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

Area of Science:

• Microbiology
• Biotechnology
• Molecular Biology

Background:

• Cell surface display systems are crucial for applications in biocatalysis and diagnostics.
• Hansenula polymorpha is a methylotrophic yeast with potential for biotechnological applications.
• Identifying robust cell wall proteins for anchoring foreign proteins is essential for effective display.

Purpose of the Study:

• To develop and characterize a novel cell surface display system in Hansenula polymorpha.
• To evaluate the efficacy of four H. polymorpha glycosylphosphatidyl-inositol (GPI)-anchored proteins (HpSED1, HpGAS1, HpTIP1, HpCWP1) as display motifs.
• To assess the surface anchoring efficiency of these proteins using reporter enzymes.

Main Methods:

• Cloning and characterization of four H. polymorpha GPI-anchored protein genes.
• Fusion of reporter genes (CMCase, GOD) to the identified H. polymorpha cell surface proteins.
• Enzymatic activity assays on intact cell fractions to confirm surface display.
• Fluorescence-activated cell sorter (FACS) analysis to quantify surface protein exposure.

Main Results:

• All four H. polymorpha genes encode typical GPI-anchored proteins with secretion and anchor signals.
• Fusion proteins exhibited significant enzymatic activity on the cell surface, confirming successful anchoring.
• HpCwp1p, HpGas1p, and a C-terminal fragment of HpTip1p demonstrated high anchoring efficiency for CMCase.
• HpCwp1p showed the highest anchoring efficiency for both CMCase and glucose oxidase (GOD).
• FACS analysis confirmed good exposure of GOD on the cell surface.

Conclusions:

• The developed H. polymorpha cell surface display system is effective for anchoring heterologous proteins.
• HpCwp1p is a highly efficient anchoring motif for cell surface display in H. polymorpha.
• This system holds promise for various biotechnological applications requiring cell surface presentation of enzymes or proteins.