Molecular Cloning and Functional Verification of Histone Deacetylase Gene cobB in Vibrio alginolyticus

doi:10.13560/j.cnki.biotech.bull.1985.2020-1361

Abstract

Abstract:

This work aims to study the function of the histone deacetylase cobB gene in Vibrio alginolyticus. The clone of cobB gene,its bioinformatics analysis,and induction,expression,purification and function of CobB protein were carried out. The relative molecular mass was 27.09750 kD,theoretical isoelectric point was 5.15,the chemical formula of the protein was C₁₁₈₆H₁₈₆₅N₃₃₉O₃₆₉S₁₀,and hydrophilic average coefficient grand aural of hydropathity（GRAVY）：-0.409,showing that it was a hydrophilic protein. CobB presented high homology with that of Vibrio diabolicus,Vibrio harveyi,Vibrio cholerae,and Vibrio parahaemolyticus,and was relatively conserved among Gram-negative bacteria. The fusion protein was about 53 kD,and the molecular weight of the CobB protein after tag removed was about 27 kD. The optimal expression condition of the CobB protein was 37℃ and inducing time 4 h with 0.4 mmol/L IPTG. Functional analysis showed that the CobB had deacetylation effect on acetylated protein.

Key words: Vibrio alginolyticus, CobB, deacetylation, protein expression

FAN Chen-long, DING Yu. Molecular Cloning and Functional Verification of Histone Deacetylase Gene cobB in Vibrio alginolyticus[J]. Biotechnology Bulletin, 2021, 37(8): 195-202.

Figures/Tables 10

Fig.1 Results of cobB full-length PCR cloning M：DNA marker;1-4：PCR amplified product of target gene

Fig.2 Bioinformatics analysis of cobB sequences A：Signal peptide prediction. B：Transmembrane domain prediction.C：Protein tertiary structure prediction

Fig.3 Phylogenetic tree based on CobB sequences

Fig.4 Schematic diagram of gel electrophoresis of CobB protein expression M：Protein marker;1：negative control without IPTG;2：negative control with IPTG;3：pGEX-6p-cobB without IPTG;4：pGEX-6p-cobB with IPTG

Fig.5 Schematic diagram of gel electrophoresis of different induction time on CobB protein expression M：Protein marker;1-7：The induction time is 1 h、2 h、3 h、4 h、5 h、6 h、7 h

Fig.6 Schematic diagram of gel electrophoresis of different IPTG concentrations on CobB protein expression M：Protein marker;1-7：the IPTG concentrations are 0.1%、0.2%、0.4%、0.6%、0.8%、1% and 2%

Fig.7 Schematic diagram of gel electrophoresis of different temperatures on CobB protein expression M：Protein maker;1：28℃ bacteria protein;2：28℃ supernatant protein;3：28℃ precipitation protein;4：37℃ bacteria protein;5：37℃ supernatant protein;6：37℃ precipitation protein

Fig.8 Schematic diagram of digestion gel electrophoresis of CobB protein purification M：Protein molecular standard;1：Bacteria protein;2：Results of CobB fusion protein purification;3：Results of CobB fusion protease cleavage results

Fig.9 Western blot of CobB deacetylated GlmU protein A：Western blot of acetyllysine-specific antibodies and SDS-PAGE;B：GlmU acetylation level quantified by Western blot. 1：No CobB protein,no NAD+ group;2：CobB protein,no NAD + group;3：no CobB protein,NAD+ group;4：CobB protein,NAD+ group

Fig.10 Western blot of CobB fusion protein and CobB protein deacetylated GlmU protein A：Western blot of acetyllysine-specific antibodies and SDS-PAGE;B：GlmU acetylation level quantified by Western blot. 1：CobB fusion protein group;2：CobB protein group;3：GST protein group

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