Identification of functional and structural amino-acid residues by parsimonious mutagenesis.
For in vitro evolution of protein function, we previously proposed using parsimonious mutagenesis (PM), a technique where mutagenic oligodeoxynucleotides (oligo) are designed to minimize coding sequence redundancy and limit the number of amino acid (aa) residues which do not retain parental structural features. For this work, PM was used to increase 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 created where 19 aa located in three of the heavy (H) and light (L) chain antigen-binding loops (L1, L3 and H2) were simultaneously mutated. After four rounds of selection, 50% of scFv had a lower dissociation rate constant (koff) than the parental scFv. The Kd of these scFv ranged from twofold (Kd=7.0 x 10(-9) M) to sixfold (Kd=2.4 x 10(-9) M) lower than the parental scFv (Kd=1.6 x 10(-8) M). In higher affinity scFv, substitutions occurred at 10/19 of the positions, with 21/28 substitutions occurring at only four positions, two in H2, and one each 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 structural role in the variable domain, either in maintaining the main chain conformation of the loop, or in packing on the H-chain variable domain. Two of the conserved aa are solvent exposed, suggesting they may play a critical role in recognition. Thus, PM identified three types of aa: structural aa, functional aa which modulate affinity, and functional aa, which are critical for recognition. Since the sequence space was not completely sampled, higher affinity scFv could be produced by subjecting functional aa which modulate affinity to a higher rate of mutation. Furthermore, PM could prove useful for modifying function in other proteins that belong to structurally related families.
Schier
R
R
Department of Anesthesia, University of California, San Francisco General Hospital 94110, USA.
Balint
R F
RF
McCall
A
A
Apell
G
G
Larrick
J W
JW
Marks
J D
JD
D000595:Amino Acid Sequence; D000596:Amino Acids; D000915:Antibody Affinity; D001435:Bacteriophages; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D017931:DNA Primers; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics* / Q000276:immunology*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology*; D013329:Structure-Activity Relationship
D000596:Amino Acids; D017931:DNA Primers; D007128:Immunoglobulin Fragments; D018719:Erb-b2 Receptor Tyrosine Kinases; C055966:immunoglobulin Fv