Expression of recombinant human aldehyde dehydrogenase 2 by using intein induced expression system
Author(s): LIU Xiangyu, YIN Jiechao, LI Jingpeng
Pages: 38-44
Year: 2016 Issue: 1
Journal: Journal of Northeast Agricultural University
Keyword: human aldehyde dehydrogenase 2; intein; recombinant expression; inclusion body; IMPACTTM-CN protein purification system;
Abstract: To achieve the recombinant human aldehyde dehydrogenase 2 proteins with high activity, the ALDH2 gene was amplified by PCR. The cloned and expressed plasmids pGEM-T-ALDH2 and pTYB11-ALDH2 were constructed. The recombinant expression plasmids was induced by IPTG. Through orthogonal experiment, the results showed that the optimal strategy was the expression conditions (OD 0.3, 1.0 mmol·L-1 IPTG, 30 ℃, 4 h). Because all the target proteins were almost expressed in the form of inclusion body, the bioactive acetaldehyde dehydrogenase was obtained by means of degeneration and renaturation for the inclusion body. Using the IMPACTTM-CN protein purification system, the similar natural ALDH2 protein was obtained from the self cleavage of fused intein protein, and the protein concentration was 0.045 mg·mL-1 by the Bradford method. The activity of purification protein was 462 U·mg-1.
Pages: 38-44
Year: 2016 Issue: 1
Journal: Journal of Northeast Agricultural University
Keyword: human aldehyde dehydrogenase 2; intein; recombinant expression; inclusion body; IMPACTTM-CN protein purification system;
Abstract: To achieve the recombinant human aldehyde dehydrogenase 2 proteins with high activity, the ALDH2 gene was amplified by PCR. The cloned and expressed plasmids pGEM-T-ALDH2 and pTYB11-ALDH2 were constructed. The recombinant expression plasmids was induced by IPTG. Through orthogonal experiment, the results showed that the optimal strategy was the expression conditions (OD 0.3, 1.0 mmol·L-1 IPTG, 30 ℃, 4 h). Because all the target proteins were almost expressed in the form of inclusion body, the bioactive acetaldehyde dehydrogenase was obtained by means of degeneration and renaturation for the inclusion body. Using the IMPACTTM-CN protein purification system, the similar natural ALDH2 protein was obtained from the self cleavage of fused intein protein, and the protein concentration was 0.045 mg·mL-1 by the Bradford method. The activity of purification protein was 462 U·mg-1.
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