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| 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 |
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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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Received: 22 October 2021
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Corresponding Authors:
* peixu@cjlu.edu.cn
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[1] 任元雪, 高鑫, 刘茜, 等. 2021. 密码子优化提高狂犬病病毒CTN-1株核蛋白在大肠埃希菌中的表达[J]. 中华微生物学和免疫学杂志, 41(05): 333-337.
(Ren Y X, Gao X, Liu Q, et al.2021. Codon optimization improves the expression of nucleoprotein of Rabies virus CTN-1 strain in E. coli[J]. Chinese Journal of Microbiology and Immunology, 41(05): 333-337.)
[2] Cao X, Yang H, Shang C, et al.2019. The roles of auxin biosynthesis YUCCA gene family in plants[J]. International Journal of Molecular Sciences, 20(24): 6343.
[3] Erlanson D A, Fesik S W, Hubbard R E, et al.2016. Twenty years on: The impact of fragments on drug discovery[J]. Nature Reviews Drug Discovery, 15(9): 605-619.
[4] Ferrer M, Chernikova T N, Yakimov M M, et al.2003. Chaperonins govern growth of Escherichia coli at low temperatures[J]. Nature Biotechnology, 21(11): 1266-1267.
[5] Folmer R H.2016. Integrating biophysics with HTS-driven drug discovery projects[J]. Drug Discovery Today, 21(3): 491-498.
[6] Goodnow R Jr.2018. DNA-encoded library technology (DELT) after a quarter century[J]. SLAS Discovery, 23(5): 385-386.
[7] He W, Brumos J, Li H, et al.2011. A small-molecule screen identifies L-kynurenine as a competitive inhibitor of TAA1/TAR activity in ethylene-directed auxin biosynthesis and root growth in Arabidopsis[J]. Plant Cell, 23(11): 3944-3960.
[8] Hunke S, Betton J M.2003. Temperature effect on inclusion body formation and stress response in the periplasm of Escherichia coli[J]. Molecular Microbiology, 50(5): 1579-1589.
[9] Ikemura T.1985. Codon usage and tRNA content in unicellular and multicellular organisms[J]. Molecular Biology and Evolution, 2(1): 13-34.
[10] Korasick D A, Enders T A, Strader L C.2013. Auxin biosynthesis and storage forms[J]. Journal of Experimental Botany, 64(9): 2541-2555.
[11] Lin E C, Pitt B M, Civen M, et al.1958. The assay of aromatic amino acid transaminations and keto acid oxidation by the enol borate-tautomerase method[J]. Journal of Biological Chemistry, 233(3): 668-673.
[12] Lv B, Yan Z, Tian H, et al.2019. Local auxin biosynthesis mediates plant growth and development[J]. Trends in Plant Science, 24(1): 6-9.
[13] Matheron M E, Moore T C.1973. Properties of an aminotransferase of pea (Pisum sativum L.)[J]. Plant Physiology, 52(1): 63-67.
[14] Niiranen L, Espelid S, Karlsen C R, et al.2007. Comparative expression study to increase the solubility of cold adapted Vibrio proteins in Escherichia coli[J]. Protein Expression and Purification, 52(1): 210-218.
[15] Rodríguez V, Asenjo J A, Andrews B A, et al.2014. Design and implementation of a high yield production system for recombinant expression of peptides[J]. Microbial Cell Factories, 13: 65.
[16] Sahdev S, Khattar S K, Saini K S, et al.2008. Production of active eukaryotic proteins through bacterial expression systems: A review of the existing biotechnology strategies[J]. Molecular and Cellular Biochemistry, 307(1-2): 249-264.
[17] Shi B, Zhou Y, Huang Y, et al.2017. Recent advances on the encoding and selection methods of DNA-encoded chemical library[J]. Bioorganic and Medicinal Chemistry Letters, 27(3): 361-369.
[18] Soeno K, Sato A, Shimada Y.2021. Investigation of auxin biosynthesis and action using auxin biosynthesis inhibitors[J]. Methods in Molecular Biology, 2213: 131-144.
[19] Stepanova A N, Robertson-Hoyt J, Yun J, et al.2008. TAA1-mediated auxin biosynthesis is essential for hormone crosstalk and plant development[J]. Cell, 133(1): 177-191.
[20] Studier F W.2005. Protein production by auto-induction in high density shaking cultures[J]. Protein Expression and Purification, 41(1): 207-234.
[21] Sun X, Li Y, He W, et al.2017. Pyrazinamide and derivatives block ethylene biosynthesis by inhibiting ACC oxidase[J]. Nature Communications, 8: 15758.
[22] Tao Y, Ferrer J L, Ljung K, et al.2008. Rapid synthesis of auxin via a new tryptophan-dependent pathway is required for shade avoidance in plants[J]. Cell, 133(1): 164-176.
[23] Tsugafune S, Mashiguchi K, Fukui K, et al.2016. Yucasin D F, a potent and persistent inhibitor of auxin biosynthesis in plants[J]. Scientific Reports, 7(1): 13992.
[24] Vera A, González-Montalbán N, Arís A, et al.2007. The conformational quality of insoluble recombinant proteins is enhanced at low growth temperatures[J]. Biotechnology and Bioengineering, 96(6): 1101-1106.
[25] Woodward A W, Bartel B.2005. Auxin: Regulation, action, and interaction[J]. Annals of Botany, 95(5): 707-735.
[26] Xu J, Li Y, Wang Y, et al.2008. Activation of MAPK kinase 9 induces ethylene and camalexin biosynthesis and enhances sensitivity to salt stress in Arabidopsis[J]. Journal of Biological Chemistry, 283(40): 26996-27006.
[27] Yamada M, Greenham K, Prigge M J, et al.2009. The TRANSPORT INHIBITOR RESPONSE2 gene is required for auxin synthesis and diverse aspects of plant development[J]. Plant Physiology, 151(1): 168-179. |
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