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KMID : 1094720000050040234
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Biotechnology and Bioprocess Engineering
2000 Volume.5 No. 4 p.234 ~ p.241
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Effect of a PMR1 disruption on the processing of heterologous glycoproteins secreted in the yeast Saccharomyces cerevisiae
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Kim Moo-Woong
Choi Eui-Sung
Kim Jeong-Yoon
Ko Su-Min
Sohn Jung-Hoon
Kang Hyun-Ah
Rhee Sang-Ki
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Abstract
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TheSaccharomyces cerevisiae PMR1 gene encodes a Ca2+-ATPase localized in the Golgi. We have investigated the effects ofPMR1 disruption inS. cerevisiae on the glycosylation and secretion of three heterologous glycoproteins, human α1-antitrypsin (α1-AT), human antithrombin III (ATHIII), andAspergillus niger glucose oxidase (GOD). Thepmr1 null mutant strain secreted larger amounts of ATHIII and GOD proteins per a unit cell mass than the wild type strain. Despite a lower growth rate of thepmr1 mutant, two-fold higher level of human ATHIII was detected in the culture supernatant from thepmr1 mutant compared to that of the wild-type strain. Thepmr1 mutant strain secreted α1-AT and the GOD proteins mostly as core-glycosylated forms, in contrast to the hyperglycosylated proteins secreted in the wild-type strain. Furthermore, the core-glycosylated forms secreted in thepmr1 mutant migrated slightly faster on SDS-PAGE than those secreted in themnn9 deletion mutant and the wild type strains. Analysis of the recombinant GOD with anti-α1,3-mannose antibody revealed that GOD secreted in thepmr1 mutant did not have terminal α1,3-linked mannoses unlike those secreted in themnn9 mutant and the wild type strains. The present results indicate that thepmr1 mutant, with the super-secretion phenotype, is useful as a host system to produce recombinant glycoproteins lacking high-mannose outer chains.
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KEYWORD
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PMR1, Saccharomyces cerevisiae, secretion, glycosylation, heterologous protein
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