ArticleIranian biomedical journal2014
A single point mutation within the coding sequence of cholera toxin B subunit will increase its expression yield.
Article in Iranian biomedical journal, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 5 papers.
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5 citing papers in PubMed.
- Article
- Expression and Functional Study of Single Mutations of Carbonic Anhydrase 8 in Neuronal Cells.Cellular and molecular neurobiology · 2021Article
- Position of human blood group O(H) and phenotype-determining enzymes in growth and infectious disease.Annals of the New York Academy of Sciences · 2018Review
- Cumulative protective efficacy of rZot and rAce combination in challenge experiments with wild type Vibrio cholerae in mouse model.Human vaccines & immunotherapeutics · 2018Article
- Review
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3 authors.
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Abstract
backgroundCholera toxin B subunit (CTB) has been extensively considered as an immunogenic and adjuvant protein, but its yield of expression is not satisfactory in many studies. The aim of this study was to compare the expression of native and mutant recombinant CTB (rCTB) in pQE vector.
methodsctxB fragment from Vibrio cholerae O1 ATCC14035 containing the substitution of mutant ctxB for amino acid S128T was amplified by PCR and cloned in pGETM-T easy vector. It was then transformed to E. coli Top 10F' and cultured on LB agar plate containing ampicillin. Sequence analysis confirmed the mature ctxB gene sequence and the mutant one in both constructs which were further subcloned to pQE-30 vector. Both constructs were subsequently transformed to E. coli M15 (pREP4) for expression of mature and mutant rCTB.
resultsSDS-PAGE analysis showed the maximum expression of rCTB in both systems at 5 hours after induction and Western-blot analysis confirmed the presence of rCTB in blotting membranes. The expression of mutant rCTB was much higher than mature rCTB, which may be the result of serine-to-threonine substitution at position 128 of mature rCTB amino acid sequence created by PCR mutagenesis. The mutant rCTB retained pentameric stability and its ability to bind to anti- cholera toxin IgG antibodies.
conclusionPoint mutation in ctxB sequence resulted in over-expression of rCTB, probably due to the increase of solubility of produced rCTB. Consequently, this expression system can be used to produce rCTB in high yield.
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