title: Cloning, expression and characterization of the CHO cell elongating factor (Cef) from Vibrio cholerae O1.
abstract: CHO cell-elongating factor (Cef) is a recently identified putative virulence factor of Vibrio cholerae. Our previous studies show that this 85 kDa protein elongates CHO cells, causes fluid accumulation in suckling mice and has esterase activity. In this study, the cef gene was cloned in Escherichia coli using a yeast vector and subsequently expressed in the yeast Pichia pastoris. The cef genes from V. cholerae candidate vaccine strains JBK 70 and CVD 103-HgR were sequenced and found to be nearly identical (100 and 99.9% respectively) with an open reading frame (ORF) from the published sequence of V. cholerae N16961. Cloned toxin was purified to homogeneity in 3 steps using anion exchange, hydrophobic interaction and gel filtration chromatography. The size of cloned Cef on SDS-PAGE gels was 114 kDa. The increased size was probably due to glycosylation by the yeast since cloned protein reacted strongly with a glycoprotein stain. The cloned protein could not be directly sequenced, but when treated with trypsin, yielded a protein fragment with an amino acid sequence that matched the sequence predicted for the Cef protein. The purified cloned protein had esterase and CHO cell activity, but no suckling mouse activity.
authors.affiliation: Division of Virulence Assessment, FDA, Washington DC, MD, USA. bmccarde@cfsan.fda.gov
authors.forename: B A
authors.initials: BA
authors.lastname: McCardell
authors.forename: V
authors.initials: V
authors.lastname: Sathyamoorthy
authors.forename: J
authors.initials: J
authors.lastname: Michalski
authors.forename: S
authors.initials: S
authors.lastname: Lavu
authors.forename: M
authors.initials: M
authors.lastname: Kothary
authors.forename: J
authors.initials: J
authors.lastname: Livezey
authors.forename: J B
authors.initials: JB
authors.lastname: Kaper
authors.forename: R
authors.initials: R
authors.lastname: Hall
mesh_terms: D000818:Animals; D000833:Animals, Suckling; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D004926:Escherichia coli / Q000235:genetics; D006031:Glycosylation; D007526:Isoelectric Point; D051379:Mice; D008813:Mice, Inbred ICR; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020539:Sequence Analysis, Protein; D014734:Vibrio cholerae / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity*; D014774:Virulence / Q000235:genetics
chemical_list: D001427:Bacterial Toxins; D011994:Recombinant Proteins; C412287:cell-elongating factor, Vibrio cholerae