Off-target glycans encountered along the synthetic biology route toward humanized N-glycans in Pichia pastoris.

The glycosylation pathways of several eukaryotic protein expression hosts are being engineered to enable the production of therapeutic glycoproteins with humanized application-customized glycan structures. In several expression hosts, this has been quite successful, but one caveat is that the new N-glycan structures inadvertently might be substrates for one or more of the multitude of endogenous glycosyltransferases in such heterologous background. This then results in the formation of novel, undesired glycan structures, which often remain insufficiently characterized.

Congenital disorder of fucosylation type 2c (LADII) presenting with short stature and developmental delay with minimal adhesion defect.

Leukocyte adhesion deficiency type II is a hereditary disorder of neutrophil migration caused by mutations in the guanosine diphosphate-fucose transporter gene (SLC35C1). In these patients, inability to generate key fucosylated molecules including sialyl Lewis X leads to leukocytosis and recurrent infections, in addition to short stature and developmental delay. We report two brothers with short stature and developmental delay who are compound heterozygotes for novel mutations in SLC35C1 resulting in partial in vivo defects in fucosylation.

CD44 and HCELL: preventing hematogenous metastasis at step 1.

Despite great strides in our knowledge of the genetic and epigenetic changes underlying malignancy, we have limited information on the molecular basis of metastasis. Over 90% of cancer deaths are caused by spread of tumor cells from a primary site to distant organs and tissues, highlighting the pressing need to define the molecular effectors of cancer metastasis. Mounting evidence suggests that circulating tumor cells (CTCs) home to specific tissues by hijacking the normal leukocyte trafficking mechanisms.

Fed-batch fermentation of GM-CSF-producing glycoengineered Pichia pastoris under controlled specific growth rate.

Background: Yeast expression systems with altered N-glycosylation are now available to produce glycoproteins with homogenous, defined N-glycans. However, data on the behaviour of these strains in high cell density cultivation are scarce.

Results: Here, we report on cultivations under controlled specific growth rate of a GlycoSwitch-Man5 Pichia pastoris strain producing Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) at high levels (hundreds of milligrams per liter).

The HAC1 gene from Pichia pastoris: characterization and effect of its overexpression on the production of secreted, surface displayed and membrane proteins.

Background: The unfolded protein response (UPR) in eukaryotes upregulates factors that restore ER homeostasis upon protein folding stress and in yeast is activated by a non-conventional splicing of the HAC1 mRNA. The spliced HAC1 mRNA encodes an active transcription factor that binds to UPR-responsive elements in the promoter of UPR target genes. Overexpression of the HAC1 gene of S.

Engineering complex-type N-glycosylation in Pichia pastoris using GlycoSwitch technology.

Here we provide a protocol for engineering the N-glycosylation pathway of the yeast Pichia pastoris. The general strategy consists of the disruption of an endogenous glycosyltransferase gene (OCH1) and the stepwise introduction of heterologous glycosylation enzymes. Each engineering step results in the introduction of one glycosidase or glycosyltransferase activity into the Pichia endoplasmic reticulum or Golgi complex and consists of a number of stages: transformation with the appropriate GlycoSwitch vector, small-scale cultivation of a number of transformants, sugar analysis and heterologous protein expression analysis.

Pichia surface display: display of proteins on the surface of glycoengineered Pichia pastoris strains.

Expression of proteins on the surface of yeasts has a wide range of applications in biotechnology, such as directed evolution of proteins for increased affinity and thermal stability, screening of antibody libraries, epitope mapping, and use as whole-cell biocatalysts. However, hyperglycosylation can interfere with overall protein accessibility on the surface. Therefore, the less elaborate hyperglycosylation in wild type Pichia pastoris and the availability of glycoengineered strains make this yeast an excellent alternative for surface display of glycoproteins.

Fishing for lectins from diverse sequence libraries by yeast surface display - an exploratory study.

The establishment of a robust technology platform for the expression cloning of carbohydrate-binding proteins remains a key challenge in glycomics. Here we explore the utility of using yeast surface display (YSD) technology in the interaction-based lectin cloning from complete cDNA libraries. This should pave the way for more detailed studies of protein-carbohydrate interactions.