Substitution of surface-exposed framework residues alters secretion of recombinant fusion protein Fv/Tumor Necrosis Factor in Escherichia coli

Substitution of hydrophobic residues with hydrophilic ones at surface-expos ed positions mag influence the yield of antibody fragment expression in Esc herichia coli by reducing its aggregation potential, We introduced such sub stitutions at 8 surface-exposed framework region residues of a fusion prote in 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 l evels of Mut 1-3, with mutations of A9S, T18K, and G41D, respectively, decr eased 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 bur ied at the variable/constant domain interface of antibody molecules but bec omes surface-exposed in recombinant Fv molecules, increased the expression level of Mut 5 by 4-fold, Our results suggest that this important substitut ion of a hydrophobic residue with a hydrophilic one may also he applied to increase the secretion of other recombinant Fv molecules in E, coli peripla sm.