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Substitution of Surface‐Exposed Framework Residues Alters Secretion of Recombinant Fusion Protein Fv/Tumor Necrosis Factor in Escherichia coli
Author(s) -
Yang Junbao,
Jia Zongchao,
Xiang Jim
Publication year - 1999
Publication title -
iubmb life
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.132
H-Index - 113
eISSN - 1521-6551
pISSN - 1521-6543
DOI - 10.1080/713803525
Subject(s) - escherichia coli , recombinant dna , fusion protein , mutant , chemistry , microbiology and biotechnology , protein engineering , biology , biochemistry , gene , enzyme
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.