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| Prokaryotic Expression, Purification and Polyclonal Antibody Preparation of Tobacco (Nicotiana tabacum) NtGCN2 |
| HAO Ying-Chen, LONG Yue, GUO Hao, LI Ning, ZHANG Song-Jie, Zhao Qi, ZHANG Song-Tao* |
| College of Tobacco Science, Henan Agricultural University / Tobacco Cultivation Key Laboratory of China Tobacco, Zhengzhou 450003, China |
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Abstract General control non-derepressible-2 (GCN2) regulates protein synthesis by phosphorylation of eukaryotic translation initiation factor 2 (eIF2α) and responding to amino acid deficiency and various stresses. At present, the action mechanism of GCN2 in yeast and animals has been relatively clear, but the regulation mechanism of plant GCN2 is not clear. In order to further study the function of GCN2, 2 special primer sequences were designed to amplify the full-length NtGCN2 and N-terminus (1~437 aa) gene sequences, and then inserted them to pET-15b and pCzn1 vectors, respectively. The prokaryotic expression vectors pET15b-NtGCN2 and pCzn1-NtGCN2 (1~437 aa) were transformed into Escherichia coli BL21-CodonPlus-(DE3)-RIPL, BL21-CodonPlus-(DE3)-RPL and BL21(DE3) Plys for expression, respectively. The full length NtGCN2 and N-terminal NtGCN2 (1~437 aa) proteins were successfully expressed in E.coli BL21-CodonPlus-(DE3)-RIPL and BL21(DE3) Plys, respectively. The expressed protein were identified by Western blot using 6×His mouse antibody as a primary antibody. The prokaryotic expression condition of full-length NtGCN2 including induction concentration of isopropyl β-D-1-thiogalactopyranoside (IPTG), culture temperature and culture time were optimized. These results showed that soluble NtGCN2 protein was obtained in the supernatant of BL21-CodonPlus-(DE3)-RIPL under the conditions of 16 ℃, 0.5 mmol/L IPTG and 13 h induction. The protein was purified by affinity chromatography on anion exchange column Hitrap Q and Ni2+-NTA agarose and the NtGCN2 protein was not well separated and purified. Furthermore, the purified NtGCN2 and NtGCN2(1~437 aa) proteins were obtained by inclusion body renaturation, and a single band of NtGCN2(1~437 aa) pure protein was obtained by further Ni2+-NTA agarose affinity chromatography. Finally, The purified recombinant protein was used as immunized rabbit antigen and polyclonal antibody against NtGCN2 was successfully prepared, the titer and specificity of the antibodies were analyzed. Enzyme-linked immune sorbent assay (ELISA) assay showed that the antibodies titer reached over 1∶50 000. Western blot analysis showed that the prepared NtGCN2 full-length and NtGCN2(1~437 aa) polyclonal antibody could specifically recognize the recombined NtGCN2 protein. In this study, the prokaryotic expression and purification of NtGCN2 protein were achieved, and the antibody against NtGCN2 was prepared, which would provide data for the further exploration of the function of plant GCN2.
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Received: 28 June 2018
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Corresponding Authors:
* , zhangsongzi@163.com
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