Small-scale, semi-automated purification of eukaryotic proteins for structure determination
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- Frederick, R.O., Bergeman, L., Blommel, P.G. et al. J Struct Funct Genomics (2007) 8: 153. doi:10.1007/s10969-007-9032-5
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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 μg/ml of culture medium, of purified [U-15N]-His8-GFP was 68 μg/ml, and of purified selenomethione-labeled AIA–GFP (His8 removed by treatment with TEV protease) was 172 μg/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 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.
KeywordsEukaryotic protein Protein production Protein purification NMR X-ray crystallography Protein Structure Initiative Maxwell
The productivity of high-throughput structural genomics endeavors is critically dependent on how efficiently the best target proteins for structure determination can be identified [1, 2]. This applies to natural proteins as well as to products of engineering strategies, such as domain subcloning [3, 4, 5], modifications of the N- and C-termini [6, 7], surface entropy reduction [8, 9], and chemical alkylation of lysines . The pipeline used by most structural genomic centers defers the evaluation of whether a target protein is amenable for structure determination until after the target protein has been successfully produced and purified in quantities of tens of milligrams. This approach is expensive, because many important characteristics that determine whether a protein is suitable for structure determination, for example its stability, whether it is well-folded in solution, and whether it can be crystallized, only become apparent during 1H–15N HSQC measurements [7, 11, 12] or crystallization screening [13, 14, 15], and this is after considerable labor and resources have been expended.
Ideally, a bioinformatics evaluation would identify proteins best suited for structure determination [16, 17], but it is still difficult to significantly improve the output of protein structures based on this approach alone [18, 19]. Consequently, experimental methods to identify the best proteins are still required, and obtaining predictive results as early in the production pipeline as possible and with minimal labor and resources used has become a focus of much effort [20, 21, 22, 23, 24, 25, 26, 27].
The Center for Eukaryotic Structural Genomics (CESG) has worked to develop simple, efficient small-scale screening methods that successfully predict the results of large-scale protein purification. In previous studies, we have examined individual steps in the protein production and structure determination process [28, 29, 30, 31]. Recently, we have begun to combine improvements in expression vector engineering , in vivo fusion proteolysis of fusion proteins , and factorial evolution of auto-induction medium  with a bench top, automated method to prepare highly purified proteins required for structural evaluations. Here we show that this combination enables rapid production of labeled proteins that can be used directly in NMR and X-ray studies.
Materials and methods
Unless otherwise stated, bacterial growth reagents, antibiotics, routine laboratory chemicals, and disposable lab supplies were from Sigma-Aldrich (St. Louis, MO), Fisher (Pittsburgh PA) or other major distributors. l-Selenomethionine was from Acros (Morris Plains, NJ); [U-15N]-NH4Cl (98%) was from Cambridge Isotope Laboratories (Andover, MA). Preparation of standard laboratory reagents was as previously described . Deep-well growth blocks were from Qiagen (Valencia, CA). AeraSeal gas permeable sealing tape (T-2421-50) was from ISC Bioexpress (Kaysville, UT).
Genes were cloned into the expression vectors pVP56 and pVP62 [32, 33] using the Flexi Vector system (Promega, Madison WI). Vector pVP56 yields a TEV protease cleavable His8-maltose binding protein (His-MBP) fused to the N-terminus of the protein target. Vector pVP62 yields an MBP fusion that can undergo in vivo proteolysis to liberate a His8-tagged target protein. Selection during cloning was provided by substitution of the gene of interest for the toxic insert of barnase and chloramphenicol acetyltransferase bounded by the SgfI and PmeI restriction sites. Plasmids containing the lethal barnase gene must be propagated in a barnase-resistant strain (e.g., Escherichia coli BR610, which is available through Technical Services, Promega Corporation).
Expression vector for in vivo proteolysis
The pRARE2 plasmid for rare codon supplementation was isolated from E. coli Rosetta2 cells (EMD Biosciences/Novagen) using a Qiagen miniprep kit.
Alternating coding and non-coding strand PCR primers used for construction of an optimized GFP gene
The synthesized gene was purified using a Qiagen PCR purification kit (Valencia, CA), digested with NdeI (New England BioLabs) and BamHI (Promega) restriction enzymes, and ligated into similarly digested pET9a (EMD Biosciences/Novagen, Madison, WI) using standard molecular biology techniques . The cloned GFP gene was subsequently used as a template for PCR amplification to prepare the GFP gene for Flexi Vector cloning into pVP62K [32, 33].
Bacterial expression strain
Structural genomics target proteins investigated
Mol. wt. (Da)
Homo sapiens MGC:16774
Homo sapiens MGC:120678
Danio rerio MGC:103638
Homo sapiens MGC:120778
Xenopus laevis MGC53931
Danio rerio MGC:110031
Danio rerio MGC:109975
Homo sapiens MGC:4153
Xenopus laevis MGC78998
Danio rerio MGC:110849
Danio rerio MGC:113305
Danio rerio MGC:110805
Mus musculus MGC:5775
Danio rerio MGC:113187
Cell growth, protein production, and cell lysis
The methods for bacterial growth, protein production, and analysis were described elsewhere [28, 29, 30]. For the 15N labeling in the auto-induction medium , 15N Celtone liquid (Spectra Stable Isotopes, Columbia, MD) was added (5% v/v) to stimulate protein expression.
Target genes were screened for production at the small-scale (0.4 ml) in 96-well growth blocks using an auto-induction medium modified from the original  to better match correlation between small- and large-scale production . Meso-scale cell cultures (10–50 ml) for structural studies were grown as replicates in the 96-well growth blocks or in 0.5 l baffled shaking flasks (50 ml of medium per flask). Cells were grown for 24 h at 25°C with shaking at ∼250 rpm on a microplate shaker (VWR, West Chester, PA). After completion of the auto-induction protocol, bacterial cell cultures were harvested by centrifugation at 4,000 rpm and 4°C for 10–15 min using either an Allegra 6R centrifuge with a GH3.8 rotor or an Avanti J30-I with a JS5.9 rotor (Beckman Coulter, Fullerton, CA).
The pelleted cells were suspended in 8 ml of lysis buffer containing 50 mM NaH2PO4, pH 7.4, 0.5 M NaCl, 20% (w/v) ethylene glycol, and 1 mg/ml lysozyme (EMD Biosciences/Novagen). Protease inhibitors (E-64, 1.0 μM, Sigma, St. Louis, MO; benzamidine, 0.5 mM, Calbiochem, La Jolla, CA) were added to the cell suspension. A second lysis buffer was used for meso-scale (50 ml) protein production and contained 50 mM HEPES, pH 7.5, and 1 mg/ml lysozyme, E-64 protease inhibitor mix, and 10 units Benzonase (EMD Biosciences/Novagen). The suspended cells were sonicated using a Misonix 3000 sonicator (Misonix, Farmingdale NY) using 10 s sonication pulses for a total of 4 min with 30 s rest intervals between pulses.
Protein production analysis by denaturing electrophoresis
Samples for total protein production analysis were prepared for LabChip90 capillary electrophoresis (Caliper Life Sciences, Hopkinton, MA) as recommended by the manufacturer and were prepared for SDS-PAGE analysis using Criterion SDS-PAGE polyacryalmide gels (4–20% gradient Tris–HCl, 1.0 mm, 26 comb, BioRad, Richmond CA) as previously reported . The soluble protein fraction was obtained by centrifuging the sample plates for 30 min at 2,200 × g. Produced protein levels were determined by LabChip90 analysis and fluorescence (AIA–GFP). SDS- PAGE gels were analyzed using the Fotodyne Imaging System (Fotodyne Inc, Hartland, WI) and Foto/Analyst PC Image version 5.0 software.
Maxwell 16 purification screening protocol
For small-scale protein purification screening using the Maxwell 16 system, E. coli B834-pRARE2 cells were grown and genes expressed in isotopically enriched auto-induction medium using 96-well growth blocks. The growth was continued for 24 h until the culture had reached saturation, and then the culture was harvested by centrifugation (2,500 rpm for 2 min). The initial auto-induction growth medium was removed, and the cells were re-suspended by vortexing with 50 mM HEPES, pH 7.5, containing a protease inhibitor cocktail  to give an OD600 of 20 in 1 ml.
The vendor’s protocol and instructions were followed for protein purification. First, the Maxwell 16 protein purification cartridges were placed into the preparation rack. The cartridge seals were removed, and one plunger was placed into well #7 of each cartridge. An elution tube was placed for each protein sample into the front of the platform, and 300 μl of elution buffer was added to each elution tube. Aliquots of the re-suspended cells were transferred into well #1 of the Maxwell 16 purification cartridge, which also contained 10× Promega chemical lysis buffer, and the purification protocol was started. The Maxwell 16 system required 45 min to complete the purification run. The final purified proteins were removed from the elution cuvettes and analyzed by denaturing SDS-PAGE using either BioRad Criterion SDS-PAGE gels (4–20% gradient Tris–HCl) or the Caliper LC90 system.
Purification of Tcl-1 for NMR studies
A 50 ml (meso-scale) overnight culture (OD600 equal 10) expressing Tcl-1 was harvested by centrifugation, and re-suspended in ∼10 ml of 50 mM HEPES pH 7.5, with protease inhibitor cocktail , 10 units of Benzonase (EMD Biosciences/Novagen) to hydrolyze nucleic acids, and 1 mg/ml of lysozyme to reduce the viscosity of the lysate. The cell suspension was sonicated using a Sonic Dismembrator 550 (Fisher Scientific) programmed to run 10 s sonication pulses for a total of 5 min (with 20 s rest stops). Aliquots of the unclarified cell lysate (1 ml) were applied to well #1 of each lane of the Maxwell 16 cartridge, which also contained the chemical lysis buffer. The Maxwell 16 protocol for His-tagged proteins was run, and the purified His8-tagged protein samples from each lane were pooled. The final purified protein was exchanged into NMR screening buffer (10 mM KHPO4, 50 mM KCl, 0.2% NaN3, pH 7.0) and concentrated to a final volume of 0.5 ml using Millipore Amicon filtration devices. The total yield of the [U-15N]-Tcl-1 was ∼0.4 mg.
Purification of GFP for NMR and X-ray studies
A 50 ml cell culture grown with auto-induction for 24 h (OD600 equal to 10) with the appropriate isotopic label was harvested by centrifugation, resuspended in 8–12 ml of 50 mM HEPES, pH 7.5, with protease inhibitors, benzonase, and 1 mg/ml of lysozyme, and sonicated as above. Aliquots (1 ml) of the unclarified cell lysate were applied to well #1 of eight lanes of the Maxwell 16 cartridge prior to the purification run. [U-15N]-His8-GFP and [Se-Met]-labeled His8-GFP purified in this manner were each exchanged into the appropriate NMR  or X-ray  screening buffer, and concentrated to final volumes of 0.5 ml. The yield of the 15N labeled His8-GFP was 3.4 mg from 50 ml of cell culture. The purified [Se-Met]-His8-GFP was treated subsequently with TEV protease  to remove the His8 tag; the His8 tag and His8-tagged TEV protease were removed by treatment with MagneHis particles; the buffer was exchanged, and the purified protein was concentrated to a final volume of 0.5 ml. The yield of selenomethionine labeled AIA–GFP was 2.8 mg.
Purified proteins were analyzed by denaturing electrophoresis in the buffer described above. Concentrations were determined using the Caliper LC90 software and the BCA method (Pierce, Rockford IL). The masses of the purified [U-15N]- and [Se-Met]-labeled proteins were determined in the University of Wisconsin Biotechnology Center by electrospray ionization mass spectrometry using an Applied Biosystems/MDS Sciex API 365 LC/MS/MS triple quadrupole mass spectrometer (Applied Biosystems, Foster City, CA, USA). The labeling efficiency was calculated from the experimentally determined and calculated molecular weights of the protein. Protein identification was carried out by tryptic proteolysis and molecular weight assignment of generated peptides using a Bruker BIFLEX III MALDI-TOF spectrometer (Bruker Daltonics, Billerica, MA, USA).
1H–15N HSQC NMR
2D 1H–15N HSQC spectra were acquired for [U-15N]-labeled Tcl-1 and [U-15N]-labeled GFP at the National Magnetic Resonance Facility at Madison (NMRFAM) on 600 and 750 MHz Bruker spectrometers equipped with cryogenic 1H, 15N, 13C triple-resonance probes. The sample temperature was regulated at 35°C. The NMR spectra consisted of 1,024 and 100–110 complex data points, respectively for 1H and 15N. For each increment, 128 scans were used for [U-15N]-labeled Tcl-1, and 16 scans were used for [U-15N]-labeled GFP.
Crystallization of AIA–GFP
AIA–GFP was screened initially using the UW192 screen. From this screen, crystals were observed within 2 days from five different conditions. One condition was optimized for structure determination.
AIA–GFP crystals were grown by hanging drop vapor diffusion. The crystals reached a size of ∼0.1 × 0.1 × 1 mm rods in approximately 3 days at 25°C in a mother liquor consisting of 100 mM HEPPS, pH 8.5, 80 mM CaCl2, and 15% PEG 4K. Hanging drops consisted of 2.5 μl of protein solution and 2.5 μl of mother liquor. Crystals were soaked in mother liquor containing increasing amounts of glycerol to a final concentration of 20% and flash cooled in a stream of liquid nitrogen. X-ray diffraction data were collected on a BrukerAXS PROTEUM R (BrukerAXS, Madison, WI) diffractometer at −180°C using CuKα radiation from a MicroStar (BrukerAXS, Madison, WI) source with a rotating anode generator operated at 44 kV and 45 mA. The X-ray data were processed and scaled with PROTEUM 2 (BrukerAXS, Madison, WI). The structure of GFP was solved by molecular replacement using PDB 2g6e as the rotation target with MOLREP from the CCP4 suite . The model was built with alternating cycles of model building and refinement with COOT  and REFMAC . Coordinates and structure factors were deposited in the Protein Data Bank with accession number 2qu1.
Met auxotrophy in E. coli B834
The strain E. coli B834 is frequently used for selenomethionine labeling . It is a conditional methionine auxotroph that can be complemented by the addition of vitamin B12. The exact cause of the auxotrophy was not known, but some believed that MetB was inactive , while others thought that MetE was . To better define the origin of the auxotrophy, genomic DNA was isolated, and the regions around the metE gene (GeneBank accession number DQ267751, encoding vitamin B12-independent homocysteine transmethylase) and the metH gene (GeneBank accession number DQ272318, encoding vitamin B12-dependent homocysteine transmethylase) were sequenced.
Human embryonic stem cell proteins and others characterized by in vivo cleavage and purification screening
Mol. wt. (Da)
MGC:20335, 2260, 2170
Chromosome 10 open reading frame 96
Control protein, synthetic gene
Control protein, synthetic gene
Maxwell 16 purification screening
Of the 14 fusion proteins tested (A1–B2, Table 2 and Fig. 3), seven were purified with yields exceeding 100 μg/ml, six were purified with yields less than 100 μg/ml, and one protein was not purified. Among the seven high yield proteins, five also received a favorable assessment for scale-up by the original scoring method; among the six low yield proteins, three also received an unfavorable assessment for scale-up by the original scoring method. Thus the decision to scale-up the purification or not was the same by either method in eight of 14 cases.
Among the six proteins that were assessed differently by the original method and the newer purification screening, there were two outcomes. One outcome, exemplified by A2, A6, A10, and B1, were proteins that had a favorable score by the original method, but that were not purified in yield >100 μg/ml by the newer purification screening protocol. Use of purification screening would eliminate unnecessary scale-up effort for a class of proteins that ultimately fail in purification. The other outcome, exemplified by A8 and A9, was a successful purification of a large amount of protein when the work would have been stopped by the original method. Since the results of Fig. 3 with A8 and A9 do not include a proteolysis step, the original screen provided additional information suggesting that these targets would ultimately fail in purification because they had an unsuitable W score for proteolysis. Indeed, when A8 and A9 obtained from the Maxwell were treated with TEV protease, neither target was successfully liberated from the fusion protein.
In order to address this deficiency in the purification screening, we developed an in vivo cleavage expression vector to couple with the Maxwell 16 purification system. The assembly of this vector and results of using this combination for purification screening of human embryonic stem cell proteins is described in the following sections.
Purification screening of human stem cell proteins
Replicate Maxwell 16 purification of Tcl-1
A similar scale-up of the initial expression results was undertaken for AIA–GFP (Fig. 4, lane 11) with either [U-15N]- or [Se-Met]-labeling. The expressed AIA–GFP had high fluorescence, and could be easily observed by visual inspection at all steps of the automated purification. The identity of purified AIA–GFP was confirmed by tryptic proteolysis and liquid chromatography/tandem mass spectrometry. The yield of [U-15N]-His8-GFP was ∼3.4 mg. This sample was pooled and concentrated, and further exchanged into a buffer suitable for NMR studies (∼2 h). For X-ray crystallography, the His8-tag was removed by treatment with TEV protease. The final yield of purified AIA–GFP was 2.8 mg, and a high level of selenomethionine was incorporated into the purified AIA–GFP as determined by electrospray ionization mass spectrometry.
Figure 6b shows the 600 MHz 1H–15N HSQC NMR spectrum of His8-GFP recorded at 25°C. This spectrum was acquired in 1 h. For this GFP, a total of about 219 cross peaks out of a possible 253 cross peaks (including amino acid chain amides) were present. The broader NMR line widths due to the larger size of GFP (238 aa, ∼27,000 Da) account for some the difference between the numbers of expected and observed cross peaks. Nevertheless, the chemical shifts are well dispersed, suggesting that the Maxwell-purified protein was well structured in solution and thus acceptable for further structural studies.
X-ray structure of AIA–GFP
Summary of crystallization conditions observed for AIA–GFP
24% MEPEG 5K
0.1 M MES, pH 6.0
160 mM CaCl2
16% MEPEG 5K
0.1 M BTP, pH 7.0
200 mM glycine
28% MEPEG 2K
0.1 M HEPES, pH 7.5
100 mM CaCl2
20% PEG 4K
0.1 M HEPPS, pH 8.5
80 mM CaCl2
0.1 M MES, pH 6.0
Summary of data collection, crystal structure, and refinement statistics for AIA–GFP
a, b, c (Å)
51.46, 61.99, 70.02
α, β, γ (°)
90, 90, 90
Mean B-value (overall)
Most favored regions
Additional allowed regions
Bond lengths (Å)
Bond angles (°)
Protein production pipelines have numerous points of attrition that limit the number of proteins available for structural analysis. This attrition adds significant expense to the overall process, particularly when multiple handling steps and larger volumes are typically required to obtain decisions. Thus the use of small- or micro-scale protein screening methods has considerable appeal [22, 24].
The focus of this work has been on eukaryotic proteins, which are generally found to be more difficult to express and purify than prokaryotic proteins. Small-scale production screening efforts have predicted the outcome of downstream large-scale protein production with up to 80% efficiency . However, these previous efforts did not effectively address either the variability in proteolysis of fusion proteins that are often used to express eukaryotic proteins in E. coli or the behavior of the liberated targets after proteolysis. By adopting this screening approach, the decision to scale-up protein production can be based on the ability to express, proteolyze, and purify the protein, and as indicated here, this decision can be extended to the ability to include other information such as acceptable 1H–15N HSQC spectra or evidence of crystallization.
Table 2 contains information on another troublesome set of eukaryotic proteins, those with pI ∼8 or greater (A2, A3, A4, A10, B1, B2). Purification screening would provide important insight into the behavior of these proteins, which often perform well as fusion proteins in total production and solubility properties, but which often fail in proteolysis or stability after proteolysis (∼70%, unpublished results). Thus, although the original pipeline screening suggested A2, A6, A10, and B1 should have been advanced to purification, each of these targets failed to achieve the desired threshold from the Maxwell purification for purified fusion protein and thus deserved a work stopped assignment. In contrast, the high pI protein A3 was purified in high yield as a fusion protein from the Maxwell and subsequently was released by TEV protease treatment (as in the original pipeline scoring), supporting the decision that this protein should be continued along the scale-up process. Further consideration of the results of these targets will be included in a broader study of the effect of the N-terminal AIA tag on protein purification and structure determination statistics, which will be reported elsewhere.
Scalability requires similar protein production behavior in small-scale screening, large-scale protein production, and, ultimately, protein purification. For proteins A8 and A9, the original small-scale screening reported these proteins were unsuitable because of a failure in TEV proteolysis, and this result was also determined after Maxwell purification. Among the four human embryonic stem cell proteins investigated, Tcl-1 was highly expressed by auto-induction, underwent efficient in vivo proteolysis from MBP, and was successfully purified with an estimated volumetric productivity of 7.5 μg/ml. Two other stem cell proteins (C10orf96 and NPM2), were also purified, but their yields were not sufficient to indicate feasibility of the scale-up as a structural target. Nevertheless, the method yielded enough purified protein that some functional studies or other analyses could be undertaken. By coupling in vivo cleavage with automated purification, failure to proteolyze the His8-target from the fusion protein and cryptic insolubility of the His8-target after proteolysis are signaled by failure in automated purification. Since both of these results are diagnostic of likely failures in large-scale purification, the purification screening approach gave valuable insight into the behavior of the human stem cell protein CCNF and the others before any significant scale-up efforts were undertaken.
We demonstrated how the amount of His8-target successfully purified from a single Maxwell 16 lane can be used to determine the scale-up factor required to prepare samples for screening either by 1H–15N NMR for folding status (∼700 μg of a 25 kDa or less protein set as the deliverable) or by microfluidic screening for crystallization (∼10 μg of protein set as the deliverable). This scaling approach was demonstrated for both Tcl-1 and AIA–GFP. Decreasing the amount of protein required for initial structural screening through the use of small NMR tubes, cryoprobes, and by nL liquid handling effectively complements the ability to produce moderate amounts of protein in the cost-effective manner described here. Automated methods for removal of the His8-tag during the Maxwell 16 run would also be desirable, and these investigations are in progress.
CESG starts all expression work on eukaryotic proteins with sequence verified clones [32, 53]. Uncertainties in gene models and errors from primer synthesis are addressed by this effort, while immediate sequence verification of a cloned gene also supports reliable transfer into other expression vectors. Expression plasmids transformed into E. coli B834 can be available for purification screening studies after 24 h, and growth from single colony transformants can be completed in 48 h using our auto-induction approach (24 h of growth in non-inducing medium followed by 24 h of growth in inducing medium). Auto-induced cultures can be immediately loaded onto the Maxwell 16 apparatus, with parallel processing of 16 samples in 45 min. Thus a complete 96-well plate of different targets (or variants of the same target) could be purified and analyzed for protein expression by the automated capillary electrophoresis in less than 7 h. In the work flow of Fig. 8, the best performing targets, provisionally defined as those obtained from in vivo cleavage and automated purification in yield of 50 μg/ml or greater, can be identified in about 4 days, with most of the elapsed time allotted to overnight culture growths or automated protein purification.
Auto-induction media are chemically defined and assembled from inexpensive components. Furthermore, the cost of labeled amino acids (15N or Se-Met) is minimal for the initial screening due to the small cell culture volume required. For the example shown in Fig. 4, the cost for all reagents for the auto-induction and automated purification of the 15N-labeled sample was less than $50. The simple instrumentation required for the auto-induction and the Maxwell 16 purification may allow wide access to this approach, and the minimal hands-on effort required to complete the analysis through to purified protein is another considerable operational advantage.
Capillary electrophoresis has several advantages relative to slab gel electrophoresis. Although the instrument is more expensive than a standard power supply, electrophoresis equipment, and gel documentation system, the average price per sample analysis (∼$0.67 per lane of analysis) using the LC90 chip is less than pre-cast polyacrylamide gels (∼$1.17 per lane of analysis). Other advantages of capillary electrophoresis include automated operation, rapid processing time, digital information capture, and quantitative analysis of electropherograms. This work shows that the quantitative analysis of protein yield from a small-scale expression can be used as a predictive tool for scale-up feasibility.
Other applications of this approach
The automated protein purification process described here has other potential uses. This process can facilitate evaluation of different vector designs and arrays of different expression hosts. For functional studies, banks of site-directed or randomly mutated proteins can be prepared and purified in amounts sufficient for catalytic screening. This may facilitate protein engineering for new traits that can be assayed such as changes in catalytic activity, thermal stability, or other desirable properties. In many cases, the amounts of protein recovered by the automated purification (Tables 2 and 3) should be adequate to initiate these functional studies. Surface entropy reduction analysis could also be facilitated through an effective sorting of protein variants that maintain sufficient stability to be purified. The delivery of small quantities of purified proteins for examination by micro-crystallization techniques or NMR analysis before significant effort is placed into purifying large quantities also has demonstrable advantages.
For eukaryotic proteins, domain engineering is an important experimental focus. It is clear that multiple changes at the N- and C-terminus may be required to identify the best performing variant. Through the use of purification screening, it is efficient to express, purify, and examine engineered domains for improved solubility properties as part of the experimental process.
The authors thank other members of the University of Wisconsin-Madison, Center for Eukaryotic Structural Genomics for their assistance and support. Special thanks are offered to Michael A. Goren, Karl Nichols, Maggie Harteau, Steve Sarles and Stuart Ballard. The authors also thank Professor James Thomson (University of Wisconsin-Madison, Department of Anatomy and Genome Center) for identification of the human embryonic stem cell proteins used in this work. This work was supported by the National Institutes of Health, Protein Structure Initiative grant U54 GM074901 (J.L.M., PI; G.N.P., Co-Investigator; B.G.F.; Co-Investigator) and a sponsored research agreement from Promega Corporation to B.G.F. J.G.M. is supported by an NHGRI training grant to the Genomic Sciences Training Program (5T32HG002760). L.J.B. is supported by NIH grant GM50853 and National Science Foundation grant MCB-0316232 to B.G.F.
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