Prokaryotic Expression, Purification and Activity Analysis of Arabidopsis Tryptophan Aminotransferase 1
LI Xiao-Fang, FANG Ping-Ping, WANG Zhuo-Yi, XU Pei*
Key Laboratory of Specialty Agri-product Quality and Hazard Controlling Technology of Zhejiang Province/College of Life Sciences, China Jiliang University, Hangzhou 310018, China
Abstract：The expression of purified and active target protein is prerequisite for the screening of protein-specific inhibitors. In this study, the prokaryotic expression, purification and activity analysis of tryptophan aminotransferase of Arabidopsis 1 (TAA1) were carried out. First, the TAA1 coding sequence was retrieved from GenBank. After codon optimization according to the preferred codon usage in Escherichia coli the target sequence was synthesized and inserted into the prokaryotic expression vector pET24 with a His tag to construct the recombinant expression vector pET24-GST-EK-His-TAA1. The vector was then transformed into the E. coli BL21(DE3) cell line followed by induction and cultivation with different methods (IPTG induction/TB medium, IPTG induction/LB medium, and auto-induction). The products were analyzed with sodium lauryl sulfate polyacrylamide gel electrophoresis to identify the size, amount and existing form of the expressed protein. It was found that the auto-inducible expression system combined with incubation at 37 ℃for 16 h yielded the greatest amount of protein product in the soluble fraction. This optimized expression method was then used for large-scale expression and purification of the proteins. The collected fusion protein was further cleaved by the enterokinase (EK) protease and purified with a nickel ion affinity column. Finally, purified recombined TAA1 protein at a purity of 78.7% and an amount of 0.5 mg was obtained. In vitro enzymatic activity assay proved that this protein had a transaminase activity. In conclusion, a high-efficient prokaryotic expression system for the Arabidopsis TAA1 protein was established successfully, from which the obtained purified and bioactive His-TAA1 protein are very useful for the screening of TAA1-specific inhibitors based on molecular interaction.
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