A single point mutation within the coding sequence of cholera toxin B subunit will increase its expression yield.
Cholera 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.



ctxB 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.



SDS-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.



Point 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.
Dept. of Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
Bita
B
Bakhshi
Dept. of Biology, Faculty of Basic Science, Science and Research Branch, Islamic Azad University, Tehran, Iran.
Mina
M
Boustanshenas
Research and Production Complex, Pasteur Institute of Iran, Tehran, Iran.
Masoud
M
Ghorbani
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002772:Cholera Toxin / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D050505:Mutant Proteins / Q000378:metabolism; D016366:Open Reading Frames / Q000235:genetics*; D017354:Point Mutation / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism
D050505:Mutant Proteins; D011994:Recombinant Proteins; D004247:DNA; D002772:Cholera Toxin