Biochemical SocietyTransactions( 1992120 1 1 1S
Expression of Active Horseradish Peroxidase in Saccharomvces cerevisiae
50 45
ALEXIOS VLAMIS-GARDIKAS, ANDREW T. SMITH, *JOHN M. CLEMENTS, and JULIAN F. BURKE
40
Department of Biochemistry, BIOLS, University of Sussex, Brighton, BN1 9QG, U.K. and *British Bio-technology Ltd. , Watlington Road, Cowley, Oxford OX4 5LY, U.K.
30
Horseradish Peroxidase (HRPC) is a monomeric glycoprotein of 308 amino acid 8 oligosaccharide residues containing chains, one mole of haem, up to two moles of calcium per mole of protein [l], and 4 disulfide bonds. It has a calculated Mr of 33922 with a further 18% contributed by the glycosylation [2]. At least 30 different HRP isoenzymes have been identified from the horseradish root [3]. Expression of a synthetic gene encoding HRPC* in E. coli gave rise to inclusion bodies. A small fraction of the expressed enzyme could be refolded correctly to give fully active recombinant HRP (HRP C*) [4]. A yeast expression system should allow correct folding, appropriate disulfide bond formation, glycosylation, and secretion of active enzyme. The amino acid sequence of HRP C* corresponds to one of five HRP cDNA clones isolated from cultured horseradish cells [5], except that the cDNA sequence encodes an additional 15 amino acid C terminal acidic propeptide, which may have a role in vacuolar targetting in the plants [6]. To express HRPC in yeast, an HRP construct was made containing the C terminal propeptide fused downstream from the HRP synthetic gene. The HRP C1 synthetic gene was fused in-frame to the prepro a-factor leader sequence to direct extracellular secretion. The vector (pLF25) carried the Leu2d selectable marker and part of the 2pm plasmid for episomal replication. A culture containing plasmid pLF25 in the yeast host strain MD40/4C was grown for 7 days. Accumulation of activity continued late into log phase (Fig.1). No activity was detected in control cultures lacking the plasmid. In an attempt to purify the secreted peroxidase, the supernatant was concentrated by ultrafiltration, subjected to ammonium sulphate precipitation and finally chromatographed on a Superose-12 gel filtration column where it was eluted with an apparent Mr of 100 kD. The most active fractions from the Superose-12 column were western blotted with anti HRP C1 polyclonal antibodies. No cross reacting material was observed on these blots (data not shown). Saccharomvces cerevisiae is known to hyperglycosylate heterologous proteins [7]. The active fractions were therefore treated with endoglycosidase H and glycopeptidase F [ 81 to remove carbohydrates that might hide the polypeptide epitopes of the expressed peroxidase. The deglycosylated samples were western blotted using an antiserum raised to the E. coli non glycosylated recombinant enzyme (41. In the samples treated with glycopeptidase F a protein band of equal Mr to that of the non glycosylated HRPC* (about
35
25 20
15 10 5
0 0
50
100
150
200
Time (hrs) 'Fig.1. Peroxidase activitv Drofile of MD40/4C (DLF25L A 6L culture of MD40/4C (pLF25) (Phosphoglycerate kinase, PGK, promoter and terminator) was grown in selective medium (Leu-) containing 15pM haem, 5mM CaC12 at 30°C for 7 days (Fig.1). The pH of the culture medium was originally adjusted to 7.0 with 50mM Na+ MOPS buffer and was maintained between pH 5.0 and pH 6.7 during growth, by the addition of NaOH at day 5. Peroxidase activity was measured by the ABTS assay [9].
34 kD) cross-reacted with the HRPC* antibodies. The samples treated with endoglycosidase H contained a cross reacting band of slightly greater Mr (data no? shown). These blots suggest that the extracellular HRP produced in S . cerevisiae is secreted in a hyperglycosylated state with the C terminal propeptide removed. Further studies will elucidate the biochemical properties of the secreted enzyme and the role of the C terminal propeptide. 1. Haschke. R.H. & Friedhoff. J.M. 119781 Biochem. Biophys. Resear. Cok. '1039: 1042 2. Welinder, K.J. (1976) FEBS Lett. 22, 19-
a,
23 __
3. Hoyle, M.C. (1977) Plant Physiol. 60, 787-793 4. Smith, A.T., Santama, N., Dacey S . , Edwards, M., Bray, R.C. , Thorneley, R.N.F. & Burke, J.F. (1990) J. Biol. Chem. 265, 13335-13343 5. Fujiyama, K., Takemura.H., Shinmyo, A., 163Okada, H. & Takano, M. (1990) Gene 169 6. Wellinder, J.K. (1991) in Plant Peroxidases 1980-1990: Progress and Prospects in Biochemistry and Physiology (Gaspar, T. Penel, C. & Groppin H., eds.) 7. George, P.L., Ferrara, A., Roskin, R., Simon, P.L. & Young, P.R. (1990) Gene 297-301 8 . Trimble, R.B. & Maley, F. (1984) Anal. Biochem. 141 512-522 9. Childs, R.E. & Bardsley, W.G. (1975) Biochem. J. 145, 93-103
m,
Expression of Active Horseradish Peroxidase in Saccharomvces cerevisiae
50 45
ALEXIOS VLAMIS-GARDIKAS, ANDREW T. SMITH, *JOHN M. CLEMENTS, and JULIAN F. BURKE
40
Department of Biochemistry, BIOLS, University of Sussex, Brighton, BN1 9QG, U.K. and *British Bio-technology Ltd. , Watlington Road, Cowley, Oxford OX4 5LY, U.K.
30
Horseradish Peroxidase (HRPC) is a monomeric glycoprotein of 308 amino acid 8 oligosaccharide residues containing chains, one mole of haem, up to two moles of calcium per mole of protein [l], and 4 disulfide bonds. It has a calculated Mr of 33922 with a further 18% contributed by the glycosylation [2]. At least 30 different HRP isoenzymes have been identified from the horseradish root [3]. Expression of a synthetic gene encoding HRPC* in E. coli gave rise to inclusion bodies. A small fraction of the expressed enzyme could be refolded correctly to give fully active recombinant HRP (HRP C*) [4]. A yeast expression system should allow correct folding, appropriate disulfide bond formation, glycosylation, and secretion of active enzyme. The amino acid sequence of HRP C* corresponds to one of five HRP cDNA clones isolated from cultured horseradish cells [5], except that the cDNA sequence encodes an additional 15 amino acid C terminal acidic propeptide, which may have a role in vacuolar targetting in the plants [6]. To express HRPC in yeast, an HRP construct was made containing the C terminal propeptide fused downstream from the HRP synthetic gene. The HRP C1 synthetic gene was fused in-frame to the prepro a-factor leader sequence to direct extracellular secretion. The vector (pLF25) carried the Leu2d selectable marker and part of the 2pm plasmid for episomal replication. A culture containing plasmid pLF25 in the yeast host strain MD40/4C was grown for 7 days. Accumulation of activity continued late into log phase (Fig.1). No activity was detected in control cultures lacking the plasmid. In an attempt to purify the secreted peroxidase, the supernatant was concentrated by ultrafiltration, subjected to ammonium sulphate precipitation and finally chromatographed on a Superose-12 gel filtration column where it was eluted with an apparent Mr of 100 kD. The most active fractions from the Superose-12 column were western blotted with anti HRP C1 polyclonal antibodies. No cross reacting material was observed on these blots (data not shown). Saccharomvces cerevisiae is known to hyperglycosylate heterologous proteins [7]. The active fractions were therefore treated with endoglycosidase H and glycopeptidase F [ 81 to remove carbohydrates that might hide the polypeptide epitopes of the expressed peroxidase. The deglycosylated samples were western blotted using an antiserum raised to the E. coli non glycosylated recombinant enzyme (41. In the samples treated with glycopeptidase F a protein band of equal Mr to that of the non glycosylated HRPC* (about
35
25 20
15 10 5
0 0
50
100
150
200
Time (hrs) 'Fig.1. Peroxidase activitv Drofile of MD40/4C (DLF25L A 6L culture of MD40/4C (pLF25) (Phosphoglycerate kinase, PGK, promoter and terminator) was grown in selective medium (Leu-) containing 15pM haem, 5mM CaC12 at 30°C for 7 days (Fig.1). The pH of the culture medium was originally adjusted to 7.0 with 50mM Na+ MOPS buffer and was maintained between pH 5.0 and pH 6.7 during growth, by the addition of NaOH at day 5. Peroxidase activity was measured by the ABTS assay [9].
34 kD) cross-reacted with the HRPC* antibodies. The samples treated with endoglycosidase H contained a cross reacting band of slightly greater Mr (data no? shown). These blots suggest that the extracellular HRP produced in S . cerevisiae is secreted in a hyperglycosylated state with the C terminal propeptide removed. Further studies will elucidate the biochemical properties of the secreted enzyme and the role of the C terminal propeptide. 1. Haschke. R.H. & Friedhoff. J.M. 119781 Biochem. Biophys. Resear. Cok. '1039: 1042 2. Welinder, K.J. (1976) FEBS Lett. 22, 19-
a,
23 __
3. Hoyle, M.C. (1977) Plant Physiol. 60, 787-793 4. Smith, A.T., Santama, N., Dacey S . , Edwards, M., Bray, R.C. , Thorneley, R.N.F. & Burke, J.F. (1990) J. Biol. Chem. 265, 13335-13343 5. Fujiyama, K., Takemura.H., Shinmyo, A., 163Okada, H. & Takano, M. (1990) Gene 169 6. Wellinder, J.K. (1991) in Plant Peroxidases 1980-1990: Progress and Prospects in Biochemistry and Physiology (Gaspar, T. Penel, C. & Groppin H., eds.) 7. George, P.L., Ferrara, A., Roskin, R., Simon, P.L. & Young, P.R. (1990) Gene 297-301 8 . Trimble, R.B. & Maley, F. (1984) Anal. Biochem. 141 512-522 9. Childs, R.E. & Bardsley, W.G. (1975) Biochem. J. 145, 93-103
m,
