Prokaryotic Expression And Protein Purification Of AtGT-3b Gene

GT elements, which are rich in nucleotides T and A, are cis-acting elements in the promoter region of plant genes. A few GT elements and their functions have been characterized in various plants. Meantime, GT factors, the GT elements binding factors could be identified in different plants. In Arabidopsis, GT factors are transcription factors for DNA-binding domains and specific binding to GT elements. The interaction of GT factors and GT elements can regulate the transcription of related genes. Transcription factor GT-3b induced by NaCl and pathogen expresses the transcription factor GT-1-like. It could interact with GT-1cis-element (GAAAAA) and then promotes the downstream genes expression. This article show AtGT-3b gene from Arabidopsis (Arabidopsis thaliana) cloning into prokaryotic expression vector pCpld TF, and expressing fusion protein in Escherichia coli BL21. Purification of AtGT-3b fusion protein is in view to applied the GT-1cis-acting elements in vitro interaction. The main contents and results are as follows:1. The Arabidopsis AtGT-3b gene was cloned into prokaryotic expression vector pCold TF to get the pCold TF-AtGT-3b expression vector,which can be used in the next step of the prokaryotic expression analysis.2. pCold TF-AtGT-3b expression vector transformed into E. coli cells, after incubating at37℃to OD600=0.6, adding1mM IPTG for induction, and15℃for22hours (OD600=1.00) incubation, AtGT-3b proteins can be expressed as well.3. Expression of AtGT-3b protein, followed by nickel affinity resin, balance of cation resin, finally using the0.8M NaCl/Buffer C elution, the purified AtGT-3b fusion protein was obtained, which can be used in follow-up studies.