Prokaryotic Expression of the PEPCK Protein of Vibrio alginolyticus and Identification of Its Acetylation and Succinylation

doi:10.13560/j.cnki.biotech.bull.1985.2020-0949

Abstract

Abstract:

The main objective of this study is to construct a prokaryotic expression vector of PEPCK protein in Vibrio alginolyticus HY9901,to optimize its expression conditions and to identify its acetylation and succinylation. Firstly,the target gene pepck was cloned via PCR. An expression vector pET-28a-pepck was then constructed and transferred into Escherichia coli BL21（DE3）. Subsequently,the culture temperature,IPTG concentration and time of the recombinant strain were optimized. In the end,the protein expression,acetylation and succinylation were analyzed by SDS-PAGE and Western blot. The final results of this study showed that the length of the pepck gene of V. alginolyticus strain HY9901 was 1 629 bp,and its His-PEPCK fusion protein with 60.9 kD was successfully expressed in the recombinant strain. The PEPCK protein was distributed in the supernatant and inclusion body at 28℃,but existed mainly as inclusion body when the temperature was at 37℃. There was no significant difference in PEPCK expression while the IPTG concentration was in the range of 0.2-1.0 mmol/L. The PEPCK protein expression increased gradually with time and tended to be stable after 8 h when the induction time was within 0-8 h. Western blot results showed that the PEPCK protein was acetylated and succinylated. In conclusion,the recombinant expression strain for V. alginolyticus PEPCK is successfully constructed,and the recombinant protein is highly expressed under induction conditions with 0.2 mmol/L IPTG at 28℃ for 8 h. The PEPCK protein is identified to be acetylated and succinylated.

Key words: Vibrio alginolyticus, PEPCK protein, prokaryotic expression, acetylation, succinylation

ZENG Fu-yuan, SU Ze-hui, ZHOU Shi-hui, XIE Miao, PANG Huan-ying. Prokaryotic Expression of the PEPCK Protein of Vibrio alginolyticus and Identification of Its Acetylation and Succinylation[J]. Biotechnology Bulletin, 2021, 37(5): 84-91.

Figures/Tables 8

Fig. 1 Cloning of pepck gene M : DL2000 DNA marker. 1-4 : Amplification results of pepck gene

Fig. 2 Construction of pET-28a-pepck vector M : DL2000 DNA marker. 1-4 : PCR products of pET-28a-pepck

Fig. 3 SDS-PAGE analysis of PEPCK protein M : 26616 protein marker. 1 : pET-28a (uninduced). 2 : pET-28a (induced). 3 : pET-28a-pepck (uninduced). 4 : pET-28a-pepck (induced)

Fig. 4 Influence of temperature on the expression of PEPCK protein M : 26616 protein Marker. 1: Total mycoprotein induced at 28℃. 2-3 : Soluble proteins and inclusion body proteins induced at 28℃. 4 : Total mycoprotein induced at 37℃. 5-6 : Soluble and inclusion body proteins induced at 37℃

Fig. 5 Influence of IPTG concentration on the expression of PEPCK protein M : 26616 protein marker. 1-6 : The induced concentration of IPTG is 0, 0.2, 0.4, 0.6, 0.8 and 1.0 mmol/L, respectively

Fig. 6 Influence of induction time on the expression of PEPCK protein M : 26616 protein marker. 1-6 : The induction time is 0 h, 2 h, 4 h, 6 h, 8 h and 10 h, respectively

Fig. 7 Purification of PEPCK protein M : 26616 protein marker. 1: Unpurified. 2: The liquid of flowing through. 3-4: The eluent concentration was 50 and 250 mmol/L, respectively

Fig. 8 Acetylation and succinylation identification of PEPCK protein (A) M : 26616 protein marker. 1-2: PEPCK protein (The primary antibody used in the western blot was Anti-acetyllysine mouse mAb, and horseradish peroxidase conjugated goat Anti-mouse IgG was used as the secondary antibody) . (B) M : 26616 protein marker. 3-4: PEPCK protein (The primary antibody used in the Western blot was anti- succinyllysine mouse mAb, and horseradish peroxidase conjugated goat anti- mouse IgG was used as the secondary antibody)

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