人釉原蛋白基因在大肠杆菌中的融合表达

Expression and purification of human amelogenin in Escherichia coli

目的建立人釉原蛋白(AMG)成熟肽基因在大肠杆菌中融合表达和纯化的技术路线。方法利用已构建并经鉴定的重组质粒pGEX-4T-1/AMG转化大肠杆菌BL21,分别对诱导时间、异丙基-β-D硫代半乳糖苷(IPTG)浓度和诱导温度进行优化,在最佳诱导表达条件下,分别对菌液上清、周质、胞质和包涵体中的目的蛋白表达进行分析,在可溶性蛋白中发现大量目的蛋白,随后利用GSTrapFF亲和层析柱进行人AMG融合蛋白的过柱纯化。结果pGEX-4T-1/AMG重组质粒的双酶切凝胶电泳鉴定结果和测序鉴定结果和预期一致。最佳诱导时间为14.5h、最佳诱导剂浓度为1.0mmol/L、最佳诱导温度为20℃,在此条件下目的蛋白的表达量达到峰值。在最佳诱导条件下,胞质蛋白和包涵体中都有大量的目的蛋白。提取大量胞质蛋白,经GSTrapFF亲和层析柱纯化,收集纯化液,进行SDS-聚丙烯酰胺凝胶电泳分析,显示成功纯化了AMG融合蛋白,在提取液洗涤2次后,可获得高纯度的融合蛋白。结论利用pGEX-4T-1/AMG原核表达体系成功获得纯化的人AMG融合蛋白。

Objective To establish the expression and purification route for the gene encoding human amelogenin （AMG） mature peptide in Escherichia coli （E.coli）. Methods Recombined plasmid pGEX-4T-1/AMG was identified by double endonuclease digestion electrophoretogram and DNA sequence analysis. The recombined plasmid was transformed to E.coli BL21. The inducing time, isopropyl-beta-D-thiogalactopyranoside （IPTG） concentration and inducing temperature were optimized for the express system. Under the optimized condition, the target fusing protein in supernatant,periplasm, plasm and inclusion body was analyzed separately. A great amount of target fusing protein was found in the dissoluble protein. AMG fusing protein was purified by the GSTrapFF affinity column. Results Double endonuclease digestion electrophoretogram and DNA sequence analysis were done to identify the recombined vector pGEX-4T-1/AMG. The results were consistent with the anticipation. The optimum inducing time was 14.5 hours. The optimum IPTG concentration was 1.0 mmol/L. The optimum inducing temperature was 20 ℃. Under this condition, the target protein was expressed to a maximum. Plentiful target protein was expressed in plasm and inclusion body under the optimized condition. A mount of plasm protein was obtained and purified by the GSTrapFF affinity column. The purified liquid was collected and analyzed by SDS-polyacrylamide gel electrophoresis （SDS- PAGE）. The protein electrophoresis map showed that AMG fusing protein was purified successfully. After twice elution, high pure fusing protein was obtained. Conclusion pGEX-4T-1/AMG system is used successfully to express human AMG fusing protein.