Secretion and surface display of green fluorescent protein using the yeast Saccharomyces cerevisiae.
Green fluorescent protein (GFP) continues to be a very useful tool in biotechnology, but soluble production of GFP and GFP-protein fusions has been difficult. In this study, we have produced yeast-enhanced green fluorescent protein (yEGFP) in Saccharomyces cerevisiae as a soluble, secreted product with a purified level of 6 mg/L. Expression was directed by the inducible GAL1-10 promoter and synthetic prepro leader sequence. The secretion of yEGFP by yeast was strongly dependent on temperature, with 20 degrees C induction being optimal. Use of 2 micro multicopy expression constructs elevated yields over a low-copy CEN-based system by approximately 2-fold. Yeast-enhanced GFP was also expressed as a fusion to the Aga2p mating agglutinin in order to test the secretory processing fidelity of yEGFP-protein fusions. When the cell surface anchoring protein, Aga1p, was co-overexpressed with the Aga2p-yEGFP fusion, the Aga2p-yEGFP protein was tethered to the yeast cell surface. Flow cytometry and fluorescence microscopy analysis indicated that the fusion was displayed on the yeast cell surface at high levels. In the absence of high level Aga1p expression, the Aga2p-yEGFP fusion protein was instead secreted in its entirety with no detectable surface display. These findings reveal that yeast is a suitable host for secretion of GFP and GFP-protein fusions and thus could enable a wide range of biochemistry and biotechnology applications.
Huang
Dagang
D
Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Wisconsin 53706, USA.
Shusta
Eric V
EV
D001483:Base Sequence; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D013696:Temperature
D017931:DNA Primers; D049452:Green Fluorescent Proteins