Biotin-Assisted Folding of Streptavidin on the Yeast Surface Kok Hong Lim Dept. of Chemical and Biological Engineering, University at Buffalo, The State University of New York, Buffalo, NY 14260 Inseong Hwang Dept. of Chemistry, Seoul National University, Seoul 151-742, Korea Sheldon Park Dept. of Chemical and Biological Engineering, University at Buffalo, The State University of New York, Buffalo, NY 14260 DOI 10.1002/btpr.721 Published online October 11, 2011 in Wiley Online Library (wileyonlinelibrary.com). Yeast surface display allows heterologously expressed proteins to be targeted to the exte- rior 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 biotinyl- ated 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 bacte- ria. 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 sub- units is not necessary, as expressing the anchored subunit alone is sufficient to form a func- tional 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 immu- noprecipitate a protein target. V V C 2011 American Institute of Chemical Engineers Biotechnol. Prog., 28: 276–283, 2012 Keywords: yeast display, streptavidin, protein folding, biotin, molecular chaperone Introduction Yeast surface display is a versatile tool for engineering pro- tein interactions and stability. The advantages of yeast display over other display platforms, such as bacterial display or phage display, have been described 1,2 and include the eukary- otic translational apparatus that is capable of folding proteins with a complex topology and a quality control system that preferentially displays well-folded proteins on the surface. 3 A variety of proteins of biomedical and biotechnological interest have been displayed on the yeast surface, including immune proteins, 4–8 fluorescent proteins, 9 engineered scaffolds, 10,11 and enzymes. 12–15 As proteins are anchored using a long flexi- ble loop, yeast display is useful for immobilizing heterologous proteins on a solid matrix with minimal structural perturba- tion. In contrast, nonspecific adsorption of proteins on a hydro- phobic surface, sometimes used during phage display, can easily result in protein denaturation. 16,17 The technique is also simpler than performing chemical modifications on the protein for subsequent crosslinking on a solid matrix. For the applica- tions, in which the host yeast is simultaneously used for its metabolic properties—for example, ethanol production— anchoring related enzymes, such as cellulosome, on the yeast surface may also help optimize the yield from the engineered chemical process. 18,19 In this study, we investigated the use of the Aga2 system to display functional streptavidin tetramer on the yeast sur- face. We selected streptavidin for investigation because the molecule binds biotin with high affinity (K d 10 14 M) 20 and is used widely in various applications that require stable noncovalent interaction. Streptavidin is also stable (T m ¼ 75  C) and can tolerate extreme buffer conditions containing high concentrations of chaotropic salts and organic sol- vents. 21,22 The molecule is commonly used to immobilize bi- otinylated compounds by crosslinking them to a biotinylated solid surface. A recent study that uses avidinated yeast sur- face to capture biotinylated protein ligands secreted by the same yeast 23 further highlights the potential benefits of dis- playing streptavidin on the yeast surface. As such, the ability to display functional streptavidin on the yeast surface is likely to lead to other novel applications based on the well- known protein–ligand interaction. Most yeast display studies reported to date have focused on engineering single chain proteins. Single chain constructs provide a straightforward mechanism to immobilize the expressed proteins by fusing them to a yeast protein. As Additional Supporting Information may be found in the online ver- sion of this article. Correspondence concerning this article should be addressed to S. Park at sjpark6@buffalo.edu. 276 V V C 2011 American Institute of Chemical Engineers