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

Abstract

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-15 N]-His8-Tcl-1 was 7.5 lg/ml of culture medium, of purified [U-15 N]-His8-GFP was 68 lg/ml, and of purified selenomethione-labeled AIA-GFP (His8 removed by treatment with TEV protease) was 172 lg/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.1 H-15 N 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 Å . 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.

Autoinduction of Protein Expression

“…Based on observed expression levels, other applications might also be supported by small-scale autoinduction experiments. For example, cultures obtained from autoinduction can yield sufficient purified protein for automated protein purification (Frederick et al, 2007), microfluidics-based crystallization screening (van der Woerd et al, 2003; Segelke, 2005), initial nanoliter-scale crystallization trials (Lion et al, 2004; Zheng et al, 2005), 15 N HSQC NMR measurements (Kennedy et al, 2002; Galvao-Botton et al, 2003), or functional and enzymatic characterizations (Kumar and Clark, 2006; Blommel and Fox, 2007). …”

“…Work from the authors’ laboratory has shown autoinduction is effective in 2-liter shaken bottles, 96-well growth blocks, and automated stirred-vessel fermenters (Blommel and Fox, 2007; Frederick et al, 2007). The different autoinduction results presented here include the use of shaken bottles (Fig.…”

Expression platforms for producing eukaryotic proteins: a comparison of E. coli cell-based and wheat germ cell-free synthesis, affinity and solubility tags, and cloning strategies

“…The E. coli strain B834-pRARE2, which is useful in SeMet labeling for phase determination in X-ray crystallography, was the expression host [16]. Small-scale trials evaluating expression, solubility, and tag cleavage were carried out using autoinduction in both chemically defined and complex medium [5].…”

A semi-automated method for purification of milligram quantities of proteins on the QIAcube

“…For a life science lab wishing to automate the purification of biomolecules such as protein and nucleic acids, there are several relatively inexpensive systems available: Promega's Maxwell 16 (16 samples) and Thermofisher’s Kingfisher (24 samples) which both use paramagnetic particle technology for protein and nucleic acid purification [6, 7], the Bio-Rad Profinia (1 sample) that can run 2 sequential columns (i.e. affinity followed by desalting) for protein purification [8] and Qiagen’s QIAcube (12 samples) for protein and nucleic acid purification [9].…”