溶藻弧菌PEPCK蛋白原核表达及其乙酰化、琥珀酰化修饰的鉴定

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

摘要/Abstract

摘要：

构建溶藻弧菌HY9901 PEPCK蛋白的原核表达载体、优化其表达条件,并鉴定该蛋白的乙酰化及琥珀酰化修饰。采用PCR克隆pepck基因,构建pET-28a-pepck并将其转入大肠杆菌BL21（DE3）,对重组菌株的培养温度、IPTG浓度及时间进行优化,采用SDS-PAGE和Western blot分析蛋白的表达及乙酰化、琥珀酰化修饰。结果显示, pepck基因全长1 629 bp,重组菌株可以正确表达分子质量约60.9 kD的蛋白。诱导温度：在28℃条件下PEPCK存在于上清和包涵体中,在37℃则主要以包涵体形式存在;IPTG浓度：0.2 mmol/L-1.0 mmol/L范围内,PEPCK的表达量无明显差异;时间：在0-8 h内PEPCK表达量随时间增长逐渐增加,诱导8 h后趋于稳定。Western blot显示PEPCK蛋白具有乙酰化修饰和琥珀酰化修饰。成功构建溶藻弧菌PEPCK重组表达菌株,其最优诱导表达条件为0.2 mmol/L IPTG,28℃诱导8 h;经鉴定PEPCK蛋白具有乙酰化修饰和琥珀酰化修饰。

关键词: 溶藻弧菌, PEPCK蛋白, 原核表达, 乙酰化, 琥珀酰化

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

曾福源, 苏泽辉, 周诗慧, 谢妙, 庞欢瑛. 溶藻弧菌PEPCK蛋白原核表达及其乙酰化、琥珀酰化修饰的鉴定[J]. 生物技术通报, 2021, 37(5): 84-91.

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.

图/表 8

图1 pepck基因的克隆 M：DL2000 DNA分子标准;1-4：pepck基因扩增结果

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

图2 构建pET-28a-pepck载体 M：DL2000 DNA分子标准;1-4：pET-28a-pepck的PCR产物

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

图3 PEPCK蛋白SDS-PAGE电泳分析 M：26616蛋白质分子标准;1：pET-28a（未诱导）;2：pET-28a（诱导）;3：pET-28a-pepck（未诱导）;4：pET-28a-pepck（诱导）

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)

图4 温度对PEPCK蛋白表达的影响 M：26616蛋白质分子标准;1：28℃下诱导的全菌蛋白;2-3：28℃下诱导的可溶性蛋白和包涵体蛋白;4：37℃下诱导的全菌蛋白;5-6：37℃下诱导的可溶性蛋白和包涵体蛋白

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℃

图5 IPTG浓度对PEPCK蛋白表达的影响 M：26616蛋白质分子标准;1-6：IPTG诱导浓度分别为0、0.2、0.4、0.6、0.8、1.0 mmol/L

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

图6 诱导时间对PEPCK蛋白表达的影响 M：26616蛋白质分子标准;1-6：诱导时间为别为0 h、2 h、4 h、6 h、8 h、10 h

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

图7 PEPCK蛋白的纯化 M：26616蛋白质分子标准;1：未纯化;2：流穿液;3-4：洗脱液浓度分别为50、250 mmol/L

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

图8 PEPCK蛋白的乙酰化（A）及琥珀酰化（B）鉴定（A） M：26616蛋白质分子标准;1-2：PEPCK蛋白（一抗使用鼠抗乙酰化赖氨酸抗体,二抗使用辣根过氧化物酶标记的山羊抗小鼠 IgG）;（B） M：26616蛋白质分子标准;3-4：PEPCK蛋白（一抗使用鼠抗琥珀酰化赖氨酸抗体,二抗使用辣根过氧化物酶标记的山羊抗小鼠IgG）

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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