N-terminal mutations in the anti-estradiol Fab 57-2 modify its hapten binding properties

Recombinant antibodies often contain N-terminal mutations arising from the use of degenerate cloning primer sets and/or the introduction of restrictio n sites in the framework 1 regions. We studied the effects of such mutation s in a recombinant anti-estradiol Fab fragment derived from the hybridoma c ell line 57-2. The 5' ends of the heavy and light chain genes were original ly modified to introduce the restriction sites XhoI and SacI, respectively, for cloning purposes. However. the affinity and specificity of the recombi nant Fab were lowered compared to the proteolytic Fab' fragment of the pare ntal hybridoma IgG. Replacing the mutated sites with authentic amino acid c oding sequences restored the binding properties as well as increased the ba cterial production levels fivefold and 10-fold at 30 and 37 degreesC, respe ctively. Local changes in the antigen binding site were probed by determini ng the affinity constants (K-a) for estradiol and four related steroids. It was found that the mutated heavy chain amino terminus specifically increas ed the K-a for testosterone whereas the mutated light chain amino terminus decreased the K-a 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 V-H, and 2 and 4 in the V-L domain were thos e responsible for the observed effects, Their general roles as structure-de termining residues for the CDR3 loops imply that similar effects can occur with other recombinant antibodies as well.