Synthesis of cholera toxin B subunit gene: cloning and expression of a functional 6XHis-tagged protein in Escherichia coli.
Cholera toxin B subunit (CTB) has been extensively studied as immunogen, adjuvant, and oral tolerance inductor depending on the antigen conjugated or coadministered. It has been already expressed in several bacterial and yeast systems. In this study, we synthesized a versatile gene coding a 6XHis-tagged CTB (359bp). The sequence was designed according to codon usage of Escherichia coli, Lactobacillus casei, and Salmonella typhimurium. The gene assembly was based on a polymerase chain reaction, in which the polymerase extends DNA fragments from a pool of overlapping oligonucleotides. The synthetic gene was amplified, cloned, and expressed in E. coli in an insoluble form, reaching levels about 13 mg of purified active pentameric rCTB per liter of induced culture. Western blot and ELISA analyses showed that recombinant CTB is strongly and specifically recognized by polyclonal antibodies against the cholera toxin. The ability to form the functional pentamers was observed in cell culture by the inhibition of cholera toxin activity on Y1 adrenal cells in the presence of recombinant CTB. The 6XHis-tagged CTB provides a simple way to obtain functional CTB through Ni(2+)-charged resin after refolding and also free of possible CTA contaminants as in the case of CTB obtained from Vibrio cholerae cultures.
Arêas
Ana Paula de Mattos
AP
Centro de Biotecnologia, Instituto Butantan, Av. Vital Brasil 1500, 05503-900 São Paulo, SP, Brazil.
Oliveira
Maria Leonor Sarno de
ML
Ramos
Celso Raul Romero
CR
Sbrogio-Almeida
Maria Elisabete
ME
Raw
Isaías
I
Ho
Paulo Lee
PL
D000311:Adrenal Glands / Q000166:cytology; D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D002772:Cholera Toxin / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D003062:Codon / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial / Q000235:genetics*; D005822:Genetic Vectors; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D014734:Vibrio cholerae / Q000737:chemistry
D003062:Codon; D004274:DNA, Recombinant; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D006639:Histidine; D002772:Cholera Toxin