A cell surface display system using novel GPI‐anchored proteins in <i>Hansenula polymorpha</i>

Yeast - Tập 19 Số 13 - Trang 1153-1163 - 2002
So‐Young Kim1,2, Jung‐Hoon Sohn2, Yu‐Ryang Pyun1, Eui‐Sung Choi2
1Department of Biotechnology, Yonsei University, Seoul, 120-749, Korea
2Microbial Genomics Laboratory, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Yusong, Taejon 305-333, Korea

Tóm tắt

AbstractA cell surface display system was developed in yeast Hansenula polymorpha.The four genes HpSED1, HpGAS1, HpTIP1and HpCWP1, encoding glycosylphosphatidyl‐inositol (GPI)‐anchored cell surface proteins from H. polymorpha, were cloned, characterized and evaluated for their efficacies as cell surface display motifs of reporter proteins. Sequence analysis of these genes revealed that each encodes a typical GPI‐anchored protein that is structurally similar to a counterpart gene in S. cerevisiae. The genes showed a high content of serine‐threonine (alanine) and harboured a putative secretion signal in the N‐terminus and the GPI‐attachment signal in the C‐terminus. The surface anchoring efficiency of these putative cell surface proteins was tested by fusion to the C‐terminal of carboxymethylcellulase (CMCase) from Bacillus subtilis. In all cases, high CMCase activities were detected in intact cell fraction, indicating anchoring of CMCase to the cell surface. HpCwp1p, HpGas1p and the 40 C‐terminal amino acids of HpTip1p from H. polymorphaexhibited a comparatively high CMCase surface anchoring efficiency. When these proteins were used as anchoring motifs for surface display of the glucose oxidase (GOD) from Aspergillus niger, most enzyme activity was detected at the cell surface. Fluorescence activated cell sorter (FACS) analysis of cells displaying GOD on the cell surface demonstrated that GOD was well exposed on the cell surface. HpCwp1p showed the highest anchoring efficiency among others. Copyright © 2002 John Wiley & Sons, Ltd.

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Tài liệu tham khảo

10.1002/yea.320100410

10.1083/jcb.130.6.1333

10.1016/S0734-9750(00)00054-9

10.1038/nbt0697-553

10.1016/S0076-6879(00)28410-3

10.1002/(SICI)1097-0061(199712)13:15<1477::AID-YEA184>3.0.CO;2-L

10.1002/(SICI)1097-0061(19990130)15:2<91::AID-YEA343>3.0.CO;2-#

10.1073/pnas.89.7.2713

10.1038/nbt0493-491

Frederick KR, 1990, Glucose oxidase from Aspergillus niger. Cloning, gene sequence, secretion from Saccharomyces cerevisiae and kinetic analysis of a yeast‐derived enzyme, J Biol Chem, 265, 3793, 10.1016/S0021-9258(19)39664-4

10.1007/s002530000464

10.1038/nbt0391-291

10.1007/s002530050781

10.1007/s004380050706

10.1074/jbc.273.41.26946

Hamada K, 1999, Amino acid residues in the −ω region participate in cellular localization of yeast glycosylphosphatidylinositol‐attached proteins, J Bacteriol, 181, 3886, 10.1128/JB.181.13.3886-3889.1999

10.1002/yea.320090609

10.1016/S0958-1669(97)80028-6

10.1016/0378-1119(86)90293-3

10.1016/S0141-0229(97)00224-X

10.1002/(SICI)1097-0061(19980315)14:4<371::AID-YEA231>3.0.CO;2-1

10.1046/j.1365-2672.2001.01233.x

10.1128/jb.179.20.6279-6284.1997

10.1046/j.1365-2958.2000.01729.x

10.1093/protein/10.11.1303

10.1111/j.1574-6968.2000.tb09394.x

Levine DW, 1973, Isolation and characterization of a thermotolerant methanol‐utilizing yeast, Appl Microbiol, 26, 982, 10.1128/am.26.6.982-990.1973

10.1002/(SICI)1097-0061(19970930)13:12<1145::AID-YEA163>3.0.CO;2-Y

10.1007/s002530051086

Murai T, 1998, Assimilation of cellooligosaccharides by a cell surface‐engineered yeast expressing β‐glucosidase and carboxymethylcellulase from Aspergillus aculeatus, Appl Environ Microbiol, 64, 4857, 10.1128/AEM.64.12.4857-4861.1998

10.1007/s002530051364

Murai T, 1997, Construction of a starch‐utilizing yeast by cell surface engineering, Appl Environ Microbiol, 63, 1362, 10.1128/aem.63.4.1362-1366.1997

10.1073/pnas.83.17.6263

10.1271/bbb1961.55.441

10.1016/S0304-4165(98)00138-X

Sambrook J, 1989, Molecular Cloning: A Laboratory Manual

10.1002/yea.320090410

10.1002/(SICI)1097-0061(199707)13:9<809::AID-YEA141>3.0.CO;2-1

Shahinian S, 1998, Involvement of protein N‐glycosyl chain glucosylation and processing in the biosynthesis of cell wall β‐1,6‐glucan of Saccharomyces cerevisiae, Genetics, 149, 843, 10.1093/genetics/149.2.843

10.1093/oxfordjournals.jbchem.a124907

Shimoi H, 1998, Sed1p is a major cell wall protein of Saccharomyces cerevisiae in the stationary phase and is involved in lytic enzyme resistance, J Bacteriol, 180, 3381, 10.1128/JB.180.13.3381-3387.1998

10.1128/jb.178.15.4420-4428.1996

10.1007/s002530051465

10.1038/nbt0896-1017

10.1084/jem.178.1.197

10.1093/nar/22.22.4673

10.1016/S0734-9750(00)00031-8

10.1128/jb.177.11.3104-3110.1995

van der Vaart JM, 1997, Comparison of cell wall proteins of Saccharomyces cerevisiae as anchors for cell surface expression of heterologous proteins, Appl Environ Microbiol, 63, 615, 10.1128/aem.63.2.615-620.1997

10.1002/yea.320080504

10.1007/BF00327024

Williams HE, 1999, Effective query filtering for fast homology searching, Pac Symp Biocomput, 214

10.1111/j.1365-2958.1989.tb00097.x

10.1016/S0958-1669(00)00233-0

10.1016/0167-4838(89)90034-4

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Yeast

Tập 19 Số 13

1153-1163

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