Yeast polypeptide fusion surface display levels predict thermal stability and soluble secretion efficiency

Journal of Molecular Biology
Eric V ShustaK D Wittrup

Abstract

Efficiency of yeast cell surface display can serve as a proxy screening variable for enhanced thermal stability and soluble secretion efficiency of mutant proteins. Several single-chain T cell receptor (scTCR) single-site mutants that enable yeast surface display, along with their double and triple mutant combinations, were analyzed for soluble secretion from the yeast Saccharomyces cerevisiae. While secretion of the wild-type scTCR was not detected, each of the single, double, and triple mutants were produced in yeast supernatants, with increased expression resulting from the double and triple mutants. Soluble secretion levels were strongly correlated with the quantity of active scTCR displayed as a fusion to Aga2p on the surface of yeast. Thermal stability of the scTCR mutants correlated directly with the secreted and surface levels of scTCR, with evidence suggesting that intracellular proteolysis by the endoplasmic reticulum quality control apparatus dictates display efficiency. Thus, yeast display is a directed evolution scaffold that can be used for the identification of mutant eucaryotic proteins with significantly enhanced stability and secretion properties.

Citations

Jan 31, 2012·Biochimica Et Biophysica Acta·Michael W TraxlmayrChristian Obinger
Apr 5, 2007·Nature Protocols·Ginger ChaoK Dane Wittrup
Sep 25, 2008·Proceedings of the National Academy of Sciences of the United States of America·Casim A SarkarAndreas Plückthun
Jul 27, 2011·Proceedings of the National Academy of Sciences of the United States of America·Douglas S JonesJennifer R Cochran
Jul 25, 2009·The Journal of Biological Chemistry·Pete HeinzelmanFrances H Arnold
Feb 16, 2012·Nucleic Acids Research·Kyle JacobyAndrew M Scharenberg
Apr 15, 2009·Protein Engineering, Design & Selection : PEDS·Csaba KissAndrew R M Bradbury
Dec 17, 2010·Protein Engineering, Design & Selection : PEDS·David H AggenDavid M Kranz
Mar 16, 2011·Protein Engineering, Design & Selection : PEDS·J A RakestrawD Lipovsek
Jul 7, 2012·Protein Engineering, Design & Selection : PEDS·Tej V PavoorEric V Shusta
Sep 4, 2012·Protein Engineering, Design & Selection : PEDS·Andrew BuchananRalph Minter
Dec 26, 2006·Applied and Environmental Microbiology·Alane E Wentz, Eric V Shusta
Feb 7, 2003·Applied and Environmental Microbiology·Thomas BulterFrances H Arnold
May 7, 2004·Applied and Environmental Microbiology·Tomoko YoshinoTadashi Matsunaga
Jan 5, 2006·Applied and Environmental Microbiology·Tomoko Yoshino, Tadashi Matsunaga
Mar 29, 2013·PloS One·Alexei KharitonenkovDavid E Moller
Dec 21, 2005·Proceedings of the National Academy of Sciences of the United States of America·K Scott WeberDavid M Kranz
Dec 16, 2004·Proceedings of the National Academy of Sciences of the United States of America·David W ColbyK Dane Wittrup
May 9, 2002·Proceedings of the National Academy of Sciences of the United States of America·Yoshihisa Hagihara, Peter S Kim
Apr 26, 2000·Proceedings of the National Academy of Sciences of the United States of America·P D HollerD M Kranz
Sep 17, 2014·Protein Engineering, Design & Selection : PEDS·Dujduan Waraho-ZhmayevMatthew P DeLisa
May 5, 2007·Expert Opinion on Biological Therapy·Itai Benhar
Sep 20, 2015·Protein Engineering, Design & Selection : PEDS·Mark C JulianPeter M Tessier
Sep 4, 2015·Protein Engineering, Design & Selection : PEDS·James A Van DeventerSachdev S Sidhu
Sep 20, 2015·Protein Engineering, Design & Selection : PEDS·Brandon W HeimerHadley D Sikes
Jan 1, 2010·Journal of Zhejiang University. Science. B·Qin GuoGuo-qing He
Mar 12, 2011·MAbs·Jonathan Fitzgerald, Alexey Lugovskoy
Jan 1, 2011·Annual Review of Chemical and Biomolecular Engineering·Eric T Boder, Wei Jiang
Apr 12, 2005·Cell·Yoshiki SekijimaJeffery W Kelly
Sep 21, 2005·Journal of Molecular Biology·Rebecca A BuonpaneDavid M Kranz
Mar 28, 2006·Protein Expression and Purification·Andrea PiatesiK Dane Wittrup
Oct 4, 2005·Protein Science : a Publication of the Protein Society·Sanjib DuttaShohei Koide
Apr 6, 2007·Biomolecular Engineering·Sarah A Richman, David M Kranz
Feb 1, 2017·Protein Science : a Publication of the Protein Society·Olga Khersonsky, Sarel J Fleishman

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