Recombinant antibodies often contain N-terminal mutations
arising from the use of degenerate cloning primer sets and/or
the introduction of restriction sites in the framework 1 regions.
We studied the effects of such mutations in a recombinant
anti-estradiol Fab fragment derived from the hybridoma cell line
57-2. The 5′ ends of the heavy and light chain genes were
originally modified to introduce the restriction sites XhoI
and SacI, respectively, for cloning purposes. However, the
affinity and specificity of the recombinant Fab were lowered compared
to the proteolytic Fab′ fragment of the parental hybridoma IgG.
Replacing the mutated sites with authentic amino acid coding sequences
restored the binding properties as well as increased the bacterial
production levels fivefold and 10-fold at 30 and 37 °C, respectively.
Local changes in the antigen binding site were probed by determining the
affinity constants (Ka) for estradiol and four
related steroids. It was found that the mutated heavy chain amino
terminus specifically increased the Ka for
testosterone whereas the mutated light chain amino terminus decreased
the Ka for all of the steroids to the same
extent; the heavy and light chain effects were additive. Analysis of a newly
determined crystal structure of the authentic Fab 57-2 in complex with
estradiol suggests that mutations in the residue 2 in VH,
and 2 and 4 in the VL domain were those responsible
for the observed effects. Their general roles as structure-determining
residues for the CDR3 loops imply that similar effects can occur with other
recombinant antibodies as well.