Small-scale, semi-automated purification of eukaryotic proteins for structure determination

The University of Wisconsin Center for Eukaryotic Structural Genomics and Department of Biochemistry, University of Wisconsin, Room 141B, 433 Babcock Drive, Madison, WI 53706, USA.
Journal of Structural and Functional Genomics 01/2008; 8(4):153-66. DOI: 10.1007/s10969-007-9032-5
Source: PubMed


A simple approach that allows cost-effective automated purification of recombinant proteins in levels sufficient for functional characterization or structural studies is described. Studies with four human stem cell proteins, an engineered version of green fluorescent protein, and other proteins are included. The method combines an expression vector (pVP62K) that provides in vivo cleavage of an initial fusion protein, a factorial designed auto-induction medium that improves the performance of small-scale production, and rapid, automated metal affinity purification of His8-tagged proteins. For initial small-scale production screening, single colony transformants were grown overnight in 0.4 ml of auto-induction medium, produced proteins were purified using the Promega Maxwell 16, and purification results were analyzed by Caliper LC90 capillary electrophoresis. The yield of purified [U-15N]-His8-Tcl-1 was 7.5 microg/ml of culture medium, of purified [U-15N]-His8-GFP was 68 microg/ml, and of purified selenomethione-labeled AIA-GFP (His8 removed by treatment with TEV protease) was 172 microg/ml. The yield information obtained from a successful automated purification from 0.4 ml was used to inform the decision to scale-up for a second meso-scale (10-50 ml) cell growth and automated purification. 1H-15N NMR HSQC spectra of His8-Tcl-1 and of His8-GFP prepared from 50 ml cultures showed excellent chemical shift dispersion, consistent with well folded states in solution suitable for structure determination. Moreover, AIA-GFP obtained by proteolytic removal of the His8 tag was subjected to crystallization screening, and yielded crystals under several conditions. Single crystals were subsequently produced and optimized by the hanging drop method. The structure was solved by molecular replacement at a resolution of 1.7 A. This approach provides an efficient way to carry out several key target screening steps that are essential for successful operation of proteomics pipelines with eukaryotic proteins: examination of total expression, determination of proteolysis of fusion tags, quantification of the yield of purified protein, and suitability for structure determination.

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    • "During the initial growth period, glucose is preferentially used as the carbon source, and protein expression is prevented through catabolic repression. When glucose is depleted, catabolic repression is relieved, thereby shifting cellular metabolism toward the import and consumption of lactose and glycerol [11]. Lactose is further generated into allolactose through ␤-galactosidase, which acts as a physiological inducer of the lac operon for the expression of recombinant proteins. "
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    • "The CESG high-throughput (HT) protein production pipeline (Aceti et al., 2003) (Figure 1) uses an economical small-scale screening (0.4 mL cultures), trials to predict the outcome of large-scale protein production (LSPP) cell-based growths (Figure 2), and to lower protein production costs. All protein targets are first produced as MBP-fusion protein in a single vector (pVP16) in the methionine auxotroph E. coli strain, B834-pRARE2 (metE-) (Studier, 2005 & Frederick et al., 2007), using seleno-methionine medium (5SM), and then purified using a generic immobilized metal-chelating chromatography (IMAC) scheme. In small-scale screening, the production of MBP-target protein fusions are analyzed by SDS-PAGE to evaluate their levels of expression, solubility, and TEV protease cleavage (Figure 1). "
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    ABSTRACT: CESG's strategy to minimize costs and maximize the output of soluble proteins suitable for purification uses small-scale screening methodologies to identify proteins targets that are statistically likely to proceed through all downstream processing stages (large-scale protein production and generic IMAC purification). Passage through the entire cell-based protein production process, beginning with gene cloning, requires ~2-3 months to deliver ~10 mg of purified, isotopically enriched protein for either NMR spectroscopy screens or X-ray crystallization trials. Small-scale screening identifies several important features of cloned eukaryotic proteins that together indicate whether the proteins will be "suitable" for structural studies. CESG small-scale methodologies include expression vector engineering, optimization and improvement of auto-induction medium using factorial evolution techniques, and automated protein production analysis. These approaches have been used to identify the best eukaryotic proteins (Arabidopsis, rice, human, yeast, zebrafish, mouse, and algae) for large-scale cell growths (2 liter), and also yield sufficient sample for functional studies and preliminary structural analysis. The CESG cell-based small-scale methodologies have proven to be rapid, simple, efficient, and cost-effective. The approaches developed provide information on the level of expression, the potential for in vivo degradation; the solubility of the MBP fusion; the ability of TEV protease to cleave the fusion, and the yield of cleaved and purified target protein.The small-scale screening method is flexible and assessments of the above mentioned properties are made prior to committing valuable resources for downstream processes. Over a 2 year period, this approach has successfully predicted proteins that are suitable for large-scale production and purification with ~80% accuracy. In summary, the CESG small-scale purification screening approach saves considerable resources and labor, and reduces expenditure of resources on proteins targets that will ultimately fail in protein purification.

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