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## Background: We have cloned an anti-TSH antibody with high affinity and good specificity to produce recombinant antibody fragments for immunoassay development. Unfortunately, expression of the antibody Fab fragment in bacteria was poor limiting the production of Fab for purposes of assay development. In this study, we have applied consensus based mutagenesis approach to improve the expression properties of this Fab fragment. Methods: The variable heavy and light chain regions encoding genes of the anti-TSH antibody were compared to the consensus sequence of the corresponding mouse germline V-gene families. Five amino acid differences were identified outside complementary-determining-regions in the heavy chain V-region, and the corresponding mutations (named A=Arg18Leu, B=Thr23Ala, C=Pro75Ala, D=Phe80Tyr and E=Thr84Ser) were individually introduced in the parental antibody Fab fragment gene by using site-directed mutagenesis. The effect of individual mutations on expression properties was studied by using immunoassay. Beneficial mutations were combined and the combinatorial clones were characterized for the yield in bacterial expression system. Results: Four single mutants (A, C,

Who reads Miscellaneous?

Author

PJ

Publisher

Walter de Gruyter GmbH

Published

2009

Field

Medicine (Health Sciences)