A COMBINATORIAL LIBRARY OF AN ALPHA-HELICAL BACTERIAL RECEPTOR DOMAIN

The construction and characterization of a combinatorial library of a solvent-exposed surface of an alpha-helical domain derived from a bact erial receptor is described. Using a novel solid-phase approach, the l ibrary was assembled in a directed and successive manner utilizing sin gle-stranded oligonucleotides containing multiple random substitutions for the variegated segments of the gene fragment. The simultaneous su bstitution of 13 residues to all 20 possible amino acids was carried o ut in a region spanning 81 nucleotides. The randomization was made in codons for amino acids that were modelled to be solvent accessible at a surface made up from two of the three alpha-helices of a monovalent Fc-binding domain of staphylococcal protein A. After cloning of the PC R-amplified library into a phagemid vector adapted for phage display o f the mutants, DNA sequencing analysis suggested a random distribution of codons in the mutagenized positions. Four members of the library w ith multiple substitutions were produced in Escherichia coli as fusion s to an albumin-binding affinity tag derived from streptococcal protei n G. The fusion proteins were purified by human serum albumin affinity chromatography and subsequently characterized by SDS-electrophoresis, CD spectroscopy and biosensor analysis. The analyses showed that the mutant protein A derivatives could all be secreted as soluble full-len gth proteins. Furthermore, the CD analysis showed that all mutants, ex cept one with a proline introduced into helix 2, have secondary struct ures in close agreement with the wild-type domain. These results prove d that members of this alpha-helical receptor library with multiple su bstitutions in the solvent-exposed surface remain stable and soluble i n E.coli. The possibility of using this library for a phenotypic selec tion strategy to obtain artificial antibodies with novel functions is discussed.