and recovered by Reversed-Phase Chromatography. For testing the physical stabilization, the refolded protein was dried up, and then redissolved. Results IN was expressed as inclusion bodies and observed about 10% of total cellular proteins. The final concentration of purified IN proteins by Ni affinity chromatography was 0.9 mg/ml. The best refolding buffer was a compound including Tris-C1 55 mmol/L, NaC1 264 mmol/L, KC1 11 mmol/L, Gu-HC1 550 mmol/L, EDTA 1.1 mmol/L and GSH, GSSG of additives. Moreover, the redissolved refolded IN protein clear, and the oligomer of IN protein is well-identified by SDS-PAGE. Conclusion We successfully construct the pET-30a-IN vector and obtain high level IN protein expression. The best refolding buffer is confirmed. The physical stabilization of refolded IN protein was tested. This study laid a foundation for developing IN function research and for anti-HIV drug.