A combinatorial library of an α-helical bacterial receptor domain

Abstract

The construction and characterization of a combinatorial library of a solvent-exposed surface of an alpha-helical domain derived from a bacterial receptor is described. Using a novel solid-phase approach, the library was assembled in a directed and successive manner utilizing single-stranded oligonucleotides containing multiple random substitutions for the variegated segments of the gene fragment. The simultaneous substitution of 13 residues to all 20 possible amino acids was carried out 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 PCR-amplified library into a phagemid vector adapted for phage display of the mutants, DNA sequencing analysis suggested a random distribution of codons in the mutagenized positions. Four members of the library with multiple substitutions were produced in Escherichia coli as fusions to an albumin-binding affinity tag derived from streptococcal protein 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-length proteins. Furthermore, the CD analysis showed that all mutants, except one with a proline introduced into helix 2, have secondary structures in close agreement with the wild-type domain. These results proved that members of this alpha-helical receptor library with multiple substitutions in the solvent-exposed surface remain stable and soluble in E. coli.(ABSTRACT TRUNCATED AT 250 WORDS)

Surface display on staphylococci: a comparative study

“…Also nonimmunoglobulin scaffolds could potentially be utilized for the presentation of randomized segments, surface displayed on Gram-positive bacteria. Particularly interesting would be combinatorial libraries based on receptor derivatives from Gram-positive bacteria such as the one described by Nord and coworkers [48], utilizing a domain from staphylococcal protein A as scaffold, which thus should be suitable for display on staphylococcal surfaces.…”

Affibody molecules: Engineered proteins for therapeutic, diagnostic and biotechnological applications

“…However, it was relatively early envisioned that the favorable properties of domain Z should be advantageous in an even broader variety of applications, resulting in the concept of engineering new so-called Affibody molecules based on the Zdomain scaffold with specific binding for theoretically any given target [6]. In contrast to monoclonal antibodies that may be generated by immunization of laboratory animals combined with hybridoma technology, isolation of new affinity proteins based on non-immunoglobulin scaffolds is performed using synthetic combinatorial libraries and in vitro selection systems (e.g.…”

Tumor cell targeted gene delivery by adenovirus 5 vectors carrying knobless fibers with antibody-binding domains

“…13,14 Affibodies are derived from the Z domain (here abbreviated Z wt , for Z wild type (WT)), which is a cystein-free, 58-amino-acid sequence arranged in a three-helix bundle structure, derived from domain B of Staphylococcal protein A (SpA). 15,16 In the present study, we describe two types of Ig-binding Ad recombinants, in which one carried two Z wt motifs in tandem in place of the knob and is known as Ad5/R7-Z wt -Z wt . 13 The other one, called Ad5/R7-C2-C2, carried fibers containing the C2 domain from Streptococcal protein G. Protein G binds to a broader range of immunoglobulins (Igs) compared to protein A for example, protein G is capable of binding strongly to human IgG3 and mouse IgG1, both of which bind weakly to protein A.…”