High-throughput Purification and Quality Assurance of Arabidopsis thaliana Proteins for Eukaryotic Structural Genomics

The Center for Eukaryotic Structural Genomics (CESG) has established procedures for the purification of Arabidopsis proteins in a high-throughput mode. Recombinant proteins were fused with (His)(6)-MBP tags at their N-terminus and expressed in Escherichia coli. Using an automated AKTApurifier system, fusion proteins were initially purified by immobilized metal affinity chromatography (IMAC). After cleavage of (His)(6)-MBP tags by TEV protease, (His)(6)-MBP tags were separated from target proteins by a subtractive 2nd IMAC. As a part of quality assurance, all purified proteins were subjected to MALDI-TOF and ESI mass spectrometry to confirm target identity and integrity, and determine incorporation of seleno-methionine (SeMet) and (15)N and (13)C isotopes. The protocols have been used successfully to provide high quality proteins that are suitable for structural studies by X-ray crystallography and NMR.     

Automation of protein purification for structural genomics

A critical issue in structural genomics, and in structural biology in general, is the availability of high-quality samples. The additional challenge in structural genomics is the need to produce high numbers of proteins with low sequence similarities and poorly characterized or unknown properties. 'Structural-biology-grade' proteins must be generated in a quantity and quality suitable for structure determination experiments using X-ray crystallography or nuclear magnetic resonance (NMR). The choice of protein purification and handling procedures plays a critical role in obtaining high-quality protein samples. The purification procedure must yield a homogeneous protein and must be highly reproducible in order to supply milligram quantities of protein and/or its derivative containing marker atom(s). At the Midwest Center for Structural Genomics we have developed protocols for high-throughput protein purification. These protocols have been implemented on AKTA EXPLORER 3D and AKTA FPLC 3D workstations capable of performing multidimensional chromatography. The automated chromatography has been successfully applied to many soluble proteins of microbial origin. Various MCSG purification strategies, their implementation, and their success rates are discussed in this paper.     

A dual protease approach for expression and affinity purification of recombinant proteins

We describe a new method for affinity purification of recombinant proteins using a dual protease protocol. Escherichia coli maltose binding protein (MBP) is employed as an N-terminal tag to increase the yield and solubility of its fusion partners. The MBP moiety is then removed by rhinovirus 3C protease, prior to purification, to yield an N-terminally His₆-tagged protein. Proteins that are only temporarily rendered soluble by fusing them to MBP are readily identified at this stage because they will precipitate after the MBP tag is removed by 3C protease. The remaining soluble His₆-tagged protein, if any, is subsequently purified by immobilized metal affinity chromatography (IMAC). Finally, the N-terminal His₆ tag is removed by His₆-tagged tobacco etch virus (TEV) protease to yield the native recombinant protein, and the His₆-tagged contaminants are removed by adsorption during a second round of IMAC, leaving only the untagged recombinant protein in the column effluent. The generic strategy described here saves time and effort by removing insoluble aggregates at an early stage in the process while also reducing the tendency of MBP to “stick” to its fusion partners during affinity purification.     

Purification and characterization of RGD tumor-homing peptide conjugated human tumor necrosis factor alpha over-expressed in Escherichia coli

A number of approaches have been investigated to enhance the selective toxicity of tumor necrosis factor alpha (TNFalpha) to permit its systemic use in cancer therapy. Because vascular targeting has been proven to be a valid strategy for improving the therapeutic index of TNFalpha, we prepared RGD-hTNF consisting of human TNF fused with the ACDCRGDCFCG peptide, a ligand of alpha(v)beta(3) and alpha(v)beta(5) integrins. Recombinant RGD-hTNF was produced in Escherichia coli as a polyhistidine fusion protein. Between polyhistidine tag and RGD-hTNF, a tobacco etch virus (TEV) protease cleavage site (ENLYFQG) was introduced to ensure the release of intact RGD-hTNF. The purification strategy consisted of the target protein capture step by immobilized metal affinity chromatography (IMAC), TEV protease cleavage of fusion protein, the subtractive depletion of removed His-tag by IMAC and the final gel filtration step. As a result, about 18 mg of intact RGD-hTNF was obtained from 1l of bacteria culture. The purified RGD-hTNF was characterized by SDS-PAGE, Western blot, mass spectroscopy and gel filtration. Since the RGD-hTNF molecule retained the cytotoxic activity of the TNF moiety and the integrin binding ability of the RGD moiety, the purification method provided material for assessing its anti-tumor activity in animal model.     

Exploring the activity of tobacco etch virus protease in detergent solutions

Tobacco etch virus (TEV) protease is generally used to remove affinity tags from target proteins. It has been reported that some detergents inhibit the activity of this protease, and therefore should be avoided when removing affinity tags from membrane proteins. The aim of this study was to explore and evaluate this further. Hence, affinity tag removal with TEV protease was tested from three membrane proteins (a Pgp synthase and two CorA homologs) in the presence of 16 different detergents commonly used in membrane protein purification and crystallization. We observed that in the presence of the same detergent (Triton X-100), TEV protease could remove the affinity tag completely from one protein (CorA) but not from another protein (Pgp synthase). There was also a large variation in yield of cleaved membrane protein in different detergents, which probably depends on features of the protein-detergent complex. These observations show that, contrary to an earlier report, detergents do not inhibit the enzymatic activity of the TEV protease.     

Two-step metal affinity purification of double-tagged (NusA-His6) fusion proteins

The present purification protocol applies to target proteins that are fused to a double tag, such as NusA-His6, through a linker that includes a protease-recognition sequence. It involves two steps of immobilized metal ion affinity chromatography (IMAC). NusA stabilizes the passenger protein during translation, whereas the His-tag enables affinity purification of the fusion. The eluate resulting from the first IMAC is buffer-exchanged to remove the imidazole and to achieve optimal conditions for the enzymatic cleavage performed by a His-tagged recombinant protease. The digested sample is loaded directly for a second IMAC step and the target protein is selectively recovered in the flow-through. The resin binds residual non-digested fusion protein, double-tagged moiety, protease and any contaminant that bound the affinity resin and was eluted from the first IMAC. The purity of the target protein usually makes a further purification step unnecessary for most of the lab applications. It takes less than 5 hours to purify the protein from a 5 g pellet.     

A simple and efficient expression and purification system using two newly constructed vectors

Structural biology places a high demand on proteins both in terms of quality and quantity. Although many protein expression and purification systems have been developed, an efficient and simple system which can be easily adapted is desirable. Here, we report a new system which combines improved expression, solubility screening and purification efficiency. The system is based on two newly constructed vectors, pEHISTEV and pEHISGFPTEV derived from a pET vector. Both vectors generate a construct with an amino-terminal hexahistidine tag (His-tag). In addition, pEHISGFPTEV expresses a protein with an N-terminal His-tagged green fluorescent protein (GFP) fusion to allow rapid quantitation of soluble protein. Both vectors have a tobacco etch virus (TEV) protease cleavage site that allows for production of protein with only two additional N-terminal residues and have the same multiple cloning site which enables parallel cloning. Protein purification is a simple two-stage nickel affinity chromatography based on the His tag removal. A total of seven genes were tested using this system. Expression was optimised using pEHISGFPTEV constructs by monitoring the GFP fluorescence and the soluble target proteins were quantified using spectrophotometric analysis. All the tested proteins were purified with sufficient quantity and quality to attempt structure determination. This system has been proven to be simple and effective for structural biology. The system is easily adapted to include other vectors, tags or fusions and therefore has the potential to be broadly applicable.     

Immobilised metal-ion affinity chromatography purification of histidine-tagged recombinant proteins: a wash step with a low concentration of EDTA

Immobilised metal-ion affinity chromatography (IMAC) is widely used for the purification of recombinant proteins in which a poly-histidine tag is introduced. However, other proteins may also bind to IMAC columns. We describe the use of a washing buffer with a low concentration of EDTA (0.5 mM) for the removal of proteins without histidine tag from IMAC columns. Four histidine-tagged recombinant proteins/protein complexes were purified to homogeneity from cell culture medium of insect cells by using an EDTA washing buffer. The presence of a low concentration of EDTA in washing buffers during IMAC may have a general application in the purification of histidine-tagged proteins.     

Recombinant Passenger Proteins Can Be Conveniently Purified by One-Step Affinity Chromatography

Fusion tag is one of the best available tools to date for enhancement of the solubility or improvement of the expression level of recombinant proteins in Escherichia coli. Typically, two consecutive affinity purification steps are often necessitated for the purification of passenger proteins. As a fusion tag, acyl carrier protein (ACP) could greatly increase the soluble expression level of Glucokinase (GlcK), α-Amylase (Amy) and GFP. When fusion protein ACP-G2-GlcK-Histag and ACP-G2-Amy-Histag, in which a protease TEV recognition site was inserted between the fusion tag and passenger protein, were coexpressed with protease TEV respectively in E. coli, the efficient intracellular processing of fusion proteins was achieved. The resulting passenger protein GlcK-Histag and Amy-Histag accumulated predominantly in a soluble form, and could be conveniently purified by one-step Ni-chelating chromatography. However, the fusion protein ACP-GFP-Histag was processed incompletely by the protease TEV coexpressed in vivo, and a large portion of the resulting target protein GFP-Histag aggregated in insoluble form, indicating that the intracellular processing may affect the solubility of cleaved passenger protein. In this context, the soluble fusion protein ACP-GFP-Histag, contained in the supernatant of E. coli cell lysate, was directly subjected to cleavage in vitro by mixing it with the clarified cell lysate of E. coli overexpressing protease TEV. Consequently, the resulting target protein GFP-Histag could accumulate predominantly in a soluble form, and be purified conveniently by one-step Ni-chelating chromatography. The approaches presented here greatly simplify the purification process of passenger proteins, and eliminate the use of large amounts of pure site-specific proteases.     

Saccharomyces cerevisiae–Based Platform for Rapid Production and Evaluation of Eukaryotic Nutrient Transporters and Transceptors for Biochemical Studies and Crystallography

To produce large quantities of high quality eukaryotic membrane proteins in Saccharomyces cerevisiae, we modified a high-copy vector to express membrane proteins C-terminally-fused to a Tobacco Etch Virus (TEV) protease detachable Green Fluorescent Protein (GFP)-8His tag, which facilitates localization, quantification, quality control, and purification. Using this expression system we examined the production of a human glucose transceptor and 11 nutrient transporters and transceptors from S. cerevisiae that have not previously been overexpressed in S. cerevisiae and purified. Whole-cell GFP-fluorescence showed that induction of GFP-fusion synthesis from a galactose-inducible promoter at 15°C resulted in stable accumulation of the fusions in the plasma membrane and in intracellular membranes. Expression levels of the 12 fusions estimated by GFP-fluorescence were in the range of 0.4 mg to 1.7 mg transporter pr. liter cell culture. A detergent screen showed that n-dodecyl-ß-D-maltopyranoside (DDM) is acceptable for solubilization of the membrane-integrated fusions. Extracts of solubilized membranes were prepared with this detergent and used for purifications by Ni-NTA affinity chromatography, which yielded partially purified full-length fusions. Most of the fusions were readily cleaved at a TEV protease site between the membrane protein and the GFP-8His tag. Using the yeast oligopeptide transporter Ptr2 as an example, we further demonstrate that almost pure transporters, free of the GFP-8His tag, can be achieved by TEV protease cleavage followed by reverse immobilized metal-affinity chromatography. The quality of the GFP-fusions was analysed by fluorescence size-exclusion chromatography. Membranes solubilized in DDM resulted in preparations containing aggregated fusions. However, 9 of the fusions solubilized in DDM in presence of cholesteryl hemisuccinate and specific substrates, yielded monodisperse preparations with only minor amounts of aggregated membrane proteins. In conclusion, we developed a new effective S. cerevisiae expression system that may be used for production of high-quality eukaryotic membrane proteins for functional and structural analysis.     

Inhibition of tobacco etch virus protease activity by detergents

Affinity tags such as polyhistidine greatly facilitate recombinant protein production. The solubility of integral membrane proteins is maintained by the formation of protein-detergent complexes (PDCs), with detergent present at concentration above its critical micelle concentration (CMC). Removal of the affinity tag necessitates inclusion of an engineered protease cleavage site. A commonly utilized protease for tag removal is tobacco etch virus (TEV) protease. TEV is available in a recombinant form (rTEV) and frequently contains its own polyhistidine affinity tag for removal after use in enzymatic digestion. Proteolytic cleavage of the tagged domain is carried out by incubation of the protein with rTEV protease. We have observed that the efficiency of rTEV digestion decreases significantly in the presence of a variety of detergents utilized in purification, crystallization, and other biochemical studies of integral membrane proteins. This reduction in protease activity is suggestive of detergent-induced inhibition of rTEV. To test this hypothesis, we examined the effects of detergents upon the rTEV proteolytic digestion of a soluble fusion protein, alpha(1) platelet activating factor acetylhydrolase (PAFAHalpha(1)). Removal of a hexahistidine amino-terminal affinity tag has been characterized in the presence of 16 different detergents at concentrations above their respective CMCs. Our data indicate that half of the detergents tested reduce the activity of rTEV and that these detergents should be avoided or otherwise accounted for during rTEV digestion of recombinant integral membrane proteins.     

Tobacco etch virus protease retains its activity in various buffers and in the presence of diverse additives

Tobacco etch virus (TEV) protease is widely used to remove tags from recombinant fusion proteins because of its stringent sequence specificity. It is generally accepted that the high concentrations of salts or other special agents in most protein affinity chromatography buffers can affect enzyme activity, including that of TEV protease. Consequently, tedious desalination or the substitution of standard TEV reaction buffer for elution buffer are often needed to ensure TEV protease activity when removing fusion tags after purifying target proteins using affinity chromatography. To address this issue, we used SOE PCR technology to synthesize a TEV protease gene with a codon pattern adapted to the codon usage bias of Escherichia coli, recovered the purified recombinant TEV protease, and examined its activity in various elution buffers commonly used in affinity chromatography as well as the effects of selected additives on its activity. Our results showed that the rTEV protease maintained high activity in all affinity chromatography elution buffers tested and tolerated high concentrations of additives commonly used in protein purification procedures, such as ethylene glycol, EGTA, Triton X-100, Tween-20, NP-40, CHAPS, urea, SDS, guanidine hydrochloride and β-mercaptoethanol. These results will facilitate the use of rTEV protease in removing tags from fusion proteins.     

1,000 structures and more from the MCSG

Background  

The Midwest Center for Structural Genomics (MCSG) is one of the large-scale centres of the Protein Structure Initiative (PSI). During the first two phases of the PSI the MCSG has solved over a thousand protein structures. A criticism of structural genomics is that target selection strategies mean that some structures are solved without having a known function and thus are of little biomedical significance. Structures of unknown function have stimulated the development of methods for function prediction from structure.     

The P1' specificity of tobacco etch virus protease

Affinity tags have become indispensable tools for protein expression and purification. Yet, because they have the potential to interfere with structural and functional studies, it is usually desirable to remove them from the target protein. The stringent sequence specificity of the tobacco etch virus (TEV) protease has made it a useful reagent for this purpose. However, a potential limitation of TEV protease is that it is believed to require a Gly or Ser residue in the P1' position of its substrates to process them with reasonable efficiency. Consequently, after an N-terminal affinity tag is removed by TEV protease, the target protein will usually retain a non-native Ser or Gly residue on its N-terminus, and in some cases this may affect its biological activity. To investigate the stringency of the requirement for Gly or Ser in the P1' position of a TEV protease recognition site, we constructed 20 variants of a fusion protein substrate with an otherwise optimal recognition site, each containing a different amino acid in the P1' position. The efficiency with which these fusion proteins were processed by TEV protease was compared both in vivo and in vitro. Additionally, the kinetic parameters K(M) and k(cat) were determined for a representative set of peptide substrates with amino acid substitutions in the P1' position. The results indicate that many side-chains can be accommodated in the P1' position of a TEV protease recognition site with little impact on the efficiency of processing.     

SOn-column Protein Refolding for Crystallization

One major bottleneck in protein production in Escherichia coli for structural genomics projects is the formation of insoluble protein aggregates (inclusion bodies). The efficient refolding of proteins from inclusion bodies is becoming an important tool that can provide soluble native proteins for structural and functional studies. Here we report an on-column refolding method established at the Berkeley Structural Genomics Center (BSGC). Our method is a combination of an ‘artificial chaperone-assisted refolding’ method previously proposed and affinity chromatography to take advantage of a chromatographic step: less time-consuming, no filtration or concentration, with the additional benefit of protein purification. It can be easily automated and formatted for high-throughput process.     

Purification of proteins containing zinc finger domains using immobilized metal ion affinity chromatography

Heterologous proteins are frequently purified by immobilized metal ion affinity chromatography (IMAC) based on their modification with a hexa-histidine affinity tag (His-tag). The terminal His-tag can, however, alter functional properties of the tagged protein. Numerous strategies for the tag removal have been developed including chemical treatment and insertion of protease target sequences in the protein sequence. Instead of using these approaches, we took an advantage of natural interaction of zinc finger domains with metal ions to purify functionally similar retroviral proteins from two different retroviruses. We found that these proteins exhibited significantly different affinities to the immobilized metal ions, despite that both contain the same type of zinc finger motif (i.e., CCHC). While zinc finger proteins may differ in biochemical properties, the multitude of IMAC platforms should allow relatively simple yet specific method for their isolation in native state.     

A rapid and universal tandem-purification strategy for recombinant proteins

A major goal in the production of therapeutic proteins, subunit vaccines, as well as recombinant proteins needed for structure determination and structural proteomics is their recovery in a pure and functional state using the simplest purification procedures. Here, we report the design and use of a novel tandem (His)(6)-calmodulin (HiCaM) fusion tag that combines two distinct purification strategies, namely, immobilized metal affinity (IMAC) and hydrophobic interaction chromatography (HIC), in a simple two-step procedure. Two model constructs were generated by fusing the HiCaM purification tag to the N terminus of either the enhanced green fluorescent protein (eGFP) or the human tumor suppressor protein p53. These fusion constructs were abundantly expressed in Escherichia coli and rapidly purified from cleared lysates by tandem IMAC/HIC to near homogeneity under native conditions. Cleavage at a thrombin recognition site between the HiCaM-tag and the constructs readily produced untagged, functional versions of eGFP and human p53 that were >97% pure. The HiCaM purification strategy is rapid, makes use of widely available, high-capacity, and inexpensive matrices, and therefore represents an excellent approach for large-scale purification of recombinant proteins as well as small-scale protein array designs.     

An automated small-scale protein expression and purification screening provides beneficial information for protein production

One of the first key steps in structural genomics is high-throughput expression and rapid screening to select highly soluble proteins, the preferred candidates for crystal production. Here we describe the methodology used at the Berkeley Structural Genomics Center (BSGC) for automated parallel expression and small-scale purification of fusion proteins using a 96-well format. Our robotic method includes cell lysis, soluble fraction separation and purification with affinity resins. For detection of His-tagged proteins in the soluble fractions and after affinity resin elution, a dot-blot procedure with an anti-His-antibody is used. The expression level and molecular mass of recombinant proteins are checked by SDS-PAGE. With this approach, we are able to obtain beneficial information to be used for large-scale protein expression and purification.     

Exploiting the interactions between poly-histidine fusion tags and immobilized metal ions

Immobilized metal affinity chromatography (IMAC) of proteins containing poly-histidine fusion tags is an efficient research tool for purifying recombinant proteins from crude cellular feedstocks at laboratory scale. Nevertheless, to achieve successful purification of large amounts of the target protein for critical therapeutic applications that demand the precise removal of fusion tags, it is important to also take into consideration issues such as protein quality, efficiency, cost effectiveness, and optimal affinity tag choice and design. Despite the many considerations described in this article, it is expected that enhanced selectivity, the primary consideration in the field of protein separation, will continue to see the use of IMAC in solving new purification challenges. In addition, the platform nature of this technology makes it an ideal choice in purifying proteins with unknown properties. Finally, the unique interaction between immobilized metal ions and poly-histidine fusion tag has enabled new developments in the areas of biosensor, immunoassay, and other analytical technologies.     

Protein Production in Yarrowia lipolytica Via Fusion to the Secreted Lipase Lip2p

We established a strategy for protein production and purification via expression in Yarrowia lipolytica as Lip2p fusion protein. To evaluate the expression system a cysteine-rich miniprotein, an antibody fragment and an enzyme showing galactose oxidase activity were chosen. These proteins have varying disulfide bond content, size, and structural complexity. Endogenous lipase Lip2p was used as a fusion partner to direct the fused proteins to the extracellular medium. A linker sequence was introduced at the junction of Lip2p and the respective fused protein that contains a hexahistidine tag followed by a TEV protease cleavage site. This allows for a specific and simple purification via IMAC for capturing the secreted proteins from the supernatant followed by a second IMAC for removing all contaminants after proteolytic release of the protein of interest. Up to 174 mg/L fusion protein was obtained using shake flask cultivation. Functionality of each of the purified proteins was confirmed by individual assays. Expression of proteins of interest via Lip2p fusion not only provides a convenient expression and purification scheme but also enables for an online monitoring of accumulation of secreted fusion proteins in the medium by exploiting the intrinsic lipase activity of the fusion.