Phage display libraries are widely used as tools for identifying, dissecting and optimizing ligands. Development of a simple method to access greater library diversities could expedite and expand the technique. This paper reports progress toward harnessing the naturally occurring diversity generating retroelement used by Bordetella bronchiseptica bacteriophage to alter its tail-fiber protein. Mutagenesis and testing identified four sites amenable to the insertion of <19-residue heterologous peptides within the variable region. Such sites allow auto-generation of peptide libraries surrounded by a scaffold with additional variations. The resultant self-made phage libraries were used successfully for selections targeting anti-FLAG antibody, immobilized metal affinity chromatography microtiter plates and HIV-1 gp41. The reported experiments demonstrate the utility of the major tropism determinant protein of B.bronchiseptica as a natural scaffold for diverse, phage-constructed libraries with heterologous self-made phage libraries.

Recombinant protein overexpression of large proteins in bacteria often results in insoluble and misfolded proteins directed to inclusion bodies. We report the application of shear stress in micrometer-wide, thin fluid films to refold boiled hen egg white lysozyme, recombinant hen egg white lysozyme, and recombinant caveolin-1. Furthermore, the approach allowed refolding of a much larger protein, cAMP-dependent protein kinase A (PKA). The reported methods require only minutes, which is more than 100 times faster than conventional overnight dialysis. This rapid refolding technique could significantly shorten times, lower costs, and reduce waste streams associated with protein expression for a wide range of industrial and research applications.