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.
Shusta
E V
EV
Department of Chemical Engineering, University of Illinois, Urbana, IL 61801, USA.
Kieke
M C
MC
Parke
E
E
Kranz
D M
DM
Wittrup
K D
KD
D017077:Culture Media, Conditioned; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006031:Glycosylation; D006207:Half-Life; D007700:Kinetics; D009154:Mutation; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011948:Receptors, Antigen, T-Cell / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics* / Q000378:metabolism*; D012995:Solubility; D013696:Temperature; D013879:Thioredoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D017077:Culture Media, Conditioned; D010455:Peptides; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins; D013879:Thioredoxins