Establishing a structural genomics platform: The Berlin-based Protein Structure Factory

Heinemann, Udo

doi:10.1002/1438-826x(200210)3:1/2<25::aid-gnfd25>3.0.co;2-w

Cited by 4 publications

(5 citation statements)

References 83 publications

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“…This may affect the soluble rate in a negative way. On the other hand, our current approach is not directed at expressing folded membrane proteins, which constitute 30% of a typical proteome (Christendat 2000;Heinemann 2002). Membrane proteins account for 31% of the C. elegans genome, but only 23% of the ORFs tested in this study.…”

Section: Discussionmentioning

confidence: 99%

“…It is generally recognized that the production of proteins in soluble form, sufficient (milligram) quantity, and homogeneity for structural analyses is the most prodigious part of a structural genomics project (Stevens and Wilson 2001;Chambers 2002;Heinemann 2002). To express all proteins from the C. elegans genome of ∼20,000 ORFs, the traditional clone-by-clone approach is inadequate, and an automated, parallelized, HTP approach is necessary.…”

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confidence: 99%

“…Therefore, any selection crite-ria based on existing knowledge would be inadequate due to lack of prior data. Secondly, in some reported studies, 50% or more selected targets were dropped prematurely due to solubility problems, in spite of having been predicted by bioinformatics analysis to be soluble and amenable to structural elucidation (Heinemann 2002). To produce large numbers of proteins from the C. elegans genome, we utilized a two-tiered approach for target screening and production.…”

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confidence: 99%

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High-Throughput Expression of C. elegans Proteins

Luan¹,

Qiu²,

Finley³

et al. 2004

Genome Res.

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Proteome-scale studies of protein three-dimensional structures should provide valuable information for both investigating basic biology and developing therapeutics. Critical for these endeavors is the expression of recombinant proteins. We selected Caenorhabditis elegans as our model organism in a structural proteomics initiative because of the high quality of its genome sequence and the availability of its ORFeome, protein-encoding open reading frames (ORFs), in a flexible recombinational cloning format. We developed a robotic pipeline for recombinant protein expression, applying the Gateway cloning/expression technology and utilizing a stepwise automation strategy on an integrated robotic platform. Using the pipeline, we have carried out heterologous protein expression experiments on 10,167 ORFs of C. elegans. With one expression vector and one Escherichia coli strain, protein expression was observed for 4854 ORFs, and 1536 were soluble. Bioinformatics analysis of the data indicates that protein hydrophobicity is a key determining factor for an ORF to yield a soluble expression product. This protein expression effort has investigated the largest number of genes in any organism to date. The pipeline described here is applicable to high-throughput expression of recombinant proteins for other species, both prokaryotic and eukaryotic, provided that ORFeome resources become available.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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High-Throughput Expression of C. elegans Proteins

Luan¹,

Qiu²,

Finley³

et al. 2004

Genome Res.

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“…Considering these numbers it seems obvious that structural genomics will fail without the development and implementation of high-throughput protein production and analysis methods and facilities . The Protein Structure Factory is committed to establishing high-throughput techniques for the NMR and crystal structure analysis of proteins from human origin.…”

Section: Structural Genomics and The Protein Structure Factorymentioning

confidence: 99%

Facilities and Methods for the High-Throughput Crystal Structural Analysis of Human Proteins

et al. 2003

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Facilities and methods for the high-throughput crystal structure analysis of human proteins are described as recently established in the Protein Structure Factory, a Berlin-area structural genomics project. Genes encoding human proteins are expressed in either recombinant Escherichia coli or yeast (Saccharomyces cerevisiae or Pichia pastoris). To facilitate and standardize protein purification, the target proteins are produced with various tags for affinity chromatography. For high-throughput crystallization, a robotic station is being set up that has the capacity to handle 960 000 experiments simultaneously. The resulting protein crystals will be subjected to X-ray diffraction experiments at the third-generation synchrotron storage ring BESSY where protein crystallography beamlines are currently under construction. The Protein Structure Factory's strategy for high-throughput production and structure analysis of human proteins is evaluated based on first results.

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“…In both cases, the cells can, and should, be grown to high densities (OD 600 of [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] in highly enriched medium47 in baffled shake flasks48 , 49. Whatever the final cell density, it is advisable to induce the expression of the T7 RNA polymerase at mid-to-late log phase of the growth curve to ensure maximal yield while avoiding the problems associated with cells going into stationary phase (for example, induction of proteases).…”

Section: Expression Conditionsmentioning

confidence: 99%

Protein production and purification

Gräslund¹,

Nordlund²,

Weigelt³

et al. 2008

Nat Methods

742

315

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In selecting a method to produce a recombinant protein, a researcher is faced with a bewildering array of choices as to where to start. To facilitate decision-making, we describe a consensus 'what to try first' strategy based on our collective analysis of the expression and purification of over 10,000 different proteins. This review presents methods that could be applied at the outset of any project, a prioritized list of alternate strategies and a list of pitfalls that trip many new investigators.

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Establishing a structural genomics platform: The Berlin-based Protein Structure Factory

Cited by 4 publications

References 83 publications

High-Throughput Expression of C. elegans Proteins

High-Throughput Expression of C. elegans Proteins

Facilities and Methods for the High-Throughput Crystal Structural Analysis of Human Proteins

Protein production and purification

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