Screening Libraries of Circularly Permuted Proteins by Phage Display to Manipulate Protein Topographies

Abstract

Active biomolecules, particularly proteins, are increasingly used in functional nanostructures. For some applications, native proteins (i.e. proteins as they occur in nature) are not effective nanodevice components because distances between the ends of the protein and some protein functional domain exceeds some critical nanoscale distance. This problem occurs in sensing architectures that detect energy or electron transfer where native protein sizes exceed critical distances for quantum energy transfer or exceed the thickness of shielding layers of counter ions, and might be addressed by alterations of protein topography. This work describes a high-throughput recombinant DNA method rapidly to identify functional circularly permuted (cp) protein variants. When appended to a sensing device or other nanometer scale distance-dependent device using chemoselective conjugation methods, cp variants offer a potential strategy to address the nanometer size limitations of native proteins.

Isolation of scFv fragments specific for monokine induced by interferon-gamma (MIG) using phage display

“…The results obtained showed that the relative eluate yield/output of binding phage particles was increased approximately 1000-fold and 100-fold from the first to the fourth round of affinity selection for libraries J (4 × 10 6 – 5 × 10 9 ) and I (1.1 × 10 6 – 3.5 × 10 8 ), respectively. During successful biopanning experiments, phage yields increase from one round to another round of selection enrichment (Eteshola et al, 2005; Eteshola et al, 2006). The results clearly indicates that isolation and enrichment of MIG binding phages was accomplished.…”

Engineering functional protein interfaces for immunologically modified field effect transistor (ImmunoFET) by molecular genetic means

“…Firstly, we isolated affinity peptides recognizing thermally grown silica (Eteshola et al 2005) that can be inserted at various positions within receptor sequences as protein fusions to derive receptors that bind silica in specific orientation determined by the position of peptide insertion. Secondly, we developed a high-throughput method to modify the topography of receptor proteins (scanning circular permutagenesis; Eteshola et al 2006). When used in conjunction with a chemoselective conjugation method to deploy proteins on surfaces, different permuted variants of a single receptor hold analyte at different distances from the sensing surface.…”

“…In a second approach, we apply a technology we developed (scanning circular permutation or SCP of proteins; Eteshola et al 2006) in combination with chemoselective conjugation. The method allows alteration of protein topography so as to manipulate the position of the ends of the protein and any point on the protein surface (such as the antigen-combining site).…”

“…The peptide and naive scFv libraries were subjected to affinity selection on the thermally oxidized silicon wafers or a biotinylated chemokine (protein) analyte, respectively. Affinity selection methods were a combination of those recommended by the supplier and methods developed in our laboratory (Eteshola et al 2005(Eteshola et al , 2006.…”