title: Biotin-assisted folding of streptavidin on the yeast surface.
abstract: Yeast surface display allows heterologously expressed proteins to be targeted to the exterior of the cell wall and thus has a potential as a biotechnology platform. In this study, we report the successful display of functional streptavidin on the yeast surface. Streptavidin binds the small molecule biotin with high affinity (K(d) ≈ 10(-14)M) and is used widely in applications that require stable noncovalent interaction, including immobilization of biotinylated compounds on a solid surface. As such, engineering functional streptavidin on the yeast surface may find novel uses in future biotechnology applications. Although the molecule does not require any post-translational modification, streptavidin is difficult to fold in bacteria. We show that Saccharomyces cerevisiae can fold the protein correctly if induced at 20°C. Contrary to a previous report, coexpression of anchored and soluble streptavidin subunits is not necessary, as expressing the anchored subunit alone is sufficient to form a functional complex. For unstable monomer mutants, however, addition of free biotin during protein induction is necessary to display a functional molecule, suggesting that biotin helps the monomer fold. To show that surface displayed streptavidin can be used to immobilize other biomolecules, we used it to capture biotinylated antibody, which is then used to immunoprecipitate a protein target.
authors: Department of Chemical and Biological Engineering, University at Buffalo, The State University of New York, Buffalo, NY 14260, USA.
authors: Kok Hong
authors: KH
authors: Lim
authors: Inseong
authors: I
authors: Hwang
authors: Sheldon
authors: S
authors: Park
mesh_terms: D000818:Animals; D001709:Biotechnology; D001710:Biotin / Q000378:metabolism*; D019921:Biotinylation / Q000379:methods; D005434:Flow Cytometry; D047468:Immunoprecipitation; D007700:Kinetics; D051379:Mice; D018832:Molecular Chaperones / Q000378:metabolism; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D019809:Streptavidin / Q000737:chemistry*
chemical_list: D018832:Molecular Chaperones; D001710:Biotin; D019809:Streptavidin