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1.  A scalable low-cost cGMP process for clinical grade production of the HIV inhibitor 5P12-RANTES in Pichia pastoris 
In the continued absence of an effective anti-HIV vaccine, approximately 2 million new HIV infections occur every year, with over 95% of these in developing countries. Calls have been made for the development of anti-HIV drugs that can be formulated for topical use to prevent HIV transmission during sexual intercourse. Because these drugs are principally destined for use in low-resource regions, achieving production costs that are as low as possible is an absolute requirement. 5P12-RANTES, an analog of the human chemokine protein RANTES/CCL5, is a highly potent HIV entry inhibitor which acts by achieving potent blockade of the principal HIV coreceptor, CCR5. Here we describe the development and optimization of a scalable low-cost production process for 5P12-RANTES based on expression in Pichia pastoris. At pilot (150 L) scale, this cGMP compliant process yielded 30 g of clinical grade 5P12-RANTES. As well as providing sufficient material for the first stage of clinical development, this process represents an important step towards achieving production of 5P12-RANTES at a cost and scale appropriate to meet needs for topical HIV prevention worldwide.
•New medicines are required to prevent the spread of HIV/AIDS in low-resource regions.•5P12-RANTES, a chemokine analog, is a promising new candidate drug.•We describe a process for producing clinical grade cGMP 5P12-RANTES in Pichia pastoris.•This is a key step to achieving production at cost and scale appropriate for use worldwide.
PMCID: PMC4725576  PMID: 26506568
Pichia; Yeast; Process development; Chemokine; HIV; Microbicide; CCR5; 5P12-RANTES; Biopharmaceutical; cGMP
2.  Characterization of genome-wide ordered sequence-tagged Mycobacterium mutant libraries by Cartesian Pooling-Coordinate Sequencing 
Nature Communications  2015;6:7106.
Reverse genetics research approaches require the availability of methods to rapidly generate specific mutants. Alternatively, where these methods are lacking, the construction of pre-characterized libraries of mutants can be extremely valuable. However, this can be complex, expensive and time consuming. Here, we describe a robust, easy to implement parallel sequencing-based method (Cartesian Pooling-Coordinate Sequencing or CP-CSeq) that reports both on the identity as well as on the location of sequence-tagged biological entities in well-plate archived clone collections. We demonstrate this approach using a transposon insertion mutant library of the Mycobacterium bovis BCG vaccine strain, providing the largest resource of mutants in any strain of the M. tuberculosis complex. The method is applicable to any entity for which sequence-tagged identification is possible.
The generation of characterized panels of specific mutants is an essential but time-consuming step of reverse genetic studies. Here Vandewalle et al. describe CP-CSeq, an easy to implement parallel sequencing method for rapid library construction.
PMCID: PMC4432585  PMID: 25960123
3.  Routine serum creatinine measurements: how well do we perform? 
BMC Nephrology  2015;16:21.
The first aim of the study was to investigate the accuracy and intra-laboratory variation of serum creatinine measurements in clinical laboratories in Flanders. The second purpose was to check the effect of this variation in serum creatinine concentration results on the calculated estimated glomerular filtration rate (eGFR) and the impact on classification of patients into a chronic kidney disease (CKD) stage.
26 routine instruments were included, representing 13 different types of analyzers from 6 manufacturers and covering all current methodologies (Jaffe, compensated Jaffe, enzymatic liquid and dry chemistry methods). Target values of five serum pools (creatinine concentrations ranging from 35 to 934 μmol/L) were assigned by the gold standard method (ID-GC/MS).
Intra-run CV (%) (n = 5) and bias (%) from the target values were higher for low creatinine concentrations. Especially Jaffe and enzymatic dry chemistry methods showed a higher error. The calculated eGFR values corresponding with the reported creatinine concentration ranges resulted in a different CKD classification in 47% of cases.
Although most creatinine assays claim to be traceable to the gold standard (ID-GC/MS), large inter-assay differences still exist. The inaccuracy in the lower concentration range is of particular concern and may lead to clinical misinterpretation when the creatinine-based eGFR of the patient is used for CKD staging. Further research to improve harmonization between methods is required.
Electronic supplementary material
The online version of this article (doi:10.1186/s12882-015-0012-x) contains supplementary material, which is available to authorized users.
PMCID: PMC4358903  PMID: 25803560
Creatinine; External quality assessment; Glomerular filtration rate
4.  Genome dynamics of the human embryonic kidney 293 lineage in response to cell biology manipulations 
Nature Communications  2014;5:4767.
The HEK293 human cell lineage is widely used in cell biology and biotechnology. Here we use whole-genome resequencing of six 293 cell lines to study the dynamics of this aneuploid genome in response to the manipulations used to generate common 293 cell derivatives, such as transformation and stable clone generation (293T); suspension growth adaptation (293S); and cytotoxic lectin selection (293SG). Remarkably, we observe that copy number alteration detection could identify the genomic region that enabled cell survival under selective conditions (i.c. ricin selection). Furthermore, we present methods to detect human/vector genome breakpoints and a user-friendly visualization tool for the 293 genome data. We also establish that the genome structure composition is in steady state for most of these cell lines when standard cell culturing conditions are used. This resource enables novel and more informed studies with 293 cells, and we will distribute the sequenced cell lines to this effect.
The human embryonic kidney 293 (HEK293) cell lineage is widely used in cell biology and biotechnology. Here, the authors apply whole genome resequencing methods to characterise genomic variation in six HEK293 cell lines and suggest that this variation could affect experiments using these cell lines.
PMCID: PMC4166678  PMID: 25182477
5.  Enhanced membrane protein expression by engineering increased intracellular membrane production 
Membrane protein research is frequently hampered by the low natural abundance of these proteins in cells and typically relies on recombinant gene expression. Different expression systems, like mammalian cells, insect cells, bacteria and yeast are being used, but very few research efforts have been directed towards specific host cell customization for enhanced expression of membrane proteins. Here we show that by increasing the intracellular membrane production by interfering with a key enzymatic step of lipid synthesis, enhanced expression of membrane proteins in yeast is achieved.
We engineered the oleotrophic yeast, Yarrowia lipolytica, by deleting the phosphatidic acid phosphatase, PAH1, which led to massive proliferation of endoplasmic reticulum (ER) membranes. For all eight tested representatives of different integral membrane protein families, we obtained enhanced protein accumulation levels and in some cases enhanced proteolytic integrity in the ∆pah1 strain. We analysed the adenosine A2AR G-protein coupled receptor case in more detail and found that concomitant induction of the unfolded protein response in the ∆pah1 strain enhanced the specific ligand binding activity of the receptor. These data indicate an improved quality control mechanism for membrane proteins accumulating in yeast cells with proliferated ER.
We conclude that redirecting the metabolic flux of fatty acids away from triacylglycerol- and sterylester-storage towards membrane phospholipid synthesis by PAH1 gene inactivation, provides a valuable approach to enhance eukaryotic membrane protein production. Complementary to this improvement in membrane protein quantity, UPR co-induction further enhances the quality of the membrane protein in terms of its proper folding and biological activity. Importantly, since these pathways are conserved in all eukaryotes, it will be of interest to investigate similar engineering approaches in other cell types of biotechnological interest, such as insect cells and mammalian cells.
PMCID: PMC3878919  PMID: 24321035
6.  Engineering Yarrowia lipolytica to Produce Glycoproteins Homogeneously Modified with the Universal Man3GlcNAc2 N-Glycan Core 
PLoS ONE  2012;7(6):e39976.
Yarrowia lipolytica is a dimorphic yeast that efficiently secretes various heterologous proteins and is classified as “generally recognized as safe.” Therefore, it is an attractive protein production host. However, yeasts modify glycoproteins with non-human high mannose-type N-glycans. These structures reduce the protein half-life in vivo and can be immunogenic in man. Here, we describe how we genetically engineered N-glycan biosynthesis in Yarrowia lipolytica so that it produces Man3GlcNAc2 structures on its glycoproteins. We obtained unprecedented levels of homogeneity of this glycanstructure. This is the ideal starting point for building human-like sugars. Disruption of the ALG3 gene resulted in modification of proteins mainly with Man5GlcNAc2 and GlcMan5GlcNAc2 glycans, and to a lesser extent with Glc2Man5GlcNAc2 glycans. To avoid underoccupancy of glycosylation sites, we concomitantly overexpressed ALG6. We also explored several approaches to remove the terminal glucose residues, which hamper further humanization of N-glycosylation; overexpression of the heterodimeric Apergillus niger glucosidase II proved to be the most effective approach. Finally, we overexpressed an α-1,2-mannosidase to obtain Man3GlcNAc2 structures, which are substrates for the synthesis of complex-type glycans. The final Yarrowia lipolytica strain produces proteins glycosylated with the trimannosyl core N-glycan (Man3GlcNAc2), which is the common core of all complex-type N-glycans. All these glycans can be constructed on the obtained trimannosyl N-glycan using either in vivo or in vitro modification with the appropriate glycosyltransferases. The results demonstrate the high potential of Yarrowia lipolytica to be developed as an efficient expression system for the production of glycoproteins with humanized glycans.
PMCID: PMC3386995  PMID: 22768188
7.  Engineering the yeast Yarrowia lipolytica for the production of therapeutic proteins homogeneously glycosylated with Man8GlcNAc2 and Man5GlcNAc2 
Protein-based therapeutics represent the fastest growing class of compounds in the pharmaceutical industry. This has created an increasing demand for powerful expression systems. Yeast systems are widely used, convenient and cost-effective. Yarrowia lipolytica is a suitable host that is generally regarded as safe (GRAS). Yeasts, however, modify their glycoproteins with heterogeneous glycans containing mainly mannoses, which complicates downstream processing and often interferes with protein function in man. Our aim was to glyco-engineer Y. lipolytica to abolish the heterogeneous, yeast-specific glycosylation and to obtain homogeneous human high-mannose type glycosylation.
We engineered Y. lipolytica to produce homogeneous human-type terminal-mannose glycosylated proteins, i.e. glycosylated with Man8GlcNAc2 or Man5GlcNAc2. First, we inactivated the yeast-specific Golgi α-1,6-mannosyltransferases YlOch1p and YlMnn9p; the former inactivation yielded a strain producing homogeneous Man8GlcNAc2 glycoproteins. We tested this strain by expressing glucocerebrosidase and found that the hypermannosylation-related heterogeneity was eliminated. Furthermore, detailed analysis of N-glycans showed that YlOch1p and YlMnn9p, despite some initial uncertainty about their function, are most likely the α-1,6-mannosyltransferases responsible for the addition of the first and second mannose residue, respectively, to the glycan backbone. Second, introduction of an ER-retained α-1,2-mannosidase yielded a strain producing proteins homogeneously glycosylated with Man5GlcNAc2. The use of the endogenous LIP2pre signal sequence and codon optimization greatly improved the efficiency of this enzyme.
We generated a Y. lipolytica expression platform for the production of heterologous glycoproteins that are homogenously glycosylated with either Man8GlcNAc2 or Man5GlcNAc2 N-glycans. This platform expands the utility of Y. lipolytica as a heterologous expression host and makes it possible to produce glycoproteins with homogeneously glycosylated N-glycans of the human high-mannose-type, which greatly broadens the application scope of these glycoproteins.
PMCID: PMC3512530  PMID: 22548968
8.  A novel strategy for the comprehensive analysis of the biomolecular composition of isolated plasma membranes 
A methodology for rapid, high-purity isolation of plasma membranes using superparamagnetic nanoparticles is described. The method is illustrated with high-resolution proteomic, glycomic and lipidomic analyses of presenilin-deficient cells.
We present a novel strategy based on cationic phospholipids-coated superparamagnetic nanoparticles (SPMNPs) to isolate plasma membranes with very high purity and at a preparative scale.The SPMNP-based isolation method is compatible with subsequent analysis of the biomolecular composition of plasma membranes, including proteomics, lipidomics and N-glycomics analysis.A comparative ‘omics' analysis of plasma membranes from wild-type, presenilin-deficient and human presenilin-1-rescued fibroblasts revealed convergent changes in proteins and lipids, suggesting an underlying endosomal transport defect.Our methodology allows for the systematic set-up of comprehensive plasma membrane inventories: alterations in the composition of the cell surface may potentially identify novel biomarkers or drug targets.
One of the major goals of this paper was to establish a robust method for plasma membrane (PM) isolation in order to perform a full analysis of their biomolecular composition. Using thermal decomposition, we manufactured superparamagnetic nanoparticles (SPMNPs) that we rendered water soluble and monodisperse by subsequent coupling of NH2 phospholipids. When incubated with cell monolayers, these cationic phospholipids-SPMNPs remain predominately localized at the cell surface. We applied these unexpected feature of phospholipids-SPMNPs to establish a novel protocol to isolate high yields of highly pure PMs. Due to the superb quality and quantity of isolated PM fractions, we could perform a comprehensive and comparative biomolecular profiling on this subcellular compartment that included proteomics, N-glycoproteomics, lipidomics and N-glycan profiling. This method was subsequently applied to compare the biomolecular composition of PMs isolated from wild-type and presenilin-deficient and human presenilin-1-rescued mouse embryonic fibroblasts (MEFs). For the first time, we succeeded in identifying convergent changes in the biomolecular composition of the cell surface caused by presenilin gene deficiency. Furthermore, the observed proteomic/cholesterol changes were restored in presenilin-deficient MEFs rescued with the human presenilin-1 ortholog. These (subtle) changes in protein and lipid composition suggest an underlying endosomal transport defect in presenilin-deficient cell lines that is the subject of ongoing research.
Moreover, and extending the versatility of our method, we could show that our PM isolations are compatible with fluorophore-assisted carbohydrate electrophoresis (FACE), allowing for N-glycan profiling of the cell surface. Hitherto, the N-glycan composition was measured on total cell extracts where the sensitivity to detect mature N-glycan chains is significantly hampered by the higher abundances of intracellular immature N-glycan intermediates. Finally, using the γ-secretase protein complex as a model, we confirm that we can study the activity and composition of active protein complexes in their native membrane environment. Our strategy incorporates for the first time most available omics analyses and this on a single isolated membrane compartment. As such, it allows for the monitoring and identification of systematic changes at the cell surface, for instance during differentiation and polarization or as a consequence of environmental insults, ER-stress, apoptotic stimuli and altered lipogenesis. The identification of alterations in the PM protein and lipid composition, as they occur as a consequence of disease, is therefore of paramount importance in several fields of experimental medicine, including immunology, cancer and stem cell research. Our methodology provides also a foundation to systematically start collecting PM ‘fingerprints' of an increasing number of cells. The resulting integrated and comprehensive databases may become the starting point of a ‘subcellular systems biology' approach of the cell's limiting membrane. Since PM proteins provide many pathological relevant biomarkers representing two-thirds of the currently used drug targets, this novel technology has great potential for biomedical and pharmaceutical applications.
We manufactured a novel type of lipid-coated superparamagnetic nanoparticles that allow for a rapid isolation of plasma membranes (PMs), enabling high-resolution proteomic, glycomic and lipidomic analyses of the cell surface. We used this technology to characterize the effects of presenilin knockout on the PM composition of mouse embryonic fibroblasts. We found that many proteins are selectively downregulated at the cell surface of presenilin knockout cells concomitant with lowered surface levels of cholesterol and certain sphingomyelin species, indicating defects in specific endosomal transport routes to and/or from the cell surface. Snapshots of N-glycoproteomics and cell surface glycan profiling further underscored the power and versatility of this novel methodology. Since PM proteins provide many pathologically relevant biomarkers representing two-thirds of the currently used drug targets, this novel technology has great potential for biomedical and pharmaceutical applications.
PMCID: PMC3261717  PMID: 22027552
eukaryotic cell systems; glycomics; lipidomics; presenilin, proteomics
9.  Disruption of the SapM locus in Mycobacterium bovis BCG improves its protective efficacy as a vaccine against M. tuberculosis 
EMBO Molecular Medicine  2011;3(4):222-234.
Mycobacterium bovis bacille Calmette-Guerin (BCG) provides only limited protection against pulmonary tuberculosis. We tested the hypothesis that BCG might have retained immunomodulatory properties from its pathogenic parent that limit its protective immunogenicity. Mutation of the molecules involved in immunomodulation might then improve its vaccine potential. We studied the vaccine potential of BCG mutants deficient in the secreted acid phosphatase, SapM, or in the capping of the immunomodulatory ManLAM cell wall component with α-1,2-oligomannoside. Both systemic and intratracheal challenge of mice with Mycobacterium tuberculosis following vaccination showed that the SapM mutant, compared to the parental BCG vaccine, provided better protection: it led to longer-term survival. Persistence of the SapM-mutated BCG in vivo resembled that of the parental BCG indicating that this mutation will likely not compromise the safety of the BCG vaccine. The SapM mutant BCG vaccine was more effective than the parental vaccine in inducing recruitment and activation of CD11c+MHC-IIintCD40int dendritic cells (DCs) to the draining lymph nodes. Thus, SapM acts by inhibiting recruitment of DCs and their activation at the site of vaccination.
PMCID: PMC3377067  PMID: 21328541
Mycobacterium; SapM; tuberculosis; vaccine; BCG
10.  Fed-batch fermentation of GM-CSF-producing glycoengineered Pichia pastoris under controlled specific growth rate 
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.
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). We demonstrate that homogenous Man5GlcNAc2 N-glycosylation of the secreted proteins is achieved at all specific growth rates tested.
Together, these data illustrate that the GlycoSwitch-Man5 P. pastoris is a robust production strain for homogenously N-glycosylated proteins.
PMCID: PMC3004841  PMID: 21092289
11.  The HAC1 gene from Pichia pastoris: characterization and effect of its overexpression on the production of secreted, surface displayed and membrane proteins 
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. cerevisiae can reportedly lead to increased production of heterologous proteins. To further such studies in the biotechnology favored yeast Pichia pastoris, we cloned and characterized the P. pastoris HAC1 gene and the splice event.
We identified the HAC1 homologue of P. pastoris and its splice sites. Surprisingly, we could not find evidence for the non-spliced HAC1 mRNA when P. pastoris was cultivated in a standard growth medium without any endoplasmic reticulum stress inducers, indicating that the UPR is constitutively active to some extent in this organism. After identification of the sequence encoding active Hac1p we evaluated the effect of its overexpression in Pichia. The KAR2 UPR-responsive gene was strongly upregulated. Electron microscopy revealed an expansion of the intracellular membranes in Hac1p-overexpressing strains. We then evaluated the effect of inducible and constitutive UPR induction on the production of secreted, surface displayed and membrane proteins. Wherever Hac1p overexpression affected heterologous protein expression levels, this effect was always stronger when Hac1p expression was inducible rather than constitutive. Depending on the heterologous protein, co-expression of Hac1p increased, decreased or had no effect on expression level. Moreover, α-mating factor prepro signal processing of a G-protein coupled receptor was more efficient with Hac1p overexpression; resulting in a significantly improved homogeneity.
Overexpression of P. pastoris Hac1p can be used to increase the production of heterologous proteins but needs to be evaluated on a case by case basis. Inducible Hac1p expression is more effective than constitutive expression. Correct processing and thus homogeneity of proteins that are difficult to express, such as GPCRs, can be increased by co-expression with Hac1p.
PMCID: PMC2905327  PMID: 20591165
12.  Open access to sequence: Browsing the Pichia pastoris genome 
The first genome sequences of the important yeast protein production host Pichia pastoris have been released into the public domain this spring. In order to provide the scientific community easy and versatile access to the sequence, two web-sites have been installed as a resource for genomic sequence, gene and protein information for P. pastoris: A GBrowse based genome browser was set up at and a genome portal with gene annotation and browsing functionality at . Both websites are offering information on gene annotation and function, regulation and structure.
In addition, a WiKi based platform allows all users to create additional information on genes, proteins, physiology and other items of P. pastoris research, so that the Pichia community can benefit from exchange of knowledge, data and materials.
PMCID: PMC2768684  PMID: 19835590
13.  O Mannosylation of α-Dystroglycan Is Essential for Lymphocytic Choriomeningitis Virus Receptor Function 
Journal of Virology  2005;79(22):14297-14308.
α-Dystroglycan (α-DG) was identified as a common receptor for lymphocytic choriomeningitis virus (LCMV) and several other arenaviruses including the human pathogenic Lassa fever virus. Initial work postulated that interactions between arenavirus glycoproteins and α-DG are based on protein-protein interactions. We found, however, that susceptibility toward LCMV infection differed in various cell lines despite them expressing comparable levels of DG, suggesting that posttranslational modifications of α-DG would be involved in viral receptor function. Here, we demonstrate that glycosylation of α-DG, and in particular, O mannosylation, which is a rare type of O-linked glycosylation in mammals, is essential for LCMV receptor function. Cells that are defective in components of the O-mannosylation pathway showed strikingly reduced LCMV infectibility. As defective O mannosylation is associated with severe clinical symptoms in mammals such as congenital muscular dystrophies, it is likely that LCMV and potentially other arenaviruses may have selected this conserved and crucial posttranslational modification as the primary target structure for cell entry and infection.
PMCID: PMC1280192  PMID: 16254364
14.  In Vivo Synthesis of Mammalian-Like, Hybrid-Type N-Glycans in Pichia pastoris 
The Pichia pastoris N-glycosylation pathway is only partially homologous to the pathway in human cells. In the Golgi apparatus, human cells synthesize complex oligosaccharides, whereas Pichia cells form mannose structures that can contain up to 40 mannose residues. This hypermannosylation of secreted glycoproteins hampers the downstream processing of heterologously expressed glycoproteins and leads to the production of protein-based therapeutic agents that are rapidly cleared from the blood because of the presence of terminal mannose residues. Here, we describe engineering of the P. pastoris N-glycosylation pathway to produce nonhyperglycosylated hybrid glycans. This was accomplished by inactivation of OCH1 and overexpression of an α-1,2-mannosidase retained in the endoplasmic reticulum and N-acetylglucosaminyltransferase I and β-1,4-galactosyltransferase retained in the Golgi apparatus. The engineered strain synthesized a nonsialylated hybrid-type N-linked oligosaccharide structure on its glycoproteins. The procedures which we developed allow glycan engineering of any P. pastoris expression strain and can yield up to 90% homogeneous protein-linked oligosaccharides.
PMCID: PMC404441  PMID: 15128513

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