Christoph Scheich

Vectors for co-expression of an unrestricted number of proteins

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.

An automated method for high-throughput protein purification applied to a comparison of His-tag and GST-tag affinity chromatography

BackgroundFunctional Genomics, the systematic characterisation of the functions of an organisms 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.ResultsWe 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.ConclusionA 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.

Structural genomics of human proteins – target selection and generation of a public catalogue of expression clones

BackgroundThe 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 ConclusionProtein 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.

An automated in vitro protein folding screen applied to a human dynactin subunit

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.

Crystal structure of Homo sapiens protein hp14.5

Babu A. Manjasetty, Heinrich Delbrück, Dinh-Trung Pham, Uwe Mueller, Martin Fieber-Erdmann, Christoph Scheich, Volker Sievert, Konrad Büssow, Frank H. Neisen, Wilhelm Weihofen, Bernhard Loll, Wolfram Saenger, and Udo Heinemann* Protein Structure Factory, c/o BESSY GmbH, Berlin, Germany Forschungsgruppe Kristallographie, Max-Delbrück-Centrum für Molekulare Medizin, Berlin, Germany Institut für Chemie/Kristallographie, Freie Universität Berlin, Germany Protein Structure Factory, Berlin, Germany Alpha-Bioverfahrenstechnik GmbH, Kleinmachnow, Germany Max-Planck-Institut für Molekulare Genetik, Berlin, Germany Universitätsklinikum Charité, Institut für Medizinische Physik & Biophysik Berlin, Germany

A catalog of human cDNA expression clones and its application to structural genomics

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.

X-ray structure of engineered human Aortic Preferentially Expressed Protein-1 (APEG-1)

BackgroundHuman 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).ResultsGood 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 ΔAPEG-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 ΔAPEG-1 was determined and was refined to sub-atomic resolution (0.96 Å). This is the best resolution for an immunoglobulin domain structure so far. The structure adopts a Greek-key β-sandwich fold and belongs to the I (intermediate) set of the immunoglobulin superfamily. The residues lying between the β-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 μM at physiological ionic strength, suggesting that APEG-1 dimerisation is only transient in the cell. The binding constant is strongly dependent on ionic strength.ConclusionOur 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 physiogically relevant.

Crystal structure of Homo sapiens PTD012 reveals a zinc-containing hydrolase fold

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 αββα four‐layer topology. A metal ion residing between the central β‐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 from the crystallization buffer. The binding of Zn2+ to PTD012 is reminiscent of zinc‐containing enzymes such as carboxypeptidase, carbonic anhydrase, and β‐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.