A cell surface display system using novel GPI‐anchored proteins in Hansenula polymorpha

Abstract

A cell surface display system was developed in yeast Hansenula polymorpha.The four genes HpSED1, HpGAS1, HpTIP1 and HpCWP1, encoding glycosylphosphatidylinositol (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 GPIanchored 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.

Carbohydrate Derived Fulvic Acid: An in vitro Investigation of a Novel Membrane Active Antiseptic Agent Against Candida albicans Biofilms

“…To further test our cell membrane hypothesis we weakened the cell wall by inhibiting chitin biosynthesis using the chitin synthase inhibitor NKZ, which has been shown to inhibit all three Chs isozymes in C. albicans , and in turn results in a lack of chitin in fungal cells (Kim et al, 2002). We reasoned that if the membrane were the active target then the weakened cell would become hyper-susceptible to CHD-FA.…”

Efficient Library Construction by In Vivo Recombination with a Telomere-Originated Autonomously Replicating Sequence of Hansenula polymorpha

“…DNA sequencing was carried out with an automatic DNA sequencer (ABI model 373A; Applied Biosystems). The plasmid pGA-GOD-CwpF (15) contained the GAPDH (glyceraldehyde-3-phosphate dehydrogenase) gene promoter (27), HARS36 for autonomous replication (28), the LEU2 gene of H. polymorpha (2), and CwpF as a surface display anchor from H. polymorpha (15). This plasmid was used as a backbone for construction of a Candida antarctica lipase B (CALB) expression vector.…”

“…H. polymorpha is also a useful biocatalyst for metabolic pathway engineering in which expression of multiple genes in different ratios is necessary (8). Recently, a surface display system in H. polymorpha using novel cell wall proteins was developed (15). Surface display plays an important role in linking the genotype and phenotype in directed evolution of useful proteins.…”

Surface display of human lactoferrin using a glycosylphosphatidylinositol-anchored protein of Saccharomyces cerevisiae in Pichia pastoris

“…Although cell surface display using TIP1p in Hansenula polymorpha was reported (Kim et al 2002), there are no reported studies in P. pastoris yet. Here we report the results of surface display of hLf using ScTIP in P. pastoris.…”