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Increased Expression of Flagellins Recombinant Soluble form by Chemical Chaperon

Bakhtiarvand, Bahador | 2014

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  1. Type of Document: M.Sc. Thesis
  2. Language: Farsi
  3. Document No: 46083 (06)
  4. University: Sharif University of Technology
  5. Department: Chemical and Petroleum Engineering
  6. Advisor(s): Yaghmaei, Soheila; Tarahomjoo, Shirin
  7. Abstract:
  8. Flagella is the organelle involved in motility of Salmonella and plays an important role in colonization of Salmonella. Flagellins is the structural subunits of flagellar filament that is known as an adjuvant. Recombinant production of flagellin of S. enteritidis is advantageous due to the removal of pathogenic microorganism from the production process and improved process safety. E.coli is an appropriate host for the expression of recombinant proteins. However, its major obstacle is the formation of inclusion bodies. Inclusion bodies are aggregated inactive proteins and the recovery of proteins in the active form from these aggregates usually has a low yield. Therefore, the objective of our research is to determine a suitable approach to enhance the expression of the flagellin in the soluble form. For this purpose, the flagellin gene of S. enteritidis SGSC2475 and two Iranian isolates of S. enteritidis was cloned in Escherichia coli BL21 (DE3) using vector pET28a. In order to express the flagellin gene, T7 promoter induction using IPTG (isopropyl-beta-D-thiogalactopyranoside) were used at a concentration of 1mM. Analysis of gene expression and solubility of proteins using SDS-PAGE and gel densitometry using Coomassie Brilliant Blue (CBB) was performed. To investigate the effect of arginine on the solubility of flagellin, various concentrations of arginine in the culture medium were used. Sequencing results showed that the gene sequence was the same in all three strains. BLAST results showed that the obtained amino acid sequence of the flagellin was the same with the most prevalent amino acid sequence of flagellin of S. enteritidis strains in the NCBI site. Our results showed that 46% of the flagellin expressed in E. coli BL21 (DE3) was in the soluble form and rest of the protein was in the form of the inclusion body. Using chemical chaperones has been shown to be an appropriate solution for increasing of production of recombinant proteins in the soluble and active form. Our results showed that using arginine as a chemical chaperon at the concentration of 0.1-0.25M increased noticeably the flagellin solubility to 91-94%
  9. Keywords:
  10. Solubility ; Flagellin Bacteries ; Recombinant Protein ; Salmonella ; Chemical Chaperon

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