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

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.

[1]  E. Kawasaki,et al.  A general method for facilitating heterodimeric pairing between two proteins: application to expression of alpha and beta T-cell receptor extracellular segments. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[2]  R. Siliciano,et al.  A soluble, single-chain T-cell receptor fragment endowed with antigen-combining properties. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[3]  C. Kaiser,et al.  A pathway for targeting soluble misfolded proteins to the yeast vacuole , 1996, The Journal of cell biology.

[4]  P. Hudson,et al.  Use of mutator cells as a means for increasing production levels of a recombinant antibody directed against Hepatitis B. , 1997, Gene.

[5]  M. Davis,et al.  Expression of T cell antigen receptor heterodimers in a lipid-linked form. , 1990, Science.

[6]  R. Glockshuber,et al.  A comparison of strategies to stabilize immunoglobulin Fv-fragments. , 1990, Biochemistry.

[7]  K D Wittrup,et al.  Selection of functional T cell receptor mutants from a yeast surface-display library. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[8]  A. Plückthun,et al.  Correctly folded T-cell receptor fragments in the periplasm of Escherichia coli. Influence of folding catalysts. , 1994, Journal of molecular biology.

[9]  P. Bryan,et al.  Stabilizing the subtilisin BPN' pro‐domain by phage display selection: How restrictive is the amino acid code for maximum protein stability? , 1998, Protein science : a publication of the Protein Society.

[10]  J. Sambrook,et al.  Protein folding in the cell , 1992, Nature.

[11]  P. Marrack,et al.  Binding of a soluble alpha beta T-cell receptor to superantigen/major histocompatibility complex ligands. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[12]  D. Kranz,et al.  Binding properties and solubility of single-chain T cell receptors expressed in E. coli. , 1996, Molecular immunology.

[13]  A. Helenius,et al.  Quality control in the secretory pathway. , 1995, Current opinion in cell biology.

[14]  A. Singer,et al.  Developmental regulation of alpha beta T cell antigen receptor expression results from differential stability of nascent TCR alpha proteins within the endoplasmic reticulum of immature and mature T cells. , 1994, The EMBO journal.

[15]  R. Klausner,et al.  High-efficiency expression and solubilization of functional T cell antigen receptor heterodimers. , 1992, Science.

[16]  E. Hostinová,et al.  High-yield production of Saccharomycopsis fibuligera glucoamylase in Escherichia coli, refolding, and comparison of the nonglycosylated and glycosylated enzyme forms. , 1996, Biochemical and biophysical research communications.

[17]  P. Martineau,et al.  Expression of an antibody fragment at high levels in the bacterial cytoplasm. , 1998, Journal of molecular biology.

[18]  H. Vihinen,et al.  The hsp150Δ‐carrier confers secretion competence to the rat nerve growth factor receptor ectodomain in Saccharomyces cerevisiae , 1996, Yeast.

[19]  P. Punt,et al.  Glucoamylase gene fusions alleviate limitations for protein production in Aspergillus awamori at the transcriptional and (post) translational levels , 1997, Applied and environmental microbiology.

[20]  K. Garcia,et al.  Alanine Scanning Mutagenesis of an αβ T Cell Receptor: Mapping the Energy of Antigen Recognition , 1998 .

[21]  K D Wittrup,et al.  Secretion efficiency in Saccharomyces cerevisiae of bovine pancreatic trypsin inhibitor mutants lacking disulfide bonds is correlated with thermodynamic stability. , 1998, Biochemistry.

[22]  A. Plückthun,et al.  Selecting proteins with improved stability by a phage-based method , 1998, Nature Biotechnology.

[23]  A Helenius,et al.  The endoplasmic reticulum as a protein-folding compartment. , 1992, Trends in cell biology.

[24]  K D Wittrup,et al.  Protein Folding Stability Can Determine the Efficiency of Escape from Endoplasmic Reticulum Quality Control* , 1998, The Journal of Biological Chemistry.

[25]  K. D. Hardman,et al.  Characterization of a single-chain T-cell receptor expressed in Escherichia coli. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[26]  A. Bothwell,et al.  Heterodimeric, disulfide‐linked α/β T cell receptors in solution , 1991 .

[27]  E. Reinherz,et al.  Crystallization of a Deglycosylated T Cell Receptor (TCR) Complexed with an Anti-TCR Fab Fragment* , 1996, The Journal of Biological Chemistry.

[28]  E. Ward.  Expression and Secretion of T‐Cell Receptor Vα and Vβ Domains using Escherichia coli as a Host , 1991 .

[29]  D. Ecker,et al.  Ubiquitin fusion augments the yield of cloned gene products in Escherichia coli. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[30]  P. Punt,et al.  Efficient production of secreted proteins by Aspergillus : progress, limitations and prospects , 1997, Applied Microbiology and Biotechnology.

[31]  Ronald T. Raines,et al.  Increasing the secretory capacity of Saccharomyces cerevisiae for production of single-chain antibody fragments , 1998, Nature Biotechnology.

[32]  A. Necker,et al.  Monoclonal antibodies raised against engineered soluble mouse T cell receptors and specific for Vα8‐, Vβ2‐ or Vβ10‐bearing T cells , 1991 .

[33]  J. Coligan,et al.  Secretion of a soluble, chimeric gamma delta T-cell receptor-immunoglobulin heterodimer. , 1992, Proceedings of the National Academy of Sciences of the United States of America.