His/GST-HDAC1在大肠杆菌中表达的研究

目的:对His/GST-HDAC1在大肠杆菌BL-21中的表达进行研究。方法:HDAC1的完整基因片断被克隆到pColdⅠ载体和pGEX载体上,并在其N末端分别联有His和GST;采用大肠杆菌BL21对HDAC1进行表达;采用亲和色谱对HDAC1进行纯化;用SDS-PAGE和蛋白质印迹来验证表达和纯化效果;对HDAC1活性进行测定。结果:多数HDAC1存在于大肠杆菌BL-21细胞裂解液的沉淀组分和纯化过程中的未结合组分中,小部分HDAC1可从细胞裂解液的上清液中得以纯化,但未显现出酶活性。用FPLC对HDAC1进行进一步分离,结果表明,HDAC1发生了分子聚集,使得它们的分子量大于正常分子量。结论:活性His/GST-HDAC1不能用大肠杆菌BL21成功表达。

Study on Expression of His/GST- HDAC1 in Escherichia coli BL21

Objective: In this study,the expression of His/GST-HDAC1 in E.coli BL21 was studied.Methods: Full-length human genes for HDAC1 were cloned into the pCold1 vector and pGEX vector containing a N-terminal His-tag and GST-tag,individually.HDAC1 was expressed by E.coli BL21 and purified by affinity chromatography.SDS-PAGE and Western blots confirmed the purified HDAC1 preparation.Results: Insoluble and inactive HDAC1 could be generated.Most HDAC1 was located in the pellet or unbounded fraction of the cell lysate.Little HDAC1 was purified from cell lysate supernatant.No activity was detected for His-HDAC1 and GST-HDAC1.Further purification of HDAC1 by FPLC demonstrated that recombinant form of HDAC1 aggregated and precipitated,making their molecular weight bigger than expected.Conclusions: Enzymatically active His-HDAC1 and GST-HDAC1 cannot be expressed in E.coli BL21.