Related Experiment Video
Updated: Jun 26, 2026
09:46
Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris
Published on: February 25, 2010
Engineering complex-type N-glycosylation in Pichia pastoris using GlycoSwitch technology
Pieter P Jacobs1, Steven Geysens, Wouter Vervecken
1Unit for Molecular Glycobiology, Department for Molecular Biomedical Research, VIB, Technologiepark 927, B-9052 Ghent (Zwijnaarde), Belgium.
Nature Protocols
|January 10, 2009
Summary
This study details a protocol for engineering the Pichia pastoris yeast N-glycosylation pathway by modifying genes and introducing new enzymes. This allows for precise control over glycoprotein structures for biotechnological applications.
Area of Science:
- Biotechnology
- Molecular Biology
- Yeast Genetics
Background:
- The N-glycosylation pathway in Pichia pastoris is crucial for protein function and stability.
- Modifying this pathway is essential for producing glycoproteins with specific structures for therapeutic and industrial applications.
Purpose of the Study:
- To provide a detailed protocol for engineering the N-glycosylation pathway in Pichia pastoris.
- To enable the production of yeast strains with defined N-glycan structures.
Main Methods:
- Disruption of the endogenous OCH1 gene.
- Stepwise introduction of heterologous glycosylation enzymes using GlycoSwitch vectors.
- Transformation, cultivation, sugar analysis, and protein expression analysis at each step.
Main Results:
- A modular protocol for sequential modification of the N-glycosylation pathway.
- Demonstration of introducing specific glycosidase and glycosyltransferase activities into the yeast ER and Golgi.
- Successful engineering of Pichia pastoris to produce specific N-glycan structures, such as Gal(2)GlcNAc(2)Man(3)GlcNAc(2)N-glycans.
Conclusions:
- The presented protocol offers a robust method for tailoring Pichia pastoris N-glycosylation.
- This engineering approach facilitates the production of recombinant glycoproteins with desired glycan structures.
- The GlycoSwitch system provides a flexible platform for complex glycoengineering in Pichia pastoris.
Related Concept Videos
01:30Production of Pharmaceuticals
Industrial insulin production uses genetically engineered E. coli expressing a proinsulin gene controlled by a tryptophan promoter and containing a methionine linker for later cleavage. The cells also carry ampicillin resistance for selective growth. Seed cultures are stored at −80 °C and production begins by thawing a small amount to inoculate starter cultures, which are progressively scaled to a 50,000-L bioreactor. In the bioreactor, E. coli grow in nutrient-rich media under sterile, tightly...
01:22Bioreactor Controls-III
Strain improvement is a foundational strategy in industrial microbiology aimed at maximizing microbial productivity, particularly because natural isolates typically yield commercially valuable products in very low concentrations. Although optimizing the culture medium and environmental conditions can improve yields, these adjustments are inherently limited by the organism’s genetic potential. As a result, the focus shifts toward genetic modifications to enhance biosynthetic capacity. The...
01:24Oligosaccharide Assembly
Protein glycosylation starts in the ER lumen and continues in the Golgi apparatus. Glycosyltransferases catalyze the addition of sugar molecules or glycosylation of proteins. Usually, these enzymes add sugars to the hydroxyl groups of selected serine or threonine residues to form O-linked glycans or the amino groups of asparagine residues to form N-linked glycans. Different positions on the same polypeptide chain can contain differently linked glycans.
Multiple sugar molecules that may or may...
Multiple sugar molecules that may or may...
01:25Protein Glycosylation
Glycosylation, the most common post-translational modification for proteins, serves diverse functions. Adding sugars to proteins makes the proteins more resistant to proteolytic digestion. Glycosylated proteins can act as markers and receptors to promote cell-cell adhesion. Additionally, they have many essential quality control functions in the cell, such as correct protein folding and facilitating transport of misfolded proteins to the cytosol, which can be degraded.
Glycosylation occurs in...
Glycosylation occurs in...
01:27Upstream Processing
Upstream processing represents a critical phase in biomanufacturing, wherein biological systems such as microorganisms, mammalian cells, or insect cells are cultivated to produce therapeutic proteins, vaccines, enzymes, or other biologically derived products. This phase encompasses all steps from the selection and genetic manipulation of the production organism to the cultivation of cells in bioreactors under tightly controlled environmental conditions.Host Selection and Genetic OptimizationThe...
01:57Protein Complexes with Interchangeable Parts
Groups of proteins may form a complex where each protein in this complex has a different role in the overall execution of the complex’s function. Often some of the proteins in the complex can be replaced by a closely related variant to give a complex that contains many of the same components yet is functionally distinct.
The SCF ubiquitin ligase is a protein complex of five individual proteins. This complex attaches ubiquitin to other target proteins to mark them for degradation. In order to...
The SCF ubiquitin ligase is a protein complex of five individual proteins. This complex attaches ubiquitin to other target proteins to mark them for degradation. In order to...