title: Substitution of surface-exposed framework residues alters secretion of recombinant fusion protein Fv/tumor necrosis factor in Escherichia coli.
abstract: Substitution of hydrophobic residues with hydrophilic ones at surface-exposed positions may influence the yield of antibody fragment expression in Escherichia coli by reducing its aggregation potential. We introduced such substitutions at 8 surface-exposed framework region residues of a fusion protein Fv/Tumor Necrosis Factor, which resulted in the expression of 10 mutant fusion proteins (Mut 1-10) in E. coli. Our results showed that expression levels of Mut 1-3, with mutations of A9S, T18K, and G41D, respectively, decreased by 4- to 8-fold, whereas expression levels of Mut 4, 9, and 10, with mutations of S60D/S70D, T28D, and G65D, respectively, were not affected. In contrast, mutation of F83A at light-chain residue 83, which is usually buried at the variable/constant domain interface of antibody molecules but becomes surface-exposed in recombinant Fv molecules, increased the expression level of Mut 5 by 4-fold. Our results suggest that this important substitution of a hydrophobic residue with a hydrophilic one may also be applied to increase the secretion of other recombinant Fv molecules in E. coli periplasm.
authors.affiliation: Saskatoon Cancer Center, Department of Microbiology, University of Saskatchewan, Canada.
authors.forename: J
authors.initials: J
authors.lastname: Yang
authors.forename: Z
authors.initials: Z
authors.lastname: Jia
authors.forename: J
authors.initials: J
authors.lastname: Xiang
mesh_terms: D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D007128:Immunoglobulin Fragments / Q000235:genetics* / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics* / Q000378:metabolism
chemical_list: D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; C055966:immunoglobulin Fv