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Jörg O Schulze,
Claudia Quedenau,
Yvette Roske,
Thomas Adam,
Herwig Schüler,
Joachim Behlke,
Andrew P Turnbull,
Volker Sievert, Christoph Scheich,
Uwe Mueller,
Udo Heinemann,
Konrad Büssow
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ABSTRACT: Iba2 is a homolog of ionized calcium-binding adapter molecule 1 (Iba1), a 17-kDa protein that binds and cross-links filamentous actin (F-actin) and localizes to membrane ruffles and phagocytic cups. Here, we present the crystal structure of human Iba2 and its homodimerization properties, F-actin cross-linking activity, cellular localization and recruitment upon bacterial invasion in comparison with Iba1. The Iba2 structure comprises two central EF-hand motifs lacking bound Ca2+. Iba2 crystallized as a homodimer stabilized by a disulfide bridge and zinc ions. Analytical ultracentrifugation revealed a different mode of dimerization under reducing conditions that was independent of Ca2+. Furthermore, no binding of Ca2+ up to 0.1 mM was detected by equilibrium dialysis. Correspondingly, Iba EF-hand motifs lack residues essential for strong Ca2+ coordination. Sedimentation experiments and microscopy detected pronounced, indistinguishable F-actin binding and cross-linking activity of Iba1 and Iba2 with induction of F-actin bundles. Fluorescent Iba fusion proteins were expressed in HeLa cells and co-localized with F-actin. Iba1 was recruited into cellular projections to a larger extent than Iba2. Additionally, we studied Iba recruitment in a Shigella invasion model that induces cytoskeletal rearrangements. Both proteins were recruited into the bacterial invasion zone and Iba1 was again concentrated slightly higher in the cellular extensions.
FEBS Journal 09/2008; 275(18):4627-40. · 3.79 Impact Factor
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ABSTRACT: A vector system is presented that allows generation of E. coli co-expression clones by a standardized, robust cloning procedure. The number of co-expressed proteins is not limited. Five 'pQLink' vectors for expression of His-tag and GST-tag fusion proteins as well as untagged proteins and for cloning by restriction enzymes or Gateway cloning were generated. The vectors allow proteins to be expressed individually; to achieve co-expression, two pQLink plasmids are combined by ligation-independent cloning. pQLink co-expression plasmids can accept an unrestricted number of genes. As an example, the co-expression of a heterotetrameric human transport protein particle (TRAPP) complex from a single plasmid, its isolation and analysis of its stoichiometry are shown. pQLink clones can be used directly for pull-down experiments if the proteins are expressed with different tags. We demonstrate pull-down experiments of human valosin-containing protein (VCP) with fragments of the autocrine motility factor receptor (AMFR). The cloning method avoids PCR or gel isolation of restriction fragments, and a single resistance marker and origin of replication are used, allowing over-expression of rare tRNAs from a second plasmid. It is expected that applications are not restricted to bacteria, but could include co-expression in other hosts such as Bacluovirus/insect cells.
Nucleic Acids Research 02/2007; 35(6):e43. · 8.03 Impact Factor
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ABSTRACT: The human protein PTD012 is the longer product of an alternatively spliced gene and was described to be localized in the nucleus. The X-ray structure analysis at 1.7 A resolution of PTD012 through SAD phasing reveals a monomeric protein and a novel fold. The shorter splice form was also studied and appears to be unfolded and non-functional. The structure of PTD012 displays an alphabetabetaalpha four-layer topology. A metal ion residing between the central beta-sheets is partially coordinated by three histidine residues. X-ray absorption near-edge structure (XANES) analysis identifies the PTD012-bound ion as Zn(2+). Tetrahedral coordination of the ion is completed by the carboxylate oxygen atom of an acetate molecule taken up from the crystallization buffer. The binding of Zn(2+) to PTD012 is reminiscent of zinc-containing enzymes such as carboxypeptidase, carbonic anhydrase, and beta-lactamase. Biochemical assays failed to demonstrate any of these enzyme activities in PTD012. However, PTD012 exhibits ester hydrolase activity on the substrate p-nitrophenyl acetate.
Protein Science 05/2006; 15(4):914-20. · 2.80 Impact Factor
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ABSTRACT: BACKGROUND: The availability of suitable recombinant protein is still a major bottleneck in protein structure analysis. The Protein Structure Factory, part of the international structural genomics initiative, targets human proteins for structure determination. It has implemented high throughput procedures for all steps from cloning to structure calculation. This article describes the selection of human target proteins for structure analysis, our high throughput cloning strategy, and the expression of human proteins in Escherichia coli host cells. RESULTS AND CONCLUSION: Protein expression and sequence data of 1414 E. coli expression clones representing 537 different proteins are presented. 139 human proteins (18%) could be expressed and purified in soluble form and with the expected size. All E. coli expression clones are publicly available to facilitate further functional characterisation of this set of human proteins.
Microbial Cell Factories 08/2005; 4:21. · 3.55 Impact Factor
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ABSTRACT: Human Aortic Preferentially Expressed Protein-1 (APEG-1) is a novel specific smooth muscle differentiation marker thought to play a role in the growth and differentiation of arterial smooth muscle cells (SMCs).
Good quality crystals that were suitable for X-ray crystallographic studies were obtained following the truncation of the 14 N-terminal amino acids of APEG-1, a region predicted to be disordered. The truncated protein (termed DeltaAPEG-1) consists of a single immunoglobulin (Ig) like domain which includes an Arg-Gly-Asp (RGD) adhesion recognition motif. The RGD motif is crucial for the interaction of extracellular proteins and plays a role in cell adhesion. The X-ray structure of DeltaAPEG-1 was determined and was refined to sub-atomic resolution (0.96 A). This is the best resolution for an immunoglobulin domain structure so far. The structure adopts a Greek-key beta-sandwich fold and belongs to the I (intermediate) set of the immunoglobulin superfamily. The residues lying between the beta-sheets form a hydrophobic core. The RGD motif folds into a 310 helix that is involved in the formation of a homodimer in the crystal which is mainly stabilized by salt bridges. Analytical ultracentrifugation studies revealed a moderate dissociation constant of 20 microM at physiological ionic strength, suggesting that APEG-1 dimerisation is only transient in the cell. The binding constant is strongly dependent on ionic strength.
Our data suggests that the RGD motif might play a role not only in the adhesion of extracellular proteins but also in intracellular protein-protein interactions. However, it remains to be established whether the rather weak dimerisation of APEG-1 involving this motif is physiologically relevant.
BMC Structural Biology 02/2005; 5:21. · 2.48 Impact Factor
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ABSTRACT: High-throughput protein structure analysis of individual protein domains requires analysis of large numbers of expression clones to identify suitable constructs for structure determination. For this purpose, methods need to be implemented for fast and reliable screening of the expressed proteins as early as possible in the overall process from cloning to structure determination.
88 different E. coli expression constructs for 17 human protein domains were analysed using high-throughput cloning, purification and folding analysis to obtain candidates suitable for structural analysis. After 96 deep-well microplate expression and automated protein purification, protein domains were directly analysed using 1D 1H-NMR spectroscopy. In addition, analytical hydrophobic interaction chromatography (HIC) was used to detect natively folded protein. With these two analytical methods, six constructs (representing two domains) were quickly identified as being well folded and suitable for structural analysis.
The described approach facilitates high-throughput structural analysis. Clones expressing natively folded proteins suitable for NMR structure determination were quickly identified upon small scale expression screening using 1D 1H-NMR and/or analytical HIC. This procedure is especially effective as a fast and inexpensive screen for the 'low hanging fruits' in structural genomics.
BMC Structural Biology 04/2004; 4:4. · 2.48 Impact Factor
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Babu A Manjasetty,
Heinrich Delbrück,
Dinh-Trung Pham,
Uwe Mueller,
Martin Fieber-Erdmann, Christoph Scheich,
Volker Sievert,
Konrad Büssow,
Frank H Niesen,
Wilhelm Weihofen,
Bernhard Loll,
Wolfram Saenger,
Udo Heinemann,
Frank H Neisen
Proteins Structure Function and Bioinformatics 04/2004; 54(4):797-800. · 3.39 Impact Factor
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ABSTRACT: The preparation of proteins for structural and functional analysis using the Escherichia coli expression system is often hampered by the formation of insoluble intracellular protein aggregates (inclusion bodies). Transferring those proteins into their native states by in vitro protein folding requires screening for the best buffer conditions and suitable additives. However, it is difficult to assess the success of such a screen if no biological assay is available. We established a fully automated folding screen and a system to detect folded protein that is based on analytical hydrophobic interaction chromatography and tryptophan fluorescence spectroscopy. The system was evaluated with two model enzymes (carbonic anhydrase II and malate dehydrogenase), and was successfully applied to the folding of the p22 subunit of human dynactin, which is expressed in inclusion bodies in E. coli. The described screen allows for high-throughput folding analysis of inclusion body proteins for structural and functional analyses.
Protein Science 03/2004; 13(2):370-80. · 2.80 Impact Factor
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Konrad Büssow,
Claudia Quedenau,
Volker Sievert,
Janett Tischer, Christoph Scheich,
Harald Seitz,
Brigitte Hieke,
Frank H Niesen,
Frank Götz,
Ulrich Harttig,
Hans Lehrach
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ABSTRACT: We describe here a systematic approach to the identification of human proteins and protein fragments that can be expressed as soluble proteins in Escherichia coli. A cDNA expression library of 10,825 clones was screened by small-scale expression and purification and 2,746 clones were identified. Sequence and protein-expression data were entered into a public database. A set of 163 clones was selected for structural analysis and 17 proteins were prepared for crystallization, leading to three new structures.
Genome biology 02/2004; 5(9):R71. · 6.63 Impact Factor
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Konrad Büssow,
Claudia Quedenau,
Volker Sievert,
Janett Tischer, Christoph Scheich,
Harald Seitz,
Brigitte Hieke,
Frank H. Niesen,
Frank Götz,
Ulrich Harttig,
Hans Lehrach
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ABSTRACT: We describe here a systematic approach to the identification of human proteins and protein fragments that can be expressed as soluble proteins in Escherichia coli. A cDNA expression library of 10,825 clones was screened by small-scale expression and purification and 2,746 clones were identified. Sequence and protein-expression data were entered into a public database. A set of 163 clones was selected for structural analysis and 17 proteins were prepared for crystallization, leading to three new structures.
Genome Biology. 01/2004; 5:R71.
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ABSTRACT: Functional Genomics, the systematic characterisation of the functions of an organism's genes, includes the study of the gene products, the proteins. Such studies require methods to express and purify these proteins in a parallel, time and cost effective manner.
We developed a method for parallel expression and purification of recombinant proteins with a hexahistidine tag (His-tag) or glutathione S-transferase (GST)-tag from bacterial expression systems. Proteins are expressed in 96-well microplates and are purified by a fully automated procedure on a pipetting robot. Up to 90 microgram purified protein can be obtained from 1 ml microplate cultures. The procedure is readily reproducible and 96 proteins can be purified in approximately three hours. It avoids clearing of crude cellular lysates and the use of magnetic affinity beads and is therefore less expensive than comparable commercial systems. We have used this method to compare purification of a set of human proteins via His-tag or GST-tag. Proteins were expressed as fusions to an N-terminal tandem His- and GST-tag and were purified by metal chelating or glutathione affinity chromatography. The purity of the obtained protein samples was similar, yet His-tag purification resulted in higher yields for some proteins.
A fully automated, robust and cost effective method was developed for the purification of proteins that can be used to quickly characterise expression clones in high throughput and to produce large numbers of proteins for functional studies.His-tag affinity purification was found to be more efficient than purification via GST-tag for some proteins.
BMC Biotechnology 08/2003; 3:12. · 2.35 Impact Factor
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ABSTRACT: The human protein PTD012 is the longer product of an alternatively spliced gene and was described to be localized in the nucleus. The X-ray structure analysis at 1.7 Å resolution of PTD012 through SAD phasing reveals a monomeric protein and a novel fold. The shorter splice form was also studied and appears to be unfolded and non-functional. The structure of PTD012 displays an four-layer topology. A metal ion residing between the central -sheets is partially coordinated by three histidine residues. X-ray absorption near-edge structure (XANES) analysis identifies the PTD012-bound ion as Zn2+. Tetrahedral coordination of the ion is completed by the carboxylate oxygen atom of an acetate molecule taken up fromthe crystallization buffer. The binding of Zn2+ to PTD012 is reminiscent of zinccontaining enzymes such as carboxypeptidase, carbonic anhydrase, and -lactamase. Biochemical assays failed to demonstrate any of these enzyme activities in PTD012. However, PTD012 exhibits ester hydrolase activity on the substrate p -nitrophenyl acetate.
