IDENTIFICATION OF FUNCTIONAL AND STRUCTURAL AMINO-ACID-RESIDUES BY PARSIMONIOUS MUTAGENESIS

For in vitro evolution of protein function, we previously proposed usi ng parsimonious mutagenesis (PM), a technique where mutagenic oligodeo xynucleotides (oligo) are designed to minimize coding sequence redunda ncy and limit the number of amino acid (aa) residues which do not reta in parental structural features. For this work, PM was used to increas e the affinity of C6.5, a human single-chain Fv (scFv) that binds the glycoprotein tumor antigen, c-erbB-2. A phage antibody library was cre ated where 19 aa located in three of the heavy (H) and light (L) chain antigen-binding loops (L1, L3 and H2) were simultaneously mutated. Af ter four rounds of selection, 50% of scFv had a lower dissociation rat e constant (k(off)) than the parental scFv. The K-d of these scFv rang ed from twofold (K-d = 7.0 x 10(-9) M) to sixfold (K-d = 2.4 x 10(-9) M) lower than the parental scFv (K-d = 1.6 x 10(-8) M). In higher affi nity scFv, substitutions occurred at 10/19 of the positions, with 21/2 8 substitutions occurring at only four positions, two in H2, and one e ach in L1 and L3. Only the wild type (wt) aa was observed at 9/19 aa, Based on a model of C6.5, seven of the nine conserved aa have a struct ural role in the variable domain, either in maintaining the main chain conformation of the loop, or in packing on the H-chain variable domai n. Two of the conserved aa are solvent exposed, suggesting they may pl ay a critical role in recognition. Thus, PM identified three types of aa, structural aa, functional aa which modulate affinity, and function al aa, which are critical for recognition. Since the sequence space wa s not completely sampled, higher affinity scFv could be produced by su bjecting functional aa which modulate affinity to a higher rate of mut ation. Furthermore, PM could prove useful for modifying function in ot her proteins that belong to structurally related families.