Publications by authors named "Joop van den Heuvel"

45 Publications

A SARS-CoV-2 neutralizing antibody selected from COVID-19 patients binds to the ACE2-RBD interface and is tolerant to most known RBD mutations.

Cell Rep 2021 Jul 7;36(4):109433. Epub 2021 Jul 7.

Städtisches Klinikum Braunschweig gGmbH, Holwedestraße 16, 38118 Braunschweig, Germany.

The novel betacoronavirus severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) causes a form of severe pneumonia disease called coronavirus disease 2019 (COVID-19). To develop human neutralizing anti-SARS-CoV-2 antibodies, antibody gene libraries from convalescent COVID-19 patients were constructed and recombinant antibody fragments (scFv) against the receptor-binding domain (RBD) of the spike protein were selected by phage display. The antibody STE90-C11 shows a subnanometer IC in a plaque-based live SARS-CoV-2 neutralization assay. The in vivo efficacy of the antibody is demonstrated in the Syrian hamster and in the human angiotensin-converting enzyme 2 (hACE2) mice model. The crystal structure of STE90-C11 Fab in complex with SARS-CoV-2-RBD is solved at 2.0 Å resolution showing that the antibody binds at the same region as ACE2 to RBD. The binding and inhibition of STE90-C11 is not blocked by many known emerging RBD mutations. STE90-C11-derived human IgG1 with FcγR-silenced Fc (COR-101) is undergoing Phase Ib/II clinical trials for the treatment of moderate to severe COVID-19.
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http://dx.doi.org/10.1016/j.celrep.2021.109433DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8260561PMC
July 2021

Reproducible and Easy Production of Mammalian Proteins by Transient Gene Expression in High Five Insect Cells.

Methods Mol Biol 2021 ;2305:129-140

Department of Structure and Function of Proteins, Helmholtz Zentrum für Infektionsforschung GmbH, Braunschweig, Germany.

The expression of mammalian recombinant proteins in insect cell lines using transient-plasmid-based gene expression enables the production of high-quality protein samples. Here, the procedure for virus-free transient gene expression (TGE) in High Five insect cells is described in detail. The parameters that determine the efficiency and reproducibility of the method are presented in a robust protocol for easy implementation and set-up of the method. The applicability of the TGE method in High Five cells for proteomic, structural, and functional analysis of the expressed proteins is shown.
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http://dx.doi.org/10.1007/978-1-0716-1406-8_6DOI Listing
January 2021

Validation of the Production of Antibodies in Different Formats in the HEK 293 Transient Gene Expression System.

Methods Mol Biol 2021 ;2247:59-76

Department of Structure and Function of Proteins, Helmholtz Zentrum für Infektionsforschung GmbH, Braunschweig, Germany.

Mammalian cells are the most commonly used production system for therapeutic antibodies. Protocols for the expression of recombinant antibodies in HEK293-6E cells in different antibody formats are described in detail. As model, antibodies against Kallikrein-related peptidase 7 (KLK7) were used. KLK7 is a key player in skin homeostasis and represents an emerging target for pharmacological interventions. Potent inhibitors can not only help to elucidate physiological and pathophysiological functions but also serve as a new archetype for the treatment of inflammatory skin disorders. Phage display-derived affinity-matured human anti-KLK7 antibodies were converted to scFv-Fc, IgG, and Fab formats and transiently produced in the mammalian HEK293-6E system. For the production of the corresponding antigen-KLK7-the baculovirus expression vector system (BEVS) and virus-free expression in Hi5 insect cells were used in a comparative approach. The target proteins were isolated by various chromatographic methods in a one- or multistep purification strategy. Ultimately, the interaction between anti-KLK7 and KLK7 was characterized using biolayer interferometry. Here, protocols for the expression of recombinant antibodies in different formats are presented and compared for their specific features. Furthermore, biolayer interferometry (BLI), a fast and high-throughput biophysical analytical technique to evaluate the kinetic binding constant and affinity constant of the different anti-KLK7 antibody formats against Kallikrein-related peptidase 7 is presented.
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http://dx.doi.org/10.1007/978-1-0716-1126-5_4DOI Listing
March 2021

Baculovirus-free insect cell expression system for high yield antibody and antigen production.

Sci Rep 2020 12 7;10(1):21393. Epub 2020 Dec 7.

Department of Biotechnology, Technische Universität Braunschweig, Spielmannstraße 7, 38106, Braunschweig, Germany.

Antibodies are essential tools for therapy and diagnostics. Yet, production remains expensive as it is mostly done in mammalian expression systems. As most therapeutic IgG require mammalian glycosylation to interact with the human immune system, other expression systems are rarely used for production. However, for neutralizing antibodies that are not required to activate the human immune system as well as antibodies used in diagnostics, a cheaper production system would be advantageous. In our study, we show cost-efficient, easy and high yield production of antibodies as well as various secreted antigens including Interleukins and SARS-CoV-2 related proteins in a baculovirus-free insect cell expression system. To improve yields, we optimized the expression vector, media and feeding strategies. In addition, we showed the feasibility of lyophilization of the insect cell produced antibodies. Furthermore, stability and activity of the antibodies was compared to antibodies produced by Expi293F cells revealing a lower aggregation of antibodies originating from High Five cell production. Finally, the newly established High Five expression system was compared to the Expi293F mammalian expression system in regard of yield and costs. Most interestingly, all tested proteins were producible in our High Five cell expression system what was not the case in the Expi293F system, hinting that the High Five cell system is especially suited to produce difficult-to-express target proteins.
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http://dx.doi.org/10.1038/s41598-020-78425-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7721901PMC
December 2020

Synthetic studies of cystobactamids as antibiotics and bacterial imaging carriers lead to compounds with high efficacy.

Chem Sci 2019 Dec 10;11(5):1316-1334. Epub 2019 Dec 10.

Department of Chemical Biology, Helmholtz Centre for Infection Research Inhoffenstrasse 7 38124 Braunschweig Germany

There is an alarming scarcity of novel chemical matter with bioactivity against multidrug-resistant Gram-negative bacterial pathogens. Cystobactamids, recently discovered natural products from myxobacteria, are an exception to this trend. Their unusual chemical structure, composed of oligomeric -aminobenzoic acid moieties, is associated with a high antibiotic activity through the inhibition of gyrase. In this study, structural determinants of cystobactamid's antibacterial potency were defined at five positions, which were varied using three different synthetic routes to the cystobactamid scaffold. The potency against could be increased ten-fold to an MIC (minimum inhibitory concentration) of 0.06 μg mL, and the previously identified spectrum gap of could be closed compared to the natural products (MIC of 0.5 μg mL). Proteolytic degradation of cystobactamids by the resistance factor AlbD was prevented by an amide-triazole replacement. Conjugation of cystobactamid's N-terminal tetrapeptide to a Bodipy moiety induced the selective localization of the fluorophore for bacterial imaging purposes. Finally, a first proof of concept was obtained in an infection mouse model, where derivative led to the reduction of bacterial loads (cfu, colony-forming units) in muscle, lung and kidneys by five orders of magnitude compared to vehicle-treated mice. These findings qualify cystobactamids as highly promising lead structures against infections caused by Gram-positive and Gram-negative bacterial pathogens.
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http://dx.doi.org/10.1039/c9sc04769gDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8148378PMC
December 2019

Crystal structure of -aconitate decarboxylase reveals the impact of naturally occurring human mutations on itaconate synthesis.

Proc Natl Acad Sci U S A 2019 10 23;116(41):20644-20654. Epub 2019 Sep 23.

Research Group Biomarkers for Infectious Diseases, TWINCORE Centre for Experimental and Clinical Infection Research, 30625 Hannover, Germany;

-Aconitate decarboxylase (CAD, also known as ACOD1 or Irg1) converts -aconitate to itaconate and plays central roles in linking innate immunity with metabolism and in the biotechnological production of itaconic acid by We have elucidated the crystal structures of human and murine CADs and compared their enzymological properties to CAD from Recombinant CAD is fully active in vitro without a cofactor. Murine CAD has the highest catalytic activity, whereas CAD is best adapted to a more acidic pH. CAD is not homologous to any known decarboxylase and appears to have evolved from prokaryotic enzymes that bind negatively charged substrates. CADs are homodimers, the active center is located in the interface between 2 distinct subdomains, and structural modeling revealed conservation in zebrafish and We identified 8 active-site residues critical for CAD function and rare naturally occurring human mutations in the active site that abolished CAD activity, as well as a variant (Asn152Ser) that increased CAD activity and is common (allele frequency 20%) in African ethnicity. These results open the way for 1) assessing the potential impact of human CAD variants on disease risk at the population level, 2) developing therapeutic interventions to modify CAD activity, and 3) improving CAD efficiency for biotechnological production of itaconic acid.
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http://dx.doi.org/10.1073/pnas.1908770116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6789909PMC
October 2019

Identifying parameters to improve the reproducibility of transient gene expression in High Five cells.

PLoS One 2019 6;14(6):e0217878. Epub 2019 Jun 6.

Department Recombinant Protein Expression, Helmholtz Centre for Infection Research, Braunschweig, Lower Saxony, Germany.

Virus-free, transient gene expression (TGE) in High Five cells was recently presented as an efficient protein production method. However, published TGE protocols have not been standardized to a general protocol. Therefore, reproducibility and implementation of the method in other labs remains difficult. The aim of this study is to analyse the parameters determining the reproducibility of the TGE in insect cells. Here, we identified that using linear 40 kDa PEI instead of 25 kDa PEI was one of the most important aspects to improve TGE. Furthermore, DNA amount, DNA:PEI ratio, growth phase of the cells before transfection, passage number, the origin of the High-Five cell isolates and the type of cultivation medium were considered. Interestingly, a correlation of the passage number to the DNA content of single cells (ploidy) and to the transfection efficacy could be shown. The optimal conditions for critical parameters were used to establish a robust TGE method. Finally, we compared the achieved product yields in High Five cells using our improved TGE method with both the baculoviral expression system and TGE in the mammalian HEK293-6E cell line. In conclusion, the presented robust TGE protocol in High Five cells is easy to establish and produces ample amounts of high-quality recombinant protein, bridging the gap in expression level of this method to the well-established mammalian TGE in HEK293 cells as well as to the baculoviral expression vector system (BEVS).
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0217878PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6553862PMC
February 2020

ER intrabody-mediated inhibition of interferon α secretion by mouse macrophages and dendritic cells.

PLoS One 2019 16;14(4):e0215062. Epub 2019 Apr 16.

Department Structure and Function of Proteins, Helmholtz Centre for Infection Research, Braunschweig, Germany.

Interferon α (IFNα) counteracts viral infections by activating various IFNα-stimulated genes (ISGs). These genes encode proteins that block viral transport into the host cell and inhibit viral replication, gene transcription and translation. Due to the existence of 14 different, highly homologous isoforms of mouse IFNα, an IFNα knockout mouse has not yet been established by genetic knockout strategies. An scFv intrabody for holding back IFNα isoforms in the endoplasmic reticulum (ER) and thus counteracting IFNα secretion is reported. The intrabody was constructed from the variable domains of the anti-mouse IFNα rat monoclonal antibody 4EA1 recognizing the 5 isoforms IFNα1, IFNα2, IFNα4, IFNα5, IFNα6. A soluble form of the intrabody had a KD of 39 nM to IFNα4. It could be demonstrated that the anti-IFNα intrabody inhibits clearly recombinant IFNα4 secretion by HEK293T cells. In addition, the secretion of IFNα4 was effectively inhibited in stably transfected intrabody expressing RAW 264.7 macrophages and dendritic D1 cells. Colocalization of the intrabody with IFNα4 and the ER marker calnexin in HEK293T cells indicated complex formation of intrabody and IFNα4 inside the ER. Intracellular binding of intrabody and antigen was confirmed by co-immunoprecipitation. Complexes of endogenous IFNα and intrabody could be visualized in the ER of Poly (I:C) stimulated RAW 264.7 macrophages and D1 dendritic cells. Infection of macrophages and dendritic cells with the vesicular stomatitis virus VSV-AV2 is attenuated by IFNα and IFNβ. The intrabody increased virus proliferation in RAW 264.7 macrophages and D1 dendritic cells under IFNβ-neutralizing conditions. To analyze if all IFNα isoforms are recognized by the intrabody was not in the focus of this study. Provided that binding of the intrabody to all isoforms was confirmed, the establishment of transgenic intrabody mice would be promising for studying the function of IFNα during viral infection and autoimmune diseases.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0215062PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6467385PMC
December 2019

Investigations on the mode of action of gephyronic acid, an inhibitor of eukaryotic protein translation from myxobacteria.

PLoS One 2018 31;13(7):e0201605. Epub 2018 Jul 31.

Department of Chemical Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany.

The identification of inhibitors of eukaryotic protein biosynthesis, which are targeting single translation factors, is highly demanded. Here we report on a small molecule inhibitor, gephyronic acid, isolated from the myxobacterium Archangium gephyra that inhibits growth of transformed mammalian cell lines in the nM range. In direct comparison, primary human fibroblasts were shown to be less sensitive to toxic effects of gephyronic acid than cancer-derived cells. Gephyronic acid is targeting the protein translation system. Experiments with IRES dual luciferase reporter assays identified it as an inhibitor of the translation initiation. DARTs approaches, co-localization studies and pull-down assays indicate that the binding partner could be the eukaryotic initiation factor 2 subunit alpha (eIF2α). Gephyronic acid seems to have a different mode of action than the structurally related polyketides tedanolide, myriaporone, and pederin and is a valuable tool for investigating the eukaryotic translation system. Because cancer derived cells were found to be especially sensitive, gephyronic acid could potentially find use as a drug candidate.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0201605PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6067752PMC
February 2019

Baculovirus-driven protein expression in insect cells: A benchmarking study.

J Struct Biol 2018 08 12;203(2):71-80. Epub 2018 Mar 12.

Max-Planck Institute of Biochemistry, Biochemistry Core Facility, Am Klopferspitz 18, 82152 Martinsried, Germany. Electronic address:

Baculovirus-insect cell expression system has become one of the most widely used eukaryotic expression systems for heterologous protein production in many laboratories. The availability of robust insect cell lines, serum-free media, a range of vectors and commercially-packaged kits have supported the demand for maximizing the exploitation of the baculovirus-insect cell expression system. Naturally, this resulted in varied strategies adopted by different laboratories to optimize protein production. Most laboratories have preference in using either the E. coli transposition-based recombination bacmid technology (e.g. Bac-to-Bac®) or homologous recombination transfection within insect cells (e.g. flashBAC™). Limited data is presented in the literature to benchmark the protocols used for these baculovirus vectors to facilitate the selection of a system for optimal production of target proteins. Taking advantage of the Protein Production and Purification Partnership in Europe (P4EU) scientific network, a benchmarking initiative was designed to compare the diverse protocols established in thirteen individual laboratories. This benchmarking initiative compared the expression of four selected intracellular proteins (mouse Dicer-2, 204 kDa; human ABL1 wildtype, 126 kDa; human FMRP, 68 kDa; viral vNS1-H1, 76 kDa). Here, we present the expression and purification results on these proteins and highlight the significant differences in expression yields obtained using different commercially-packaged baculovirus vectors. The highest expression level for difficult-to-express intracellular protein candidates were observed with the EmBacY baculovirus vector system.
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http://dx.doi.org/10.1016/j.jsb.2018.03.004DOI Listing
August 2018

Characterization and structural determination of a new anti-MET function-blocking antibody with binding epitope distinct from the ligand binding domain.

Sci Rep 2017 08 21;7(1):9000. Epub 2017 Aug 21.

Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge, CB2 1GA, UK.

The growth and motility factor Hepatocyte Growth Factor/Scatter Factor (HGF/SF) and its receptor, the product of the MET proto-oncogene, promote invasion and metastasis of tumor cells and have been considered potential targets for cancer therapy. We generated a new Met-blocking antibody which binds outside the ligand-binding site, and determined the crystal structure of the Fab in complex with its target, which identifies the binding site as the Met Ig1 domain. The antibody, 107_A07, inhibited HGF/SF-induced cell migration and proliferation in vitro and inhibited growth of tumor xenografts in vivo. In biochemical assays, 107_A07 competes with both HGF/SF and its truncated splice variant NK1 for MET binding, despite the location of the antibody epitope on a domain (Ig1) not reported to bind NK1 or HGF/SF. Overlay of the Fab-MET crystal structure with the InternalinB-MET crystal structure shows that the 107_A07 Fab comes into close proximity with the HGF/SF-binding SEMA domain when MET is in the "compact", InternalinB-bound conformation, but not when MET is in the "open" conformation. These findings provide further support for the importance of the "compact" conformation of the MET extracellular domain, and the relevance of this conformation to HGF/SF binding and signaling.
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http://dx.doi.org/10.1038/s41598-017-09460-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5567289PMC
August 2017

Not Limited to E. coli: Versatile Expression Vectors for Mammalian Protein Expression.

Methods Mol Biol 2017 ;1586:313-324

Helmholtz Zentrum für Infektionsforschung GmbH, Inhoffenstrasse 7, 38124, Braunschweig, Germany.

Recombinant protein expression is not limited to E. coli or other prokaryotic systems. It is inevitable to use eukaryotic systems in order to express challenging mammalian proteins. Eukaryotic systems are able to perform complex posttranslational modifications like protein processing, phosphorylation, glycosylation, which are essential for stability and functionality of many proteins. Different eukaryotic protein expression systems employing yeast, insect, or mammalian cell lines are established with each having its own advantages and disadvantages. Often it is quite difficult to decide which will be the most optimal expression system as this depends highly on the protein itself. Expression in stable cell lines requires substantial screening of expressible constructs prior to developing a stable expression cell line. To achieve fast screening by transient expression in multiple hosts, versatile vectors can be applied. In this chapter, we present an overview of the most common multi-host vectors, which allow for fast expression analysis without tedious (re)cloning of the gene of interest in several different protein production systems. The protocols in this chapter describe the latest methods for fast transient expression in insect and mammalian cell lines.
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http://dx.doi.org/10.1007/978-1-4939-6887-9_20DOI Listing
February 2018

Discovery of LRE1 as a specific and allosteric inhibitor of soluble adenylyl cyclase.

Nat Chem Biol 2016 10 22;12(10):838-44. Epub 2016 Aug 22.

Department of Pharmacology, Weill Cornell Medical College, New York, New York, USA.

The prototypical second messenger cAMP regulates a wide variety of physiological processes. It can simultaneously mediate diverse functions by acting locally in independently regulated microdomains. In mammalian cells, two types of adenylyl cyclase generate cAMP: G-protein-regulated transmembrane adenylyl cyclases and bicarbonate-, calcium- and ATP-regulated soluble adenylyl cyclase (sAC). Because each type of cyclase regulates distinct microdomains, methods to distinguish between them are needed to understand cAMP signaling. We developed a mass-spectrometry-based adenylyl cyclase assay, which we used to identify a new sAC-specific inhibitor, LRE1. LRE1 bound to the bicarbonate activator binding site and inhibited sAC via a unique allosteric mechanism. LRE1 prevented sAC-dependent processes in cellular and physiological systems, and it will facilitate exploration of the therapeutic potential of sAC inhibition.
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http://dx.doi.org/10.1038/nchembio.2151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5030147PMC
October 2016

Assembling Multi-subunit Complexes Using Mammalian Expression.

Adv Exp Med Biol 2016 ;896:225-38

Department of Structure and Function of Proteins, Helmholtz Centre for Infection Research, Braunschweig, Germany.

In this chapter conventional and emerging new technologies for the production of complex biologics in mammalian expression systems are summarized. The essential features of the most relevant methods to generate stable production cell lines for the expression of recombinant multi-protein complexes are described. Especially the promising multiple targeted integration strategy by Flp or CRISPR/Cas9 mediated recombination and their future impact on multi-protein expression are highlighted.
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http://dx.doi.org/10.1007/978-3-319-27216-0_15DOI Listing
September 2016

Bithionol Potently Inhibits Human Soluble Adenylyl Cyclase through Binding to the Allosteric Activator Site.

J Biol Chem 2016 Apr 9;291(18):9776-84. Epub 2016 Mar 9.

From the Department of Biochemistry, University of Bayreuth, 95440 Bayreuth, Germany,

The signaling molecule cAMP regulates functions ranging from bacterial transcription to mammalian memory. In mammals, cAMP is synthesized by nine transmembrane adenylyl cyclases (ACs) and one soluble AC (sAC). Despite similarities in their catalytic domains, these ACs differ in regulation. Transmembrane ACs respond to G proteins, whereas sAC is uniquely activated by bicarbonate. Via bicarbonate regulation, sAC acts as a physiological sensor for pH/bicarbonate/CO2, and it has been implicated as a therapeutic target, e.g. for diabetes, glaucoma, and a male contraceptive. Here we identify the bisphenols bithionol and hexachlorophene as potent, sAC-specific inhibitors. Inhibition appears mostly non-competitive with the substrate ATP, indicating that they act via an allosteric site. To analyze the interaction details, we solved a crystal structure of an sAC·bithionol complex. The structure reveals that the compounds are selective for sAC because they bind to the sAC-specific, allosteric binding site for the physiological activator bicarbonate. Structural comparison of the bithionol complex with apo-sAC and other sAC·ligand complexes along with mutagenesis experiments reveals an allosteric mechanism of inhibition; the compound induces rearrangements of substrate binding residues and of Arg(176), a trigger between the active site and allosteric site. Our results thus provide 1) novel insights into the communication between allosteric regulatory and active sites, 2) a novel mechanism for sAC inhibition, and 3) pharmacological compounds targeting this allosteric site and utilizing this mode of inhibition. These studies provide support for the future development of sAC-modulating drugs.
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http://dx.doi.org/10.1074/jbc.M115.708255DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4850313PMC
April 2016

Identification of Essential Genetic Baculoviral Elements for Recombinant Protein Expression by Transactivation in Sf21 Insect Cells.

PLoS One 2016 2;11(3):e0149424. Epub 2016 Mar 2.

Department of Structure and Function of Proteins, Research Group Recombinant Protein Expression, Helmholtz Centre for Infection Research, Braunschweig, Germany.

The Baculovirus Expression Vector System (BEVS) is widely used to produce high amounts of recombinant proteins. Nevertheless, generating recombinant baculovirus in high quality is rather time-consuming and labor-intensive. Alternatively, virus-free expression in insect cells did not achieve similar expression levels for most proteins so far. The transactivation method is a promising approach for protein expression in Sf21 cells. It combines advantages of BEVS and plasmid-based expression by activating strong virus-dependent promoters on a transfected plasmid by baculoviral coinfection. Here, we identified expression elements required for transactivation. Therefore, we designed several vectors comprising different viral promoters or promoter combinations and tested them for eGFP expression using the automated BioLector microcultivation system. Remarkably, only the combination of the very late promoter p10 together with the homologous region 5 (hr5) could boost expression during transactivation. Other elements, like p10 alone or the late viral promoter polH, did not respond to transactivation. A new combination of hr5 and p10 with the strongest immediate early OpMNPV viral promoter OpIE2 improved the yield of eGFP by ~25% in comparison to the previous applied hr5-IE1-p10 expression cassette. Furthermore, we observed a strong influence of the transcription termination sequence and vector backbone on the level of expression. Finally, the expression levels for transactivation, BEVS and solely plasmid-based expression were compared for the marker protein eGFP, underlining the potential of transactivation for fast recombinant protein expression in Sf21 cells. In conclusion, essential elements for transactivation could be identified. The optimal elements were applied to generate an improved vector applicable in virus-free plasmid-based expression, transactivation and BEVS.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0149424PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4774975PMC
July 2016

Fast plasmid based protein expression analysis in insect cells using an automated SplitGFP screen.

Biotechnol Bioeng 2016 09 10;113(9):1975-83. Epub 2016 Mar 10.

Recombinant Protein Expression, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124 Braunschweig, Germany.

Recombinant protein expression often presents a bottleneck for the production of proteins for use in many areas of animal-cell biotechnology. Difficult-to-express proteins require the generation of numerous expression constructs, where popular prokaryotic screening systems often fail to identify expression of multi domain or full-length protein constructs. Post-translational modified mammalian proteins require an alternative host system such as insect cells using the Baculovirus Expression Vector System (BEVS). Unfortunately this is time-, labor-, and cost-intensive. It is clearly desirable to find an automated and miniaturized fast multi-sample screening method for protein expression in such systems. With this in mind, in this paper a high-throughput initial expression screening method is described using an automated Microcultivation system in conjunction with fast plasmid based transient transfection in insect cells for the efficient generation of protein constructs. The applicability of the system is demonstrated for the difficult to express Nucleotide-binding Oligomerization Domain-containing protein 2 (NOD2). To enable detection of proper protein expression the rather weak plasmid based expression has been improved by a sensitive inline detection system. Here we present the functionality and application of the sensitive SplitGFP (split green fluorescent protein) detection system in insect cells. The successful expression of constructs is monitored by direct measurement of the fluorescence in the BioLector Microcultivation system. Additionally, we show that the results obtained with our plasmid-based SplitGFP protein expression screen correlate directly to the level of soluble protein produced in BEVS. In conclusion our automated SplitGFP screen outlines a sensitive, fast and reliable method reducing the time and costs required for identifying the optimal expression construct prior to large scale protein production in baculovirus infected insect cells. Biotechnol. Bioeng. 2016;113: 1975-1983. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/bit.25956DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5069567PMC
September 2016

A method for specifically targeting two independent genomic integration sites for co-expression of genes in CHO cells.

Methods 2016 Feb 2;95:3-12. Epub 2015 Dec 2.

Helmholtz Centre for Infection Research, Recombinant Protein Expression, Inhoffenstr. 7, 38124 Braunschweig, Germany. Electronic address:

Stable mammalian production cell lines in suspension culture enable the reproducible expression of target genes in any desired scale using bioreactor technology. Targeted integration methods have been developed to cut down timelines for the generation of stable producer cell lines. The powerful Flp recombinase mediated cassette exchange (RMCE) technique allows fast integration of target genes in preselected and optimized high expression loci in so called master cell lines. Up to now, these cells only enable the expression from a single locus. Here, we describe the set-up required for the generation of multiple tagged master cell lines on the example of a binary RMCE expression system in the glycosylation mutant CHO Lec3.2.8.1 cell line. We show how this technology is used for the expression of proteins from multiple loci by generating a binary RMCE expression system. The tools and strategy for the construction of binary master cell lines with different combinations of expression level are described in detail. The binary production cell lines show independent expression of the individual exchange loci of the producer cell lines. The expression level for the model protein tdTomato is the cumulative expression for the chosen combination of the expression loci of the master cell line. This binary RMCE expression system can be further developed to a multi RMCE expression system for co-expression of protein complex subunits with predetermined expression ratio of each individual exchange locus.
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http://dx.doi.org/10.1016/j.ymeth.2015.11.022DOI Listing
February 2016

Genomic Analysis and Isolation of RNA Polymerase II Dependent Promoters from Spodoptera frugiperda.

PLoS One 2015 11;10(8):e0132898. Epub 2015 Aug 11.

Department of Structure and Function of Proteins, Research Group Recombinant Protein Expression, Helmholtz Centre for Infection Research, Braunschweig, Germany.

The Baculoviral Expression Vector System (BEVS) is the most commonly used method for high expression of recombinant protein in insect cells. Nevertheless, expression of some target proteins--especially those entering the secretory pathway--provides a severe challenge for the baculovirus infected insect cells, due to the reorganisation of intracellular compounds upon viral infection. Therefore, alternative strategies for recombinant protein production in insect cells like transient plasmid-based expression or stable expression cell lines are becoming more popular. However, the major bottleneck of these systems is the lack of strong endogenous polymerase II dependent promoters, as the strong baculoviral p10 and polH promoters used in BEVS are only functional in presence of the viral transcription machinery during the late phase of infection. In this work we present a draft genome and a transcriptome analysis of Sf21 cells for the identification of the first known endogenous Spodoptera frugiperda promoters. Therefore, putative promoter sequences were identified and selected because of high mRNA level or in analogy to other strong promoters in other eukaryotic organism. The chosen endogenous Sf21 promoters were compared to early viral promoters for their efficiency to trigger eGFP expression using transient plasmid based transfection in a BioLector Microfermentation system. Furthermore, promoter activity was not only shown in Sf21 cells but also in Hi5 cells. The novel endogenous Sf21 promoters were ranked according to their activity and expand the small pool of available promoters for stable insect cell line development and transient plasmid expression in insect cells. The best promoter was used to improve plasmid based transient transfection in insect cells substantially.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0132898PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4532503PMC
May 2016

Cryo-EM structure of Hepatitis C virus IRES bound to the human ribosome at 3.9-Å resolution.

Nat Commun 2015 Jul 8;6:7646. Epub 2015 Jul 8.

Department of Biology, Institute of Molecular Biology and Biophysics, Otto-Stern-Weg 5, ETH Zürich, Zürich 8093, Switzerland.

Hepatitis C virus (HCV), a widespread human pathogen, is dependent on a highly structured 5'-untranslated region of its mRNA, referred to as internal ribosome entry site (IRES), for the translation of all of its proteins. The HCV IRES initiates translation by directly binding to the small ribosomal subunit (40S), circumventing the need for many eukaryotic translation initiation factors required for mRNA scanning. Here we present the cryo-EM structure of the human 40S ribosomal subunit in complex with the HCV IRES at 3.9 Å resolution, determined by focused refinement of an 80S ribosome-HCV IRES complex. The structure reveals the molecular details of the interactions between the IRES and the 40S, showing that expansion segment 7 (ES7) of the 18S rRNA acts as a central anchor point for the HCV IRES. The structural data rationalizes previous biochemical and genetic evidence regarding the initiation mechanism of the HCV and other related IRESs.
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http://dx.doi.org/10.1038/ncomms8646DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4510694PMC
July 2015

Functional knock down of VCAM1 in mice mediated by endoplasmatic reticulum retained intrabodies.

MAbs 2014 ;6(6):1394-401

a Technische Universität Braunschweig; Institute of Biochemistry; Biotechnology and Bioinformatics ; Braunschweig , Germany.

Functional knockdowns mediated by endoplasmatic reticulum-retained antibodies (ER intrabodies) are a promising tool for research because they allow functional interference on the protein level. We demonstrate for the first time that ER intrabodies can induce a knock-down phenotype in mice. Surface VCAM1 was suppressed in bone marrow of heterozygous and homozygous ER intrabody mice (iER-VCAM1 mice). iER-VCAM1 mice did not have a lethal phenotype, in contrast to the constitutive knockout of VCAM1, but adult mice exhibited physiological effects in the form of aberrant distribution of immature B-cells in blood and bone marrow. The capability to regulate knock-down strength may spark a new approach for the functional study of membrane and plasma proteins, which may especially be valuable for generating mouse models that more closely resemble disease states than classic knockouts do.
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http://dx.doi.org/10.4161/mabs.34377DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4622715PMC
September 2015

Identification of whole pathogenic cells by monoclonal antibodies generated against a specific peptide from an immunogenic cell wall protein.

J Microbiol Methods 2015 Jan 15;108:61-9. Epub 2014 Nov 15.

Helmholtz Centre for Infection Research (HZI), Inhoffenstr. 7, 38124 Braunschweig, Germany. Electronic address:

We selected the immunogenic cell wall ß-(1,3)-glucosyltransferase Bgl2p from Candida albicans as a target protein for the production of antibodies. We identified a unique peptide sequence in the protein and generated monoclonal anti- C. albicans Bgl2p antibodies, which bound in particular to whole C. albicans cells.
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http://dx.doi.org/10.1016/j.mimet.2014.11.003DOI Listing
January 2015

Structural analysis of human soluble adenylyl cyclase and crystal structures of its nucleotide complexes-implications for cyclase catalysis and evolution.

FEBS J 2014 Sep 28;281(18):4151-64. Epub 2014 Jul 28.

Department of Biochemistry, University of Bayreuth, Germany.

Unlabelled: The ubiquitous second messenger cAMP regulates a wide array of functions, from bacterial transcription to mammalian memory. It is synthesized by six evolutionarily distinct adenylyl cyclase (AC) families. In mammals, there are two AC types: nine transmembrane ACs (tmACs) and one soluble AC (sAC). Both AC types belong to the widespread cyclase class III, which has members in numerous organisms from archaeons to mammals. Class III also contains all known guanylyl cyclases (GCs), which synthesize the cAMP-related messenger cGMP in many eukaryotes and possibly some prokaryotes. Among mammalian ACs, sAC is uniquely regulated by bicarbonate, and has been proposed to be more closely related to a bacterial AC subfamily than to mammalian ACs, on the basis of sequence comparisons. Here, we used crystal structures of human sAC catalytic domains to analyze its relationships with other class III ACs and GCs, and to study its substrate selection mechanisms. Structural comparisons revealed a similarity within an sAC-like subfamily but no family-specific structure elements, and an unexpected sAC similarity to eukaryotic GCs and a potential bacterial GC. We further solved novel crystal structures of sAC catalytic domains in complex with a substrate analog, unprocessed ATP substrate, and product after soaking with ATP or GTP. The structures show a novel ATP-binding conformation, and suggest mechanisms for substrate association and recognition. Our results could explain the limited substrate specificity of sAC, suggest how specificity is increased in other cyclases, and indicate evolutionary relationships among class III enzymes, with sAC being close to a putative 'ancestor' cyclase.

Database: Coordinates and structure factors for the novel sAC-cat structures described have been deposited with the Worldwide PDB (www.pdb.org): ApCpp soak (entry 4usu), ATP + Ca(2+) soak (entry 4usv), GTP + Mg(2+) soak (entry 4ust), ATP soak (entry 4usw).
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http://dx.doi.org/10.1111/febs.12913DOI Listing
September 2014

Expression, purification, crystallization and preliminary X-ray diffraction analysis of a mammalian type 10 adenylyl cyclase.

Acta Crystallogr F Struct Biol Commun 2014 Apr 25;70(Pt 4):467-9. Epub 2014 Mar 25.

Department of Biochemistry, University of Bayreuth, 95445 Bayreuth, Germany.

The second messenger cAMP is synthesized in mammals by ten differently regulated adenylyl cyclases (AC1-10). These ACs are grouped into nucleotidyl cyclase class III based on homologies in their catalytic domains. The catalytic domain of AC10 is unique, however, in being activated through direct interaction with calcium and bicarbonate. Here, the production, crystallization and X-ray diffraction analysis of the catalytic domain of human AC10 are described as a basis for structural studies of regulator binding sites and mechanisms. The recombinant protein had high specific AC activity, and crystals of AC10 in space group P63 diffracted to ∼2.0 Å resolution on a synchrotron beamline. A complete diffraction data set revealed unit-cell parameters a = b = 99.65, c = 98.04 Å, indicating one AC10 catalytic domain per asymmetric unit, and confirmed that the obtained crystals are suitable for structure solution and mechanistic studies.
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http://dx.doi.org/10.1107/S2053230X14004014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3976064PMC
April 2014

Crystal structures of human soluble adenylyl cyclase reveal mechanisms of catalysis and of its activation through bicarbonate.

Proc Natl Acad Sci U S A 2014 Mar 24;111(10):3727-32. Epub 2014 Feb 24.

Department of Biochemistry, University of Bayreuth, 95440 Bayreuth, Germany.

cAMP is an evolutionary conserved, prototypic second messenger regulating numerous cellular functions. In mammals, cAMP is synthesized by one of 10 homologous adenylyl cyclases (ACs): nine transmembrane enzymes and one soluble AC (sAC). Among these, only sAC is directly activated by bicarbonate (HCO3(-)); it thereby serves as a cellular sensor for HCO3(-), carbon dioxide (CO2), and pH in physiological functions, such as sperm activation, aqueous humor formation, and metabolic regulation. Here, we describe crystal structures of human sAC catalytic domains in the apo state and in complex with substrate analog, products, and regulators. The activator HCO3(-) binds adjacent to Arg176, which acts as a switch that enables formation of the catalytic cation sites. An anionic inhibitor, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, inhibits sAC through binding to the active site entrance, which blocks HCO3(-) activation through steric hindrance and trapping of the Arg176 side chain. Finally, product complexes reveal small, local rearrangements that facilitate catalysis. Our results provide a molecular mechanism for sAC catalysis and cellular HCO3(-) sensing and a basis for targeting this system with drugs.
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http://dx.doi.org/10.1073/pnas.1322778111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3956179PMC
March 2014

Multi-host expression system for recombinant production of challenging proteins.

PLoS One 2013 17;8(7):e68674. Epub 2013 Jul 17.

Department of Molecular Structural Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany.

Recombinant production of complex eukaryotic proteins for structural analyses typically requires a profound screening process to identify suitable constructs for the expression of ample amounts of properly folded protein. Furthermore, the evaluation of an optimal expression host has a major impact on protein yield and quality as well as on actual cost of the production process. Here we present a novel fast expression system for multiple hosts based on a single donor vector termed pFlp-Bac-to-Mam. The range of applications of pFlp-Bac-to-Mam comprises highly efficient transient transfection of HEK293-6E in serum-free suspension culture and subsequent large-scale production of challenging proteins expressing in mg per Liter level using either the baculoviral expression vector system or stable CHO production cell lines generated by Flp-mediated cassette exchange. The success of the multi-host expression vector to identify the optimal expression strategy for efficient production of high quality protein is demonstrated in a comparative expression study of three model proteins representing different protein classes: intracellular expression using a fluorescent protein, secretion of a single-chain-Fv-hIgG1Fc fusion construct and production of a large amount of highly homogeneous protein sample of the extracellular domain of a Toll-like receptor. The evaluation of the production efficiency shows that the pFlp-Bac-to-Mam system allows a fast and individual optimization of the expression strategy for each protein class.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0068674PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3714276PMC
March 2014

Biochemical characterization of molybdenum cofactor-free nitrate reductase from Neurospora crassa.

J Biol Chem 2013 May 28;288(20):14657-14671. Epub 2013 Mar 28.

Department of Plant Biology, Braunschweig University of Technology, 38106 Braunschweig, Germany.

Nitrate reductase (NR) is a complex molybdenum cofactor (Moco)-dependent homodimeric metalloenzyme that is vitally important for autotrophic organism as it catalyzes the first and rate-limiting step of nitrate assimilation. Beside Moco, eukaryotic NR also binds FAD and heme as additional redox active cofactors, and these are involved in electron transfer from NAD(P)H to the enzyme molybdenum center where reduction of nitrate to nitrite takes place. We report the first biochemical characterization of a Moco-free eukaryotic NR from the fungus Neurospora crassa, documenting that Moco is necessary and sufficient to induce dimer formation. The molybdenum center of NR reconstituted in vitro from apo-NR and Moco showed an EPR spectrum identical to holo-NR. Analysis of mutants unable to bind heme or FAD revealed that insertion of Moco into NR occurs independent from the insertion of any other NR redox cofactor. Furthermore, we showed that at least in vitro the active site formation of NR is an autonomous process.
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http://dx.doi.org/10.1074/jbc.M113.457960DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3656317PMC
May 2013

Arranged sevenfold: structural insights into the C-terminal oligomerization domain of human C4b-binding protein.

J Mol Biol 2013 Apr 28;425(8):1302-17. Epub 2012 Dec 28.

Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Petersenstraße 22, 64287 Darmstadt, Germany.

The complement system as a major part of innate immunity is the first line of defense against invading microorganisms. Orchestrated by more than 60 proteins, its major task is to discriminate between host cells and pathogens and to initiate immune response. Additional recognition of necrotic or apoptotic cells demands a fine-tune regulation of this powerful system. C4b-binding protein (C4BP) is the major inhibitor of the classical complement and lectin pathway. The crystal structure of the human C4BP oligomerization domain in its 7α isoform and molecular simulations provide first structural insights of C4BP oligomerization. The heptameric core structure is stabilized by intermolecular disulfide bonds. In addition, thermal shift assays indicate that layers of electrostatic interactions mainly contribute to the extraordinary thermodynamic stability of the complex. These findings make C4BP a promising scaffold for multivalent ligand display with applications in immunology and biological chemistry.
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http://dx.doi.org/10.1016/j.jmb.2012.12.017DOI Listing
April 2013

High affinity peptide inhibitors of the hepatitis C virus NS3-4A protease refractory to common resistant mutants.

J Biol Chem 2012 Nov 10;287(46):39224-32. Epub 2012 Sep 10.

Research Group Directed Evolution, Helmholtz Centre for Infection Research (HZI), 38124 Braunschweig, Germany.

Hepatitis C virus (HCV) NS3-4A protease is essential for viral replication. All current small molecular weight drugs against NS3-4A are substrate peptidomimetics that have a similar binding and resistance profile. We developed inhibitory peptides (IPs) capping the active site and binding via a novel "tyrosine" finger at an alternative NS3-4A site that is of particular interest for further HCV drug development. The peptides are not cleaved due to a combination of geometrical constraints and impairment of the oxyanion hole function. Selection and optimization through combinatorial phagemid display, protein crystallography, and further modifications resulted in a 32-amino acid peptide with a K(i) of 0.53 nm. Inhibition of viral replication in cell culture was demonstrated by fusion to a cell-penetrating peptide. Negligible susceptibility to known (A156V and R155K) resistance mutations of the NS3-4A protease was observed. This work shows for the first time that antiviral peptides can target an intracellular site and reveals a novel druggable site on the HCV protease.
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http://dx.doi.org/10.1074/jbc.M112.393843DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3493962PMC
November 2012

Profiling ubiquitin linkage specificities of deubiquitinating enzymes with branched ubiquitin isopeptide probes.

Chembiochem 2012 Jul 11;13(10):1416-20. Epub 2012 Jun 11.

Department of Molecular Structural Biology, Cellular Proteomics, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124 Braunschweig, Germany.

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http://dx.doi.org/10.1002/cbic.201200261DOI Listing
July 2012
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