Expression of flagellin FLjB derived from Salmonella enterica serovar typhimurium in Escherichia coli BL21

Abstract

Flagellin FljB composes flagellar antigen (H:1,2) of S. Typhimurium. This kind of antigen increases immunogenicity of any conjugated antigen upon administration. Thus, it is supposed to have an enormous potentiality for vaccine development against bacterial infections and cancer diseases. fljB gene (1515 nucleotides) coding for mature FljB was amplified by PCR from genomic DNA of S. Typhimurium and inserted into pET32a(+) for expression in E. coli BL21. The protein FljB was well expressed under the fusion form with Trx, Stag at N terminal and hexahistidine at C terminal, thus the recombinant protein was abbreviated to TrxFljB. Study on the impact of temperature on the gene expression showed that TrxFljB was synthesized at lower level at 37 o C comparing to the levels at 22 o C and 25 o C. 13% of the protein synthesized at 37 o C was inclusion body. Lower temperatures used during induction phase increased the solubility of the recombinant protein. About 97% of TrxFljB synthesized at 25 o C was soluble. IPTG concentration had a strong effect on the growth of freshly transformed cells but did not affect on the growth of stored and re-cultivated cells. The increase of IPTG concentration resulted in the decrease of the growth of freshly transformed cells and the TrxFljB productivity. However, 0.05 mM IPTG concentration was found to gain the full TrxFljB expression. TrxFljB productivity declined during storage of cells at 4 o C and re-cultivation. At optimal condition, volumetric productivity of TrxFljB was about 300 mg/ l broth.

High-level expression of Aerococcus viridans pyruvate oxidase in Escherichia coli by optimization of vectors and induction conditions

“…For example Huyen et al . () found that IPTG concentrations were inversely related to flagellin productivity using pET32a.…”

“…Note that different IPTG concentrations were required for different pET vectors for soluble protein expression because of the metabolic burden release of recombinant E. coli (Lecina et al 2013). For example Huyen et al (2015) found that IPTG concentrations were inversely related to flagellin productivity using pET32a.…”

Generation of a Library of Carbohydrate-Active Enzymes for Plant Biomass Deconstruction

“…In contrast, solubilization and refolding of protein from inclusion bodies is a common strategy although it requires denaturing conditions and a subsequent renaturing step, usually resulting in poor soluble protein recoveries [ 32 , 33 ]. Different approaches have been developed to prevent the accumulation of inclusion bodies in E. coli , such as the optimization of culture conditions, co-expression with molecular chaperones [ 34 , 35 ], lower growth temperature during gene induction [ 35 , 36 ], induction expression in early-log phase culture [ 37 ], and induction with lower levels of inducer concentration, such as Isopropyl β-D-1-thiogalactopyranoside (IPTG) [ 38 ]. In addition, several fusion tags have been developed to increase the solubility of overexpressed proteins, although with variable degrees of success.…”