Viral preprotoxin signal sequence allows efficient secretion of green fluorescent protein by Candida glabrata, Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe

Abstract

Besides its importance as model organism in eukaryotic cell biology, yeast species have also developed into an attractive host for the expression, processing, and secretion of recombinant proteins. Here we investigated foreign protein secretion in four distantly related yeasts (Candida glabrata, Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe) by using green fluorescent protein (GFP) as a reporter and a viral secretion signal sequence derived from the K28 preprotoxin (pptox), the precursor of the yeast K28 virus toxin. In vivo expression of GFP fused to the N-terminal pptox leader sequence and/or expression of the entire pptox gene was driven either from constitutive (PGK1 and TPI1) or from inducible and/or repressible (GAL1, AOX1, and NMT1) yeast promoters. In each case, GFP entered the secretory pathway of the corresponding host cell; confocal fluorescence microscopy as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western analysis of cell-free culture supernatants confirmed that GFP was efficiently secreted into the culture medium. In addition to the results seen with GFP, the full-length viral pptox was correctly processed in all four yeast genera, leading to the secretion of a biologically active virus toxin. Taken together, our data indicate that the viral K28 pptox signal sequence has the potential for being used as a unique tool in recombinant protein production to ensure efficient protein secretion in yeast.

FIG. 1.

(A) Kex2p-mediated pptox processing to the α/β heterodimeric K28 virus toxin in yeast. Internal cleavage sites of the ER lumenal signal peptidase (SP) and of the late Golgi endopeptidase Kex2p are indicated. The three N-glycosylation sites within the pptox γ-sequence are indicated by circles. S, secretion signal at the pptox N terminus. (B) Schematic outlines of the constructed vectors that allow constitutive or regulated expression of K28 pptox and/or GFP in the yeasts S. cerevisiae, C. glabrata, P. pastoris, and S. pombe. In each vector, the pptox and/or GFP open reading frame is under the transcriptional control of the indicated promoter and transcription termination combinations; the origins of replication (ARS) and low- or high-copy numbers of the vectors (CEN and 2μm [2μ]) as well as marker genes for the selection of yeast transformants (HIS4, URA3, and LEU2) are indicated.

FIG. 2.

Constitutive or thiamine-methanol-regulated K28 pptox expression in S. pombe (upper left panel), S. cerevisiae (upper right panel), P. pastoris (lower left panel), and C. glabrata (lower right panel). Yeast transformants harboring the indicated K28 pptox expression plasmid were grown under induced and/or repressed culture conditions (in the absence [−] or presence [+] of thiamine and/or methanol), and K28 toxin production levels were determined on methylene blue agar plates (pH 4.7) against the sensitive S. cerevisiae strain 192.2d. Toxin activity levels in pptox-expressing P. pastoris transformants were determined by pipetting a 100-μl aliquot of a cell-free culture supernatant into a 10-mm-diameter well that had been cut into the agar. After the plates were incubated for 4 days at 20°C, a cell-free zone of growth inhibition around the pprox-expressing yeast strain and/or the well indicated toxin secretion and killer activity.

FIG. 3.

Western blot analysis of K28 toxin secretion in C. glabrata, P. pastoris, S. pombe, and S. cerevisiae after in vivo expression of K28 pptox. Cell-free culture supernatants (600 μl each) of the corresponding yeast transformants grown under pptox-inducing culture conditions were concentrated by ethanol precipitation, separated by SDS-PAGE under nonreducing conditions, and probed with a polyclonal antibody against the toxin's β-subunit. Lane 1, K28 toxin (positive control); lane 2, S. cerevisiae(pSL-K28); lane 3, C. glabrata(pGRB2.2-K28); lane 4, P. pastoris(pPIC3.5-K28) after cultivation for 96 h under inducing conditions; lane 5, S. cerevisiae(pYX242) (negative control); lane 6, C. glabrata(pGBR2.2) (negative control); lane 7, P. pastoris(pPIC3.5) (negative control); lane 8, P. pastoris(pPIC3.5-K28) after cultivation for 120 h under inducing conditions; lane 9, S. pombe(pTZ) (empty vector control); lane 10, S. pombe(pTZ-K28) grown under repressed conditions in the presence of thiamine; lane 11, S. pombe(pTZ-K28) grown under induced conditions in the absence of thiamine. The position and size of the correctly processed α/β heterodimeric K28 virus toxin is indicated.

FIG. 4.

pptox-driven GFP secretion in yeast. Fluorescence microscopy (upper panel in each pair of panels) and Western analysis (lower panel in each pair of panels) of the indicated yeast transformant after in vivo expression of GFP fused to the amino-terminal secretion signal of K28 pptox. The inset in the bottom right panel shows a stronger magnification of the confocal laser scanning micrograph, illustrating localization of GFP within the secretory pathway (ER and Golgi) of fission yeast. In each case, SDS-PAGE and Western analysis of a cell-free culture supernatant probed with a monoclonal anti-GFP antibody were performed as described in the legend to Fig. 3. Lanes 1, GFP marker (positive control; 5 ng); lanes 2, prestained marker protein standard; lane 3, GFP secreted by C. glabrata(pGRB2.2-pptoxGFP); lane 4, C. glabrata(pGRB2.2) (empty vector control); lane 5, GFP secreted by P. pastoris(pPIC3.5-pptoxGFP) grown for 96 h under inducing conditions in the presence of methanol; lane 6, P. pastoris(pPIC3.5-pptoxGFP) grown under noninducing conditions in the absence of methanol; lane 7, GFP secreted by S. cerevisiae(pFB-pptoxGFP) grown under inducing conditions on galactose; lane 8, S. cerevisiae(pFB-pptoxGFP) grown under repressed culture conditions on glucose; lane 9, S. pombe(pTZ-pptox) (empty vector control); lane 10, S. pombe(pTZ-pptoxGFP) grown under repressed conditions in the presence of thiamine; lane 11, S. pombe(pTZ-pptoxGFP) grown under induced conditions in the absence of thiamine.