A cell surface display system using novel GPI-anchored proteins in Hansenula polymorpha.
A 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. polymorpha exhibited 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.
Kim
So-Young
SY
Microbial Genomics Laboratory, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Yusong, Taejon 305-333, Korea.
Sohn
Jung-Hoon
JH
Pyun
Yu-Ryang
YR
Choi
Eui-Sung
ES
D020816:Amino Acid Motifs; D002473:Cell Wall / Q000378:metabolism*; D002480:Cellulase; D003001:Cloning, Molecular; D005434:Flow Cytometry; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D005949:Glucose Oxidase / Q000235:genetics / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism*; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D010843:Pichia / Q000235:genetics*
D005656:Fungal Proteins; D017261:Glycosylphosphatidylinositols; D005949:Glucose Oxidase; D006026:Glycoside Hydrolases; D002480:Cellulase; C019826:carboxymethylcellulase