Selection for mutants improving expression of an anti-MAP kinase monoclonal antibody by filamentous phage display.
It has recently been reported that single amino acid residues can strongly influence the expression of recombinant antibody fragments in Escherichia coli. Prediction of these critical positions can be difficult even with prior knowledge of the primary sequence and the three-dimensional folded structure of the antibody. To circumvent this, a Fab phage display library containing random point mutations was generated from a hybridoma specific for activated p44/p42 mitogen-activated protein (MAP) kinases. Clones that express Fab were selected by panning against the target antigen. It was found that a cysteine-to-serine substitution at position 91 in the CDR3 of the light chain was responsible for allowing expression of Fab. Site-directed mutagenesis was performed to effect this substitution and others at cysteine 91 on a nonexpressing clone. Mutants containing serine, glycine or alanine at position 91 expressed Fab and bound to target antigen. In contrast, tyrosine mutants had moderate Fab expression but no detectable binding to antigen. These results demonstrate that by using phage display, one can select for the expression of antibody fragments while retaining biological activity.
Tuckey
Corinna D
CD
New England Biolabs, 32 Tozer Rd, Beverly, MA 01915, USA.
Noren
Christopher J
CJ
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000235:genetics*; D015870:Gene Expression; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis / Q000235:genetics*; D017912:Inovirus; D051379:Mice; D008807:Mice, Inbred BALB C; D019950:Mitogen-Activated Protein Kinase 1 / Q000276:immunology*; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000276:immunology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019151:Peptide Library
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D019151:Peptide Library; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases