Prokaryotic Expression of AATF-Che1 Superfamily Gene and Its Fusion Protein Purification

Abstract

Objective To construct the apoptosis antagonizing transcription factor (AATF)-Che1 recombinant prokaryotic expression vector pGEX-4T-1-AATF-Che1 in vitro,optimize prokaryotic recombinant vector induced expression conditions,and get soluble purified glutathione S-transferase(GST) fusion proteins.Methods The AATF-Che1 gene was amplificated from the pGEX-4T-1-AATF recombinant plasmid by polymerase chain reaction(PCR).The prokaryotic expression vector pGEX-4T-1 was digested with double enzymes,and recycled purpose fragments.The directional connection of AATF-Che1 gene and linearization prokaryotic expression vector pGEX-4T-1 were through the T4 ligase,to obtain recombinant protein AATF-Che1 prokaryotic expression plasmid pGEX-4T-1-AATF-Che1.Correct plasmid was transformated to escherichia coli cells BL21 (DE3),and used isopropy1β-D-1-thiogalactopyranoside (IPTG) to induce AATF-Che1 protein expression.Through exploring the influence of the IPTG concentration,induction time and temperature to the expression quantity and form of the recombinant protein,optimized the best induction conditions.Using glutathione sepharose beads purificated soluble GST fusion protein,at last to identificate of the protein by Western blot (WB) mothd.Results The results of enzyme digestion,PCR identification and sequencing were correct,and the prokaryotic expression plasmid was successfully constructed.The electrophoresis showed that a new protein band appear at about 65 kD,which was the fusion protein of AATF-Che1 and GST.The fusion protein was no obvious change with different induction time and IPTG concentration at 37℃ or 16℃.The recombinant protein mainly existed in the form of inclusion body at 37 ℃,while soluble from in the supernatant at 16℃.Soluble purificated GST fusion protein was obtained,and by WB method identified antigenicity of fusion protein was good.Conclusions The prokaryotic expression plasmid was successfully constructed in vitro.The optimized recombinant prokaryotic expression conditions were found and purificated GST fusion protein was obtained.It maybe lay the foundation for the further study on the role of AATF-Che1 in tumor. Key words: AATF-Che1 superfamily domain; Prokaryotic expression; Fusion protein purification