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In partnership with Research Corporation Technologies (RCT), BioGrammatics is now offering the first Pichia expression vectors, strains and services to modify glycosylation in Pichia with GlycoSwitch®. GlycoSwitch® technology originates from collaborations with Roland Contreras, Ph.D., Nico Callewaert, Ph.D., and colleagues at Ghent University and the Flanders Institute for Biotechnology (VIB) in Belgium, as well as, more recent contributions by BioGrammatics. Glycosylation can influence protein folding, stability and protein-protein interactions; so GlycoSwitch® was engineered to generate proteins with more “human-like” glycosylation to produce better Bio-pharmaceuticals, as well as, improve other classes of recombinant proteins.
The core GlycoSwitch® strains have a central glycan processing enzyme (OCH1) disrupted to prevent glycan elongation, and express a foreign mannosidase to trim existing glycans to a more uniform Man5 structure. These SuperMan5 strains are available for shipment to your lab, as are expression vectors to further modify the glycosylation pathway. Contract research and development services using the GlycoSwitch® technology are now also possible with all of the Pichia expertise available at BioGrammatics to tailor glycoproteins for your specific applications.
GlycoSwitch® Strains and Vectors.
The GlycoSwitch® SuperMan5 strains are more stable derivatives of the core Man5 Pichia pastoris Och1 mutant strain expressing the T. reesei alpha-1, 2-mannosidase created at the VIB. The SuperMan5 strains limit hyper-glycosylation, and generate a more uniform Man5 glycosylation at N-linked glycosylation sites like the original Man5 strain, however, the SuperMan5 strains are more stable. DNA duplications that resulted from engineering the original Man5 strains have been removed to prevent homologous recombination. The SuperMan5 strains are available as a non-auxotroph strain, SuperMan5, as well as, the histidine auxotroph SuperMan5(his-).
Both SuperMan5 strains are fully compatible with BioGrammatics expression vectors. Work with the non-auxotroph, SuperMan5 (His+), prevents subsequent requirements for histidine and is the preferred strain for “end-point” expression testing. However, further glycan pathway engineering with the current GlycoSwitch® vectors may require the histidine auxotroph strain, SuperMan (his-). GlycoSwitch® strains with additional glycan modifying enzymes, the more “fully - humanized” strains, are currently NOT available; however, the vectors to build subsequent glycan processing steps into the SuperMan5 strains are available.
GlycoSwitch® products, including the SuperMan5 strains and vectors with glycan modifying enzymes are provided under the RCT Limited Use Label License (see the Limit Use Label License in Purchaser Notifications).
GlycoSwitch® SuperMan5 Strains.
SuperMan5, (HIS4+, Och1-disruption with a pGAP-mannosidase expression cassette, blasticidin resistant) GS115 with the mutation at the HIS4 gene reverted to wild type (HIS4+). The alpha 1,2-mannosidase from T. reesei regulated by the GAP promoter on a plasmid with the Blasticidin resistance gene disrupting the Och1 gene in the SuperMan5 genome.
SuperMan5 (his-), Och1-disruption with a pGAP-mannosidase expression cassette, blasticidin resistant). GS115 (his4-), with the alpha 1,2-mannosidase from T. reesei regulated by the GAP promoter on a plasmid with the blasticidin resistance gene disrupting the Och1 gene in the SuperMan5 genome.
GlycoSwitch® Vectors. The GlycoSwitch® vectors currently offered by BioGrammatics are a core set of vectors to express glycan modifying enzymes. All are sequence verified and compared to the published sequences. Additional GlycoSwitch® vectors are available upon request: info@bioGrammatics.com.
A detailed description of the GlycoSwitch® technology, with protocols and examples of further engineering of the N-glycosylation pathway, is presented in the following manuscript: Pieter P Jacobs, Steven Geysens, Wouter Vervecken, Roland Contreras and Nico Callewaert (2008). Engineering complex-type N-glycosylation in Pichia pastoris using GlycoSwitch technology. Nature Protocols 4(1), 58 - 70 (2009). (Link: http://www.ncbi.nlm.nih.gov/pubmed/19131957)
Cellular glycosylation, one of the most complex post-translational modifications, attaches glycans (oligosaccharides) to proteins. Predominant in eukaryotic organisms, the core aspects of glycosylation are conserved from yeast to humans and can be a primary consideration in the selection of a protein expressions system.
Most proteins that enter the secretory pathway undergo glycosylation to function properly in membranes, in the cell wall or outside the cell. Glycosylation can play an important role in protein folding, stability and protein interactions. Similarly, the glycosylation of recombinant proteins expressed in Pichia can significantly influence their structure and function, as well as, the level of expression.
N-linked glycosylation is the most common type of glycan addition. In the lumen of the ER, N-linked glycans are attached to the nitrogen in the side chain of the amino acid asparagine when the asparagine in an Asn-X-Ser (N-X-S), or Asn-X-Thr (N-X-T) sequence, where X is any amino acid except proline. O-linked glycosylation is also common. O-linked glycans are assembled in the golgi, one sugar at a time on the amino acids serine or threonine; however, unlike with N-linked glycans, no consensus sequence is known for O-linked glycosylation.
N-linked glycoengineering. In eukaryotes the initial step for N-linked glycosylation occurs co-translationally at the luminal side of the ER membrane where a Glc3Man9GlcNAc2 oligosaccharide is transferred to the nitrogen of an accessible Asp in a growing polypeptide chain. The initial processing of this structure by a series of glycosidases and glycosyltransfereases is well conserved beween Pichia and higher eukaryotes and leads to a correctly folded N-glycosylated proteins with Man8GlcNAc2 glycans. Here, the glycoproteins exit the ER to cisternae in the golgi apparatus where subsequent glycans processing is species, as well as, cell-type specific. This addition and processing of glycans is non-templated and relies on segregating enzymes and access to substrates into different cellular compartments, therefore, glycosylation is a site-specific modification.
In mammals, Man8GlcNAc2 glycans are trimmed by alpha-1,2-mannosidases resulting in Man5GlcNAc glycans that are further modified by GlcNAc transferase I (GnT-I, addition of a beta-1,2-GlcNAc). Subsequent removal of two mannoses by mannosidase II (Man-II) allows a second beta-1,2-GlcNAc to be added by GlcNAc transferase II (GnT-II). Glycan structures with both core mannose residues modified by at least one GlcNAc are called “complex type” N-glycans to which galactose and sialic acid residues can be added by galactosyltransferases and sialyltransferases and additional branching can also be initiated by additional GlcNAc transferases.
Pichia, however, does not trim the Man8GlcNAc2 glycans in the Golgi as in higher eukaryotes. Instead the Man8GlcNAc2 glycans are modified by the addition of an alpha-1,6-mannose, a reaction catalyzed by the OCH1 protein. Initiating mannoses can then be further elongated by mannosyltransferases and can result in a backbone of alpha-1,6 mannoses of up to several dozen with shorter side branches (hyper-glycosylation, or hyper mannosyl-type structures). Although hyper-glycosylation is not as prevalent in Pichia as it is in other yeast like S. cerevisiae, disruption of the Pichia Och1 gene prevents addition of the initiating mannose, and therefore further “hyperglycosylation”.
The GlycoSwitch® Technology consists of the disruption of the endogenous Pichia glycosyltransferase gene (OCH1) and the stepwise introduction of heterologous glycosidase and glycosyltransferase activities. Furthermore, the expression of the alpha-1,2-mannosidase in the SuperMan5 strains results in Man5GlcNAc glycans. Therefore the SuperMan5 strains have more uniform, limited glycosylation and are in a position to acquire modifying enzymes for subsequent glycan engineering.
Link to N. Callewaert presentation PDF
|Pichia pastoris SuperMan5 strain (HIS+)||GlycoSwitch® strain, Man5 N-linked oligosaccharide structures||Details|
|Pichia pastoris SuperMan5 strain (his—)||GlycoSwitch® strain, Man5 N-linked oligosaccharide structures (histidine auxotroph)||Details|
|GlycoSwitch Expression Vector Kit||Details|
|pGlycoSwitch®–GnT–I–HIS||Histidine selection vector for expression of GlcNAc transferase I activity in Pichia pastoris||Details|
|pGlycoSwitch®–ManII/1||G418 selection vector for expression of Mannosidase II activity in Pichia pastoris||Details|
|pGlycoSwitch®–GnT–II||Hygromycin selection vector for expression of GlcNAc transferase II activity in Pichia pastoris||Details|
|pGlycoSwitch®–GalT/1||Nourseothricin selection vector for expression of Gal transferase I activity in Pichia pastoris||Details|
|Pichia pastoris SuperMan5 strain (HIS+, pep4Δ)||GlycoSwitch® strain, Man5 N-linked oligosaccharide structures (PEP4 protease deletion)||Details|
|pGlycoSwitch®–GnT–I||Zeocin selection vector for expression of GlcNAc transferase I activity in Pichia pastoris||Details|