title: Expression and purification of GST-FHL2 fusion protein.
abstract: Escherichia coli is the most widely used host for the production of recombinant proteins. However, most eukaryotic proteins are typically obtained as insoluble, misfolded inclusion bodies that need solubilization and refolding. The interactions between human FHL2 protein and many types of proteins, including structural proteins, kinases, and several classes of transcription factor, have been found to have important roles in a variety of fundamental processes, including arrhythmia, hypertrophy, atherosclerosis, and angiogenesis. To achieve high-level expression of soluble recombinant human FHL2 protein in E. coli, we have constructed a recombinant expression plasmid, pGEX-4T-1-FHL2, in which we merged FHL2 cDNA with the glutathione S-transferase (GST) coding sequence downstream of the tac inducible promoter. Using this plasmid, we have achieved high expression of soluble FHL2 as a GST fusion protein in E. coli BL21. We have used the engineered plasmid (pGEX-4T-1-FHL2) and the modified E. coli strain to overcome the problem of removing the GST moiety while expressing soluble FHL2. Our results show that: 1) the recombinant plasmid was successfully constructed. Sequencing results showed that FHL2 and GST are in the same reading frame; 2) at 23°C, soluble GST-FHL2 fusion protein was highly expressed after induction with 0.1 mM IPTG; and 3) GST-FHL2 can be detected by Western blotting using mouse monoclonal anti-GST antibody. Our data are the first to show that high yields of soluble FHL2 tagged with GST can be achieved in E.coli.
authors.affiliation: Department of Cardiology, Guangdong General Hospital, Guangdong Provincial Cardiovascular Institute, Guangdong Academy of Medical Sciences, Guangzhou, China.
authors.forename: H
authors.initials: H
authors.lastname: Yu
authors.forename: Q
authors.initials: Q
authors.lastname: Ma
authors.forename: J
authors.initials: J
authors.lastname: Lin
authors.forename: Y F
authors.initials: YF
authors.lastname: Sun
authors.forename: F
authors.initials: F
authors.lastname: Zheng
mesh_terms: D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D060850:LIM-Homeodomain Proteins / Q000096:biosynthesis / Q000235:genetics*; D009124:Muscle Proteins / Q000096:biosynthesis / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics*; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics*
chemical_list: C105987:FHL2 protein, human; D060850:LIM-Homeodomain Proteins; D009124:Muscle Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D005982:Glutathione Transferase