Volker Sievert

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.

A Community Standard Format for the Representation of Protein Affinity Reagents

Protein affinity reagents (PARs), most commonly antibodies, are essential reagents for protein characterization in basic research, biotechnology, and diagnostics as well as the fastest growing class of therapeutics. Large numbers of PARs are available commercially; however, their quality is often uncertain. In addition, currently available PARs cover only a fraction of the human proteome, and their cost is prohibitive for proteome scale applications. This situation has triggered several initiatives involving large scale generation and validation of antibodies, for example the Swedish Human Protein Atlas and the German Antibody Factory. Antibodies targeting specific subproteomes are being pursued by members of Human Proteome Organisation (plasma and liver proteome projects) and the United States National Cancer Institute (cancer-associated antigens). ProteomeBinders, a European consortium, aims to set up a resource of consistently quality-controlled protein-binding reagents for the whole human proteome. An ultimate PAR database resource would allow consumers to visit one on-line warehouse and find all available affinity reagents from different providers together with documentation that facilitates easy comparison of their cost and quality. However, in contrast to, for example, nucleotide databases among which data are synchronized between the major data providers, current PAR producers, quality control centers, and commercial companies all use incompatible formats, hindering data exchange. Here we propose Proteomics Standards Initiative (PSI)-PAR as a global community standard format for the representation and exchange of protein affinity reagent data. The PSI-PAR format is maintained by the Human Proteome Organisation PSI and was developed within the context of ProteomeBinders by building on a mature proteomics standard format, PSI-molecular interaction, which is a widely accepted and established community standard for molecular interaction data. Further information and documentation are available on the PSI-PAR web site.

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

Establishing the yeast Saccharomyces cerevisiae as a system for expression of human proteins on a proteome-scale

Structural genomics requires the application of a standardised process for overexpression of soluble proteins that allows high-throughput purification and analysis of protein products. We have developed a highly parallel approach to protein expression, including the simultaneous expression screening of a large number of cDNA clones in an appropriate vector system and the use of a protease-deficient host strain. A set of 221 human genes coding for proteins of various sizes with unknown structures was selected to evaluate the system. We transferred the cDNAs from an E. coli vector to the yeast expression vector by recombinational cloning, avoiding time-consuming recloning steps and the use of restriction enzymes in the cloning process. The subcloning yield was 95%, provided that a PCR fragment of the correct size could be obtained. Sixty percent of these proteins were expressed as soluble products at detectable levels and 48% were successfully purified under native conditions using the His6 tag fusion.The advantages of the developed yeast-based expression system are the ease of manipulation and cultivation of S. cerevisiae in the same way as with prokaryotic hosts and the ability to introduce post-translational modifications of proteins if required, thus being an attractive system for heterologous expression of mammalian proteins. The expression clones selected in this screening process are passed on to the fermentation process in order to provide milligram amounts of proteins for structure analysis within the ‘Berlin Protein Structure Factory’. All data generated is stored in a relational database and is available on our website(http://www.proteinstrukturfabrik.de).

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.

V-gene amplification revisited - An optimised procedure for amplification of rearranged human antibody genes of different isotypes.

For studying human antibody variable (V)-gene usage in any group of individuals or for the generation of recombinant human antibody libraries for phage display, quality and yield of the amplified V-gene repertoire is of utmost importance. Key parameters affecting the amplification of full antibody repertoires are V-gene specific primer design, complementary DNA (cDNA) synthesis from total RNA extracts of peripheral blood mononuclear cells (PBMCs) and ultimately the polymerase chain reaction (PCR). In this work we analysed all these factors; we performed a detailed bioinformatic analysis of V-gene specific primers based on VBASE2 and evaluated the influence of different commercially available reverse transcriptases on cDNA synthesis and polymerases on PCR efficiency. The primers presented cover near to 100% of all functional and putatively functional V-genes in VBASE2 and the final protocol presents an optimised combination of commercial enzymes and reaction additives for cDNA synthesis and PCR conditions for V-gene amplification. Finally, applying this protocol in combination with different immunoglobulin (Ig) chain specific reverse primers we were able to amplify rearranged antibody genes of different isotypes under investigation.

X-ray structure of human gankyrin, the product of a gene linked to hepatocellular carcinoma

Babu A. Manjasetty,,* Claudia Quedenau, Volker Sievert, Konrad Büssow, Frank Niesen, Heinrich Delbrück, and Udo Heinemann Protein Structure Factory, c/o BESSY GmbH, Albert-Einstein-Straße 15, 12489 Berlin, Germany Forschungsgruppe Kristallographie, Max-Delbrück-Centrum für Molekulare Medizin, 13092 Berlin, Germany Protein Structure Factory, 14059 Berlin, Germany Max-Planck-Institut für Molekulare Genetik, 14195 Berlin, Germany Universitätsklinikum Charité, Institut für Medizinische Physik and Biophysik, 10096 Berlin, Germany Institut für Chemie/Kristallographie, Freie Universität, 14195 Berlin, Germany

Onset of Immune Senescence Defined by Unbiased Pyrosequencing of Human Immunoglobulin mRNA Repertoires

The immune system protects us from foreign substances or pathogens by generating specific antibodies. The variety of immunoglobulin (Ig) paratopes for antigen recognition is a result of the V(D)J rearrangement mechanism, while a fast and efficient immune response is mediated by specific immunoglobulin isotypes obtained through class switch recombination (CSR). To get a better understanding on how antibody-based immune protection works and how it changes with age, the interdependency between these two parameters need to be addressed. Here, we have performed an in depth analysis of antibody repertoires of 14 healthy donors representing different gender and age groups. For this task, we developed a unique pyrosequencing approach, which is able to monitor the expression levels of all immunoglobulin V(D)J recombinations of all isotypes including subtypes in an unbiased and quantitative manner. Our results show that donors have individual immunoglobulin repertoires and cannot be clustered according to V(D)J recombination patterns, neither by age nor gender. However, after incorporating isotype-specific analysis and considering CSR information into hierarchical clustering the situation changes. For the first time the donors cluster according to age and separate into young adults and elderly donors (>50). As a direct consequence, this clustering defines the onset of immune senescence at the age of fifty and beyond. The observed age-dependent reduction of CSR ability proposes a feasible explanation why reduced efficacy of vaccination is seen in the elderly and implies that novel vaccine strategies for the elderly should include the “Golden Agers”.

X-ray structure of fumarylacetoacetate hydrolase family member Homo sapiens FLJ36880.

Abstract The human protein FLJ36880 belongs to the fumarylacetoacetate hydrolase family. The X-ray structure of FLJ36880 has been determined to 2.2 Å resolution employing the semi-automated high-throughput structural genomics approach of the Protein Structure Factory. FLJ36880 adopts a mixed β-sandwich roll fold and forms homodimers in crystals as well as in solution. One Mg2+ ion is bound to each subunit of the dimeric protein by coordination to three carboxylate oxygens and three water molecules. These metal binding sites are accessible from the same surface of the dimer, partly due to the disorder of the undecapeptide stretch D29 to L39. The overall structure and metal binding site of FLJ36880 bear clear similarities to the C-terminal domain of the bifunctional enzyme HpcE from Escherichia coli C, fumarylacetoacetate hydrolase from Mus musculus and to YcgM (Apc5008) from E. coli 1262. These similarities provide a framework for suggesting biochemical functions and evolutionary relationships of FLJ36880. It appears highly probable that the metal binding sites are involved in an enzymatic activity related to the catabolism of aromatic amino acids. Two point mutations in the active-site of FAH, responsible for the metabolic disease hereditary tyrosinemia type I (HTI) in humans, affect residues that are structurally conserved in FLJ36880 and located in the putative catalytic site.