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 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 degrees C, respectively. Local changes in the antigen binding site were probed by determining the affinity constants (Kd) for estradiol and four related steroids. It was found that the mutated heavy chain amino terminus specifically increased the Kd for testosterone whereas the mutated light chain amino terminus decreased the Kd 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 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.
Saviranta PJauria
P
P
Department of Biotechnology, University of Turku, Finland. petri.saviranta@utu.fi
Lamminmäki
U
U
Hellman
J
J
Eriksson
S
S
Lövgren
T
T
D019943:Amino Acid Substitution; D000915:Antibody Affinity / Q000235:genetics; D000918:Antibody Specificity / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D022801:Complementarity Determining Regions / Q000378:metabolism; D004958:Estradiol / Q000276:immunology* / Q000378:metabolism; D006241:Haptens / Q000378:metabolism*; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013256:Steroids / Q000276:immunology / Q000378:metabolism
D022801:Complementarity Determining Regions; D006241:Haptens; D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins; D013256:Steroids; D004958:Estradiol