SHIGELLA DYSENTERY STXA MUTANT (R170L-A231D-G234E) GENE DESIGN AND OPTIMIZATION OF RECOMBINANT PROTEIN EXPRESSION AND PURIFICATION

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OLAD GH.R.; TAVALAE M.; MOHAMMAD HASSAN Z.; EBRAHIMI F.; SALIMIAN J.; NAZAREAN SH.; RAHIMI A.A.

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Journal: Journal of Shahrekord University of Medical Sciences Year:2012 | Volume:13 | Issue:5 Page(s): 93-102

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Title

SHIGELLA DYSENTERY STXA MUTANT (R170L-A231D-G234E) GENE DESIGN AND OPTIMIZATION OF RECOMBINANT PROTEIN EXPRESSION AND PURIFICATION

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Keywords

EXPRESSION OPTIMIZATIONQ2

MUTANT STXAQ2

RECOMBINANT VACCINEQ2

SHIGA TOXINQ2

SHIGELLA DYSENTERYQ2

Abstract

Background and aims: SHIGELLA DYSENTERY is one of the most important human pathogenic intestinal bacteria. Entrance of the shigella toxin into the epithelial cells inhibits protein synthesis leading to cell death. In spite of great investigations on vaccine production against S. dysentery, studying to achieve significant stxA recombinant protein still remains important. The objective of this study was to determine the appropriate mutation loci and designing stxA subunit synthetic gene, its further expression and optimization and ultimately assaying purification method for further immunization studies.Methods: Three MUTANT STXA gene including (R170L-A231D-G234E) were designed and the synthetic gene in pET28a plasmid was obtained and confirmed by PCR. Thereafter the plasmid was transformed into the host cell E. coli BL21 DE3 after which gene expression was optimized and protein purity assay was then performed.Results: Preliminary studies led to mutant stop gene design, after which it was confirmed by synthetic plasmid and PCR. Expression and optimization were then performed which resulted in large amount of protein inclusion bodies. Purification of inclusion bodies and protein which resulted solubilization was done with a combinatorial method.Conclusion: With regard to the mechanism of SHIGA TOXIN effect and favorable mutation design with new arrangement, less toxicity of expressing protein is predicted than previous other mutants, posing a better vaccine candidate.

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APA: Copy

OLAD, GH.R., TAVALAE, M., MOHAMMAD HASSAN, Z., EBRAHIMI, F., SALIMIAN, J., NAZAREAN, SH., & RAHIMI, A.A.. (2012). SHIGELLA DYSENTERY STXA MUTANT (R170L-A231D-G234E) GENE DESIGN AND OPTIMIZATION OF RECOMBINANT PROTEIN EXPRESSION AND PURIFICATION. JOURNAL OF SHAHREKORD UNIVERSITY OF MEDICAL SCIENCES, 13(5), 93-102. SID. https://sid.ir/paper/58605/en

Vancouver: Copy

OLAD GH.R., TAVALAE M., MOHAMMAD HASSAN Z., EBRAHIMI F., SALIMIAN J., NAZAREAN SH., RAHIMI A.A.. SHIGELLA DYSENTERY STXA MUTANT (R170L-A231D-G234E) GENE DESIGN AND OPTIMIZATION OF RECOMBINANT PROTEIN EXPRESSION AND PURIFICATION. JOURNAL OF SHAHREKORD UNIVERSITY OF MEDICAL SCIENCES[Internet]. 2012;13(5):93-102. Available from: https://sid.ir/paper/58605/en

IEEE: Copy

GH.R. OLAD, M. TAVALAE, Z. MOHAMMAD HASSAN, F. EBRAHIMI, J. SALIMIAN, SH. NAZAREAN, and A.A. RAHIMI, “SHIGELLA DYSENTERY STXA MUTANT (R170L-A231D-G234E) GENE DESIGN AND OPTIMIZATION OF RECOMBINANT PROTEIN EXPRESSION AND PURIFICATION,” JOURNAL OF SHAHREKORD UNIVERSITY OF MEDICAL SCIENCES, vol. 13, no. 5, pp. 93–102, 2012, [Online]. Available: https://sid.ir/paper/58605/en

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