Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

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Plant Biotechnology meets Immunology : plant-based expression of immunologically relevant proteins
Wilbers, R.H.P. - \ 2015
Wageningen University. Promotor(en): Jaap Bakker, co-promotor(en): Arjen Schots; Geert Smant. - Wageningen : Wageningen University - ISBN 9789462574335 - 229
plantenbiotechnologie - immunologie - planten - eiwitten - farmaceutische eiwitten - interleukine 10 - ontstekingsremmers - biologische activiteit - cytokinen - genexpressie - transforming growth factor - wormen - recombinant eiwitten - glycoproteïnen - plant biotechnology - immunology - plants - proteins - pharmaceutical proteins - interleukin 10 - antiinflammatory agents - biological activity - cytokines - gene expression - helminths - recombinant proteins - glycoproteins

The incidence of inflammatory disorders in industrialized countries has dramatically increased over the last decennia, which is believed to result from a change in life-style. Treatment of these inflammatory disorders relies on the intervention in immune responses thereby restoring homeostasis. For now, many inflammatory disorders are treated with broad-acting immunosuppressive drugs or monoclonal antibodies that specifically target pro-inflammatory molecules of the immune system. An alternative therapeutic approach would be to use immunomodulatory proteins that are naturally involved in re-establishing immune homeostasis. This thesis describes the plant-based expression of a variety of immunomodulatory cytokines that may be used as biopharmaceutical proteins in the future. Furthermore, this thesis contains a pioneering chapter on the plant-based expression of immunomodulatory helminth-secreted glycoproteins.

In Chapter 2 we describe the plant-based expression of the immune-regulatory cytokine human transforming growth factor β1 (TGF-β1). By co-expressing human furin with latent TGF-β1 we were able to engineer the post-translational proteolytic processing of TGF-β1, which enabled the production of biologically active TGF-β1. In Chapter 3 we reveal that aggregation is a major production bottleneck for the anti-inflammatory cytokine interleukin-10 (IL-10). By protein engineering we were able to prevent aggregation and created a biologically active fusion protein of IL-10. In Chapter 4 we express biologically active IL-22 in plants. We reveal that, in contrast to current literature, its activity is independent of the presence of N-glycans or their composition. This chapter further reveals that plants offer a powerful tool to allow investigation into the role of N-glycans in protein folding and biological activity of glycoproteins. In Chapter 5 we further explore the potential of glyco-engineering in plants by engineering helminth-like N-glycans. We produce large quantities of two major egg antigens from Schistosoma mansoni and successfully engineer Lewis X, LDN and LDNF N-glycan structures. These plant biotechnological research lines are a showcase for the potential of engineering proteins as well as post-translational modifications in plants with special emphasis on N-glycan engineering. Altogether, the results presented in the first four chapters reveal the remarkable flexibility of plants as a production platform for recombinant proteins. It showcases the potential of engineering proteins as well as post-translational modifications in plants, but it especially highlights the engineering of tailor made N-glycans in plants. This, combined with the speed of transient expression by means of agroinfiltration, makes transient expression in Nicotiana benthamiana a powerful tool to study the role of N-glycans on glycoprotein function.

In parallel to these plant biotechnological research lines, we also developed an in vitro model system based on mouse bone marrow-derived cells to study immunological responses. We used this model to obtain clues on why IL-10 therapy has not been as successful as previously anticipated. In Chapter 6 we have set-up biological activity assays based on bone marrow-derived cells and reveal that IL-10 activity is dependent on both IL-10R1 and IL-10R2, but not IL-10R2-associated signalling via Tyk2. We also show that interactions between IL-10R1 and IL-10R2 (both intracellular and extracellular) reduce cellular binding of IL-10, but are crucial to initiate IL-10 mediated signalling. Furthermore, we observed that macrophages and dendritic cells respond differently to IL-10. This was further investigated in Chapter 7 where we reveal that GM-CSF (the cytokine used to differentiate dendritic cells) is responsible for negatively regulating early IL-10-mediated responses. Strikingly, GM-CSF does not strongly affect the IL-10-induced activation of the transcription factor STAT3. Instead, GM-CSF induces strong constitutive phosphorylation of GSK-3β, a signalling component downstream of the PI3K/Akt pathway. These immunological chapters give novel insights on the mechanism of initiating IL-10-induced signalling and on the possible integration of signal transduction pathways elicited by different cytokines. Ultimately this knowledge could provide us with new therapeutic strategies to treat inflammatory disorders.

Protein biomarker-based screening for detection of recombinant bovine somatotropin abuse in dairy cows
Ludwig, S.K.J. - \ 2014
Wageningen University. Promotor(en): Michel Nielen, co-promotor(en): Leen van Ginkel. - Wageningen : Wageningen University - ISBN 9789462570146 - 248
somatotropine - melkvee - besmetters - biomarkers - recombinant eiwitten - toxicologie - detectie - somatotropin - dairy cattle - contaminants - recombinant proteins - toxicology - detection

Recombinant bovine somatotropin (rbST) is a 22 kDa proteohormone, which can be used to increase milk production in dairy cows. It has been marketed since 1994 and while its use in food production is approved in several countries, such as the US, it is banned in the EU since 2000. To enforce the ban on rbST in the EU and to control for ‘rbST-free’ –labelling in the US, detection methods are required that identify whether rbST has been used. Existing rbST detection methods focus on the detection of rbST itself in bovine serum. The recombinant form of the hormone has one amino acid exchanged at the N-terminus of the protein. RbST can therefore be potentially discriminated from the endogenous bST by mass spectrometric methods. Other methods employ sandwich enzyme-linked immunosorbent assays (ELISAs) with antibodies having a higher affinity to rbST than bST. These methods, however mainly lack sensitivity, reproducibility or selectivity for rbST and are therefore not widely applied. Hence, no method has been implemented so far to monitor rbST abuse in dairy farming. Screening methods developed for veterinary drug residue control in the EU have to perform according to Commission Decision 2002/657/EC and have to identify at least 95 % of the treated animals.

An alternative approach for rbST abuse detection is the analysis of rbST-dependent biomarkers. A biomarker is defined as an indicator of normal physiological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention. Therefore, the levels of rbST-dependent protein biomarkers are either up- or downregulated after administration of rbST and rbST-specific biomarker profiles can be used to detect its abuse. RbST exerts similar physiological actions in the cow’s body as the endogenous bST. Therefore, proteins involved in the regulatory circuit of bST have been chosen as candidate biomarkers, such as insulin-like growth factor-1 (IGF-1), IGF binding protein 2 (IGFBP2) and osteocalcin. Additionally to that, the administration of rbST induces anti-rbST antibodies in the cow’s body, which can be detected as biomarkers. This approach is according to the growth hormone (GH) abuse detection in sports doping control, where solely protein biomarker profiles are used to identify the abuse.

Chapter 2 introduces protein biomarkers and how biomarkers can be used in sports doping and veterinary control to detect the abuse of illegal substances. The advantages of using biomarkers are that the biological effect of a substance usually lasts longer than the substance itself can be detected and therewith, the window of detection is expanded. Moreover, since different substances exert similar effects on physiological machineries for growth or production enhancement, biomarker-based-detection methods have the potential to detect a whole class of substances. Furthermore, low-dose mixtures of different banned substances, which might escape from direct detection of each individual substance used, could be still detected by the combined effect they exert. In this chapter, protein biomarker-based detection strategies are discussed against generic challenges in biomarker discovery and method development.

Part I of the thesis concerns biomarker analysis in serum and plasma samples from cattle, which are analysed using laboratory-based equipment.A triplex flow cytometric immunoassay (FCIA), which combines the detection of three rbST-dependent biomarkers, viz. IGF-1, IGFBP2 and anti-rbST antibodies is demonstrated in Chapter 3. Serum samples from treated and untreated dairy cows from a single animal study were analysed using this triplex FCIA. Characteristic treatment-dependent responses for all three individual biomarkers were shown. These results were combined using the statistical model k-nearest neighbours (kNN). This model discriminated rbST-treated from untreated cows with a truepositive rate of 89.1 % and a true-negative rate of 97.7 %.

This triplex FCIA was further extended with the biomarker osteocalcin and the resulting fourplex FCIA was used for biomarker profiling in serum samples from rbST-treated and untreated cows from two independent rbST treatment studies. In Chapter 4, different data analysis approaches were tested with the aim to detect the highest possible number of true-positive samples. The statistical model kNN was used on all 11 possible biomarker combinations and the combination of the biomarkers osteocalcin and endogenously produced antibodies against rbST proved to be very reliable and correctly predicted 95 % of the samples of treated cows starting from the second rbST injection until the end of the treatment period and even thereafter. With the same biomarker combination, only 12 % of the samples of untreated animals appeared false-positive. This reliability meets the requirements of Commission Decision 2002/657/EC for screening methods in veterinary control.

It can be expected that rbST-dependent biomarkers also show a response upon other treatments. Therefore in Chapter 5, the fourplex FCIA for rbST abuse detection was applied to bovines treated with steroids, such as estradiol, dexamethasone and prednisolone. Each treatment resulted in a specific plasma biomarker profile for IGF-1, IGFBP2, osteocalcin and anti-rbST antibodies, which could be distinguished from the profile of untreated animals. Therefore, the fourplex biomarker FCIA is, apart from rbST, also capable of detecting treatment with other growth-promoting agents and clearly shows the potential of biomarker profiling as a screening method in veterinary control.

Part II of the thesis focusses on protein biomarker analysis in milk samples and the change from laboratory-based to on-site analysis. In Chapter 6, the detection of anti-rbST antibodies in raw milk samples was demonstrated, which discriminated rbST-treated from untreated cows with a 67 % true-positive and 94 % true-negative rate. The laboratory-based assay was also applied to simulated tank milk and pasteurized milk samples. Using milk as a sample matrix for detection has the advantages of non-invasive sampling, and for tank milk analysis at the farm only one milk sample is needed to screen the whole farm for rbST (ab)use.

As a next step in Chapter 7, this assay was translated to an on-site pre-screening platform including a cellphone. Using this on-site platform, samples can be tested at the point where they were taken. Only samples that are suspect are transported to a laboratory for further analysis. To this end, a cellphone-based fluorescence imaging platform for the detection of anti-rbST antibodies in milk extracts was developed, which is based on a microsphere fluorescence immunoassay. After performing the assay, the fluorescence is excited by UV LEDs embedded in a dedicated cellphone attachment and the emitted fluorescence light is imaged by the cellphone camera. The fluorescence micro-images were analysed using a custom-developed Android application running on the same cellphone and milk samples from rbST-treated and untreated cows were discriminated.

Also in milk samples, the simultaneous detection of several biomarkers is advantageous as they can increase the confidence of a positive finding. Therefore in Chapter 8, a protein microarray-based platform for multiple rbST biomarker detection on a cellphone is presented, which detects anti-rbST antibodies and IGF-1 in milk samples. The 48 microspots on the microarray were labelled with Quantum Dots depending on the biomarker levels in the sample. Quantum Dot fluorescence was detected by the cellphone camera and the same opto-mechanical attachment as in Chapter 7 and images were analysed by custom software. RbST-treated clearly showed a treatment-dependent biomarker profile in milk that could be discriminated from the profile of untreated cows.

Future research should focus on the simultaneous detection of different targets of interest in milk samples, such as hormones, allergens, antibiotics, contaminants and other substances, all at the same time using the microarray platform on the cellphone. Moreover, sample handling can be facilitated by the use of pre-fabricated microfluidic devices including all required assay reagents. With the work presented in this thesis, screening for rbST abuse in serum and milk becomes possible: in the laboratory and on-site. The future implementation of these testing platforms for rbST abuse detection is a major leap forward concerning the enforcement of the rbST ban in the EU and concerning the value of protein biomarker-based approaches in veterinary control.

Understanding of the self- and co-assembly behavior of recombinant protein polymers : from design to implementation
Golinska, M.D. - \ 2014
Wageningen University. Promotor(en): Martien Cohen Stuart, co-promotor(en): Renko de Vries; Frits de Wolf. - Wageningen : Wageningen University - ISBN 9789461738134 - 165
biopolymeren - eiwitten - zelf-assemblage - recombinant eiwitten - elastine - collageen - enzymatische cross-linking - glutaminase - biopolymers - proteins - self assembly - recombinant proteins - elastin - collagen - enzymatic cross-linking

A key part of the growing field of biomedical sciences deals with the development of new, controlled and biocompatible biomaterials. In this thesis we present results on the design, production, purification and characterization of stimuli responsive protein polymers that could ultimately be used in that field. Protein-polymers are composed of two or three blocks that are able to self- and co-assemble. An important theme in the thesis is to highlight the specific advantages of our new protein polymer for future biomedical applications.

We have used recombinant DNA techniques and expression in methylotrophic yeast Pichia pastorisfor protein production. DNAs encoding various polypeptide blocks were designed, produced using general molecular biology techniques and combined into synthetic genes for protein polymers. Synthetic genes were cloned into P. pastorisexpression vector pPIC9 that integrates into the yeast genome. Yields were high, typically reaching gram-per-liter (of medium).

In Chapter 2we study dual-stimuli (pH, temperature) responsive silk-elastin-like protein polymers (SELPs). These polymers were designed to self- and co-assembly, controlled by both pH and temperature. The first protein is a diblock S24E40composed of 24 silk-like (S) repeats and 40 elastin-like (E) repeats. The other protein is a triblock S12C4E40, in which the Sand Eblocks are separated by a random coil block (C4) that serves as an inert ‘spacer’.

A C2SHSHC2protein polymer, which consists of a pH responsive, positively charged silk-like middle block SH, flanked by two random coil collagen-like blocks C2was studied in Chapter 3.

For this protein have studied fibril formation and gelling properties at pH values close to neutral, that are crucial for biomedical applications. We find that at physiological pH, these proteins form self-healing physical gels that fulfill many requirements for use in biomedical applications.

In Chapter 4we test the influence of enzymatic cross-linking on elasticity andmechanical properties ofhydrogels that include collagen-like domains, using microbial transglutaminase (mTGase) as an enzymatic crosslinker that catalyzes the coupling of glutamines to lysines. We show that even though the collagen-like blocks are not particularly good substrates for the mTGase, the few cross-links that are made have a strong effect on the physical properties of the protein-polymer hydrogels. For silk-collagen fiber gels, the elastic moduli can be increased by a factor of five, and for thermosensitive collagen hydrogels, the enzymatic cross-linking induces qualitatively new behavior, namely shape-memory of hydrogels.

Finally, we study the co-assembly of very asymmetric diblock copolymers with oppositely charged sodium poly(acrylic acid) (NaPAA) with a range of molar masses (Chapter 5). This asymmetric diblock consists of a cationic block of 12 lysines connected to a long (400 amino acid) collagen-like block with a net charge that is nearly zero. For shorter Na-PAA chains, spherical complex coacervates micelles are formed, as have been studied before in our lab. But, for long Na-PAA chains a new self-assembled structure is found: a single (Na-PAA) chain pearl-necklace of complex-coacervate micelles.

The general discussion of the thesis in Chapter 6, focuses on recombinant and natural hydrogels as biomaterials. We point out the specific advantages of recombinant proteins and also indicate where these still need to be improved in order to be used in biomedical applications. Finally, we make some suggestions for further research in this area.

Molecular farming in tobacco hairy roots by triggering the secretion of a pharmaceutical antibody
Häkkinen, S.T. ; Raven, N. ; Henquet, M.G.L. ; Laukkanen, M.L. ; Anderlei, T. ; Pitkänen, J.P. ; Twyman, R.M. ; Bosch, H.J. ; Oksman-Caldentey, K.M. ; Schillberg, S. ; Ritala, A. - \ 2014
Biotechnology and Bioengineering 111 (2014)2. - ISSN 0006-3592 - p. 336 - 346.
enzyme replacement therapy - cell-suspension-culture - monoclonal-antibody - recombinant proteins - hyoscyamus-muticus - transgenic plants - tissue-cultures - nitrogen - optimization - glucocerebrosidase
Recombinant pharmaceutical proteins expressed in hairy root cultures can be secreted into the medium to improve product homogeneity and to facilitate purification, although this may result in significant degradation if the protein is inherently unstable or particularly susceptible to proteases. To address these challenges, we used a design of experiments approach to develop an optimized induction protocol for the cultivation of tobacco hairy roots secreting the full-size monoclonal antibody M12. The antibody yield was enhanced 30-fold by the addition of 14¿g/L KNO3, 19¿mg/L 1-naphthaleneacetic acid and 1.5¿g/L of the stabilizing agent polyvinylpyrrolidone. Analysis of hairy root cross sections revealed that the optimized medium induced lateral root formation and morphological changes in the inner cortex and pericycle cells, indicating that the improved productivity was at least partially based on the enhanced efficiency of antibody secretion. We found that 57% of the antibody was secreted, yielding 5.9¿mg of product per liter of induction medium. Both the secreted and intracellular forms of the antibody could be isolated by protein A affinity chromatography and their functionality was confirmed using vitronectin-binding assays. Glycan analysis revealed three major plant complex-type glycans on both forms of the antibody, although the secreted form was more homogeneous due to the predominance of a specific glycoform. Tobacco hairy root cultures therefore offer a practical solution for the production of homogeneous pharmaceutical antibodies in containment. Biotechnol. Bioeng. 2013;9999: 1–11.
Single Mutation in Shine-Dalgarno-Like Sequence Present in the Amino Terminal of Lactate Dehydrogenase of Plasmodium Effects the Production of an Eukaryotic Protein Expressed in a Prokaryotic System
Cicek, M. ; Mutlu, O. ; Erdemir, A. ; Ozkan, E. ; Saricay, Y. ; Turgut-Balik, D. - \ 2013
Molecular Biotechnology 54 (2013)2. - ISSN 1073-6085 - p. 602 - 608.
human malaria parasite - escherichia-coli - messenger-rna - recombinant proteins - bacillus-subtilis - ribosomal-rna - q-beta - falciparum - genome - gene
One of the most important step in structure-based drug design studies is obtaining the protein in active form after cloning the target gene. In one of our previous study, it was determined that an internal Shine-Dalgarno-like sequence present just before the third methionine at N-terminus of wild type lactate dehydrogenase enzyme of Plasmodium falciparum prevent the translation of full length protein. Inspection of the same region in P. vivax LDH, which was overproduced as an active enzyme, indicated that the codon preference in the same region was slightly different than the codon preference of wild type PfLDH. In this study, 5'-GGAGGC-3' sequence of P. vivax that codes for two glycine residues just before the third methionine was exchanged to 5'-GGAGGA-3', by mimicking P. falciparum LDH, to prove the possible effects of having an internal SD-like sequence when expressing an eukaryotic protein in a prokaryotic system. Exchange was made by site-directed mutagenesis. Results indicated that having two glycine residues with an internal SD-like sequence (GGAGGA) just before the third methionine abolishes the enzyme activity due to the preference of the prokaryotic system used for the expression. This study emphasizes the awareness of use of a prokaryotic system to overproduce an eukaryotic protein.
Pichia pastoris as a cell factory for the secreted production of tunable collagen-inspired gel-forming proteins
Silva, C.I.F. da - \ 2013
Wageningen University. Promotor(en): Gerrit Eggink, co-promotor(en): Frits de Wolf. - S.l. : s.n. - ISBN 9789461734570 - 163
pichia pastoris - eiwitten - collageen - gelering - biologische productie - recombinant eiwitten - proteins - collagen - gelation - biological production - recombinant proteins

It is the ability to establish triple helices and assemble into supramolecular structures, which makes collagen and its denature counterpart, gelatine, interesting for the food and biomedical industry. Collagen and gelatine array of applications is quite extensive, ranging from gelling agents in food, emulsifiers in photographic films, fillers in cosmetics and structural networks in drug delivery and tissue engineering systems. Despite their vast use, the animal origin of these protein materials, mainly bovine and porcine tissues, poses the risk of pathogen transmission or allergic reactions. However, mammalian tissues contain more than one type of collagen resulting in a lack of reproducibility and heterogeneity between batches. The variability in composition and structure of animal-derived collagen and gelatine presents a significant challenge for those using these proteins in medical applications, where reliable, predictable and traceable materials are essential. Additionally, the harvest of collagen and gelatine from animal tissues restrains us to the use of what is already available in nature and in commercial quantities. Thanks to the development of DNA recombinant technologies, the capability to design genes and obtain proteins with custom made conformations and functionalities has become a current day activity. In accordance, a myriad of recombinant systems have been developed to produce high yields of high quality heterologous proteins. However, there is no universal recombinant system that can be used by default. The choice between recombinant systems depends of the protein being expressed, the need for post translational modifications, the concentration of protein required and the costs associated to its production. In the present thesis, Pichia pastoris was the recombinant system of choice due to its many advantages. It is able to reach high cell densities on defined low-cost media, has a tight regulated AOX methanol induced promoter and can produce properly folded proteins with correct disulphide bond formation and other eukaryotic posttranslational modifications. The ability to secrete high amounts of heterologous proteins and low amounts of endogenous proteases is further considered as a plus by the biomedical industry, as downstream processing costs can be considerably reduced when compared to other industrial strains as Saccharomyces cerevisae or Escherichia coli. The possibility to custom design collagen and gelatine-inspired biomaterials and use a reliable production system like P. pastoris, is highly appealing for the safety demanding biomedical field. Our group has previously used this system for the secreted production of collagen-inspired proteins formed by two triple helix-forming (Pro-Gly-Pro)9 end blocks and one long random coil middle block. The triblock system allows the formation of gel networks with defined properties and porosities since i) triple helix is exclusively established by the (Pro-Gly-Pro) end blocks, connected by ii) a random coil middle block of known length. It was shown that the middle block can be independently tuned to originate gel networks with predictable rheological properties and drug delivery profiles and had no effect over the triple helices melting temperature. Only the collagen-inspired (Pro-Gly-Pro)9 end block domain seemed to contribute to the triple helix Tm which was ~ 41°C at a 1.1.mM protein concentration. Despite the possibility to establish triple helices (Tm ~ 41°C) at P. pastoris growth temperature (30°C), the secreted expression of these proteins was not impaired. Also, the elongation of the middle block proved to be no burden for P. pastoris secretory capability, since similar secretion yields were found for triblocks with middle blocks of ~37 or ~73 kDa. These positive results, paved the way to the design of a series of triblock polymers with different end block lengths and hence Tm. However, every system as a limit and P. pastoris is no exception. In Chapter 2, it was demonstrated that the elongation of the collagen-inspired (Pro- Gly-Pro)9 domain to (Pro-Gly-Pro)16 resulted in a considerable lower secretion yield and partial protein degradation by protease Yapsin 1. This result could have been explained by i) higher prolyl-tRNA turnover requirements, ii) higher hydrophobic content resulting in hydrophobic interactions or iii) incapacity to process highly stable triple helical conformations (Tm of ~74°C). The restitution of the secretion yields and intact product secretion by designing a polymer with noncollagen- like end blocks, revealed that the protein secretion impairment was due to intracellular triple helix formation. The non-collagen-like endblocks have the same amino acid composition but randomized sequence as to avoid triple helix formation. The intact gel-forming polymer could only be obtained by recurring to a P. pastoris strain deficient in the periplasmic protease Yapsin 1. Although the use of Yapsin 1 knockout strain allowed the secretion of intact polymer it did not restore the high secretion yields. To know where the secretion bottleneck was occurring, a closer look at P. pastoris cells was conducted. In Chapter 3, electron microscopy was performed to observe where triple helix formation was occurring. ER expansion and vesicle formation were observed only when gel-/triple helix-forming proteins with a high Tm were being expressed. Such observations were absent in cells expressing proteins with mutant end-blocks, endblocks with equal amino acid composition but unable to establish triple helices. The presence of extra organelles could have resulted from protein aggregation due to triple helix formation or ER volume enlargement as an attempt to minimize protein aggregation. This result suggests that triple helix formation should be avoided in order to achieve high secretion yields of proteins able to establish highly stable triple helices. In Chapter 4, it was shown that the thermomechanical stability of the triple helices could be tuned by varying the length of the (Pro-Gly-Pro) end blocks through genetic engineering. An increase of the triple helix Tm could be achieved by increasing the number of hydrogen bonds involved without the need to resort to chemical (covalent) crosslinking. All triblock copolymers studied formed stable hydrogels, and, an increase of the end block length resulted in higher stabilities under mechanical stress. The variation of the thermostability could be understood in terms of i) a simple linear relationship between the length of the end block and the free energy of helix formation, or ii) its constituting entropic and enthalpic components. The comparison with triple helices formed by free (Pro-Gly-Pro)n peptides seemed to indicate that this relationship was not influenced by the nature of the middle blocks. Regarding the use of P. pastoris as a cell factory, it was observed that there is an inverse relation between triple helix stability and secreted production yield. Proteins able to establish triple helices with high thermostablity were secreted at a lower yield. In Chapter 5, it was shown that denaturing SDS-PAGE gels can monitor the ability of collagen-inspired proteins to form supramolecular assemblies. During the destaining process proteins that cannot establish triple helices diffuse out of the acrylamide gel, while triple helix-forming collagen-inspired proteins are still visible after destaining over-night. Furthermore, it was observed that the diffusion speed from the gel is related to the triple helix Tm. Proteins that can establish triple helices with high thermostability diffuse slower from the gel. In addition, the migration of the middle blocks in a SDS-PAGE gel revealed that subtle differences in migration speed can expose changes in the amino acid sequence of random coil proteins. During the course of this project several attempts were made to increase the secretion yield of collagen-like proteins with high thermo stability. While not all experiments were successful, their results do yield additional hypothesis for further research that could lead to a further improvement of P. pastoris as a cell factory for the production of collagen-like proteins. These experiments are discussed in detail in Chapter 6.

Characterization of PmHS2 glycosyltransferases for the controlled synthesis of heparosan : a precursor of heparin and heparan sulfate
Chavaroche, A.A.E. - \ 2011
Wageningen University. Promotor(en): Gerrit Eggink. - S.l. : s.n. - ISBN 9789085858805 - 169
industriële microbiologie - heparine - synthese - recombinant eiwitten - industrial microbiology - heparin - synthesis - recombinant proteins

Heparin (Hep), a highly sulfated and complex glycosaminoglycan polysaccharides, is worldwide used as an anticoagulant compound to prevent blood clotting during surgery. Heparin and its analogue, heparan sulfate (HS), have a large potential for medical applications. Nevertheless, the utilization of Hep/HS-based drugs in new therapeutic settings requires the synthesis of well-defined heparin and heparan sulfate-like molecules. Since neither the extraction from animal derivatives, nor the chemical synthesis, are suitable for the production of a large variety of defined Hep/HS polymers, there is a general interest in developing alternative systems enabling to tightly control Hep/HS synthesis. During the synthesis of heparin and heparan sulfate, the polymerization of the polysaccharide backbone, known as heparosan, determines the chain length and the size distribution of these polymers. Here, the Pasteurella multocida heparosan synthase PmHS2, a bacterial enzyme catalyzing the formation of heparosan polymers, was studied in detail in order to develop methods to control the polymer elongation.

Secreted production of an elastin-like polypeptide by Pichia pastoris
Schipperus, R. ; Teeuwen, R.L.M. ; Werten, M.W.T. ; Eggink, G. ; Wolf, F.A. de - \ 2009
Applied Microbiology and Biotechnology 85 (2009)2. - ISSN 0175-7598 - p. 293 - 301.
protein-based polymer - inverse temperature transitions - recombinant proteins - fermentation process - escherichia-coli - drug-delivery - in-vitro - expression - gene - purification
Elastin-like polypeptides (ELPs) are biocompatible designer polypeptides with inverse temperature transition behavior in solution. They have a wide variety of possible applications and a potential medical importance. Currently, production of ELPs is done at lab scale in Escherichia coli shake flask cultures. With a view to future large scale production, we demonstrate secreted production of ELPs in methanol-induced fed-batch cultures of Pichia pastoris and purification directly from the culture medium. The production of ELPs by P. pastoris proved to be pH dependent within the experimental pH range of pH 3 to 7, as an increasing yield was found in cultures grown at higher pH. Because ELP produced at pH 7 was partly degraded, a pH optimum for production of ELP was found at pH 6 with a yield of 255 mg of purified intact ELP per liter of cell-free medium
Design strategies for integrated protein purification processes: challenges, progress and outlook
Nfor, B. ; Ahamed, T. ; Dedem, G. ; Wielen, L. van der; Sandt, E. van de; Eppink, M.H.M. ; Ottens, M. - \ 2008
Journal of Chemical Technology and Biotechnology 83 (2008)2. - ISSN 0268-2575 - p. 124 - 132.
expert-system - physicochemical properties - exchange chromatography - biochemical processes - recombinant proteins - purify proteins - optimization - selection - bioseparations - bioprocesses
The key to successful and efficient protein purification is the selection of the most appropriate purification techniques and their combination in a logical way to obtain the desired purification in the minimum number of steps. However, the rationalization of protein purification process development is faced with a number of challenges. In this paper, the challenges in protein purification process development are captured. The state-of-the-art in protein purification process synthesis and design is reviewed and the strengths and weaknesses of the current strategies highlighted. Finally, views on the future directions of protein purification process development are presented
Tagging of MADS domain proteins for chromatin immunoprecipitation
Folter, S. de; Urbanus, S.L. ; Zuijlen, L. ; Kaufmann, K. ; Angenent, G.C. - \ 2007
BMC Plant Biology (2007). - ISSN 1471-2229 - p. 7 - 47.
floral homeotic gene - arabidopsis fruit-development - box gene - recombinant proteins - regulatory elements - negative regulation - ectopic expression - flower development - organ identity - purification
Most transcription factors fulfill their role in complexes and regulate their target genes upon binding to DNA motifs located in upstream regions or introns. To date, knowledge about transcription factor target genes and their corresponding transcription factor binding sites are still very limited. Two related methods that allow in vivo identification of transcription factor binding sites are chromatin immunoprecipitation (ChIP) and chromatin affinity purification (ChAP). For ChAP, the protein of interest is tagged with a peptide or protein, which can be used for affinity purification of the protein-DNA complex and hence, the identification of the target gene.
Improving and assessing viral vectors for recombinant protein production in plants
Lacorte, C.C. - \ 2006
Wageningen University. Promotor(en): R.W. Goldbach, co-promotor(en): M.W. Prins. - [S.l.] : S.n. - ISBN 9085045398 - 119
planten - recombinant eiwitten - genetische vectoren - genexpressie - aardappelvirus x - tabaksmozaïekvirus - merkergenen - recombinant vaccins - plants - recombinant proteins - genetic vectors - gene expression - potato virus x - Tobacco mosaic virus - marker genes - recombinant vaccines
Productie van farmaceutische eiwitmoleculen in paddestoelen : kansen voor de de Nederlandse paddestoelensector?
Baars, J.J.P. ; Sonnenberg, A.S.M. - \ 2005
Horst : Praktijkonderzoek Plant & Omgeving, Sector Paddestoelen - 46
teeltsystemen - paddestoelen - medicinale fungi - genetische modificatie - recombinant eiwitten - aangrijpingspunten voor medicijnen - geneesmiddelenontwikkeling - geneesmiddelen - cropping systems - mushrooms - medicinal fungi - genetic engineering - recombinant proteins - drug targets - drug development - drugs
Onderzoek naar alternatieve gebruiksvomen van paddestoelen in de medicinale sfeer. Gedurende de laatste jaren wordt een nieuwe klasse geneesmiddelen ontwikkeld; medicijnen op basis van eiwitmoleculen. Aangezien van deze medicijnen relatief grote hoeveelheden nodig zijn, verwacht men dat de productiecapaciteit in de toekomst niet toereikend zal zijn. Het gebruik van genetisch gemodificeerde paddestoelen kan dan voor de productie van medicinale eiwitten een uitkomst bieden. Gezien het feit dat momenteel nog geen enkel recombinant eiwit door paddestoelvormende schimmels wordt geproduceerd, zal het naar verwachting ruwweg 2 decennia duren voordat grootschalige productie van medicinale eiwitten in genetisch gemodificeerde paddenstoelen op teeltbedrijven aan de orde is. Als paddenstoelen als productieplatform gebruikt gaan worden voor de productie van medicinale eiwitten, is er een ander teeltsysteem vereist dan het huidige. De initiatieven en ideeën met betrekking tot systeeminnovatie in de paddenstoelteelt sluiten hierop al voor een gedeelte aan.
Recombinant gelatin and collagen
Wolf, F.A. de - \ 2003
In: Industrial proteins in perspective / Aalbersberg, W.Y., Hamer, R.J., Jasperse, P., Amsterdam : Elsevier (Progress in Biotechnology 23) - ISBN 0444513949 - p. 215 - 216.
gelatine - collageen - biotechnologie - recombinant eiwitten - gelatin - collagen - biotechnology - recombinant proteins
Gelatins from yeast provide novel possibilities
Wolf, F.A. de; Werten, M.W.T. - \ 2001
Industrial Proteins 9 (2001)2. - ISSN 1381-0022 - p. 9 - 12.
gelatine - stabiliseermiddelen - collageen - eigenschappen - gisten - methanol - recombinant eiwitten - eiwitten - gelatin - stabilizers - collagen - properties - yeasts - recombinant proteins - proteins
Recombinant gelatinen (natuurlijk of tailor made) zijn eeen goed alternatief ter voorkoming van prion besmetting die bse veroorzaakt
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