当前位置:首页 >> 自然科学 >>

NATURE最新的蛋白表达纯化的综述


REVIEW

Protein production and purification
? 2008 Nature Publishing Group http://www.nature.com/naturemethods

Structural Genomics Consortium1–3, Architecture et Fonction des Macromolécules Biologiques4, Berkeley Structural Genomics Center5, China Structural Genomics Consortium6,7, Integrated Center for Structure and Function Innovation8, Israel Structural Proteomics Center9, Joint Center for Structural Genomics10,11, Midwest Center for Structural Genomics12, New York Structural GenomiX Research Center for Structural Genomics13–17, Northeast Structural Genomics Consortium18,19, Oxford Protein Production Facility20, Protein Sample Production Facility, Max Delbrück Center for Molecular Medicine21, RIKEN Structural Genomics/ Proteomics Initiative22 & SPINE2-Complexes23,25

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.
Recombinant proteins are used throughout biological and biomedical science. Their production was once the domain of experts, but the development of simple, commercially available systems has made the technology more widespread. As a result, also more widespread is an appreciation of the difficult, strategic choices inherent to the process. Commonly confronted questions include: should the protein(s) be expressed in bacteria, in yeast, in insect cells or in human cells? Which expression vector should be used? If bacterial expression is used, which strain(s) should be chosen? Should one express the fulllength protein or a fragment thereof? Should the protein be tagged, and which affinity tag is the best? What is a good purification strategy, and what are the common pitfalls? Unfortunately, because every protein is different, there can be no ‘right’ answer to any of these questions a priori, and purification protocols and strategies must be worked out for each individual protein and with an eye to its intended use. This said, each project must begin somewhere, and purification strategies can now be

Oxford, Old Road Campus, Roosevelt Drive, Headington, Oxford OX3 7DQ, UK. 3University of Toronto, 100 College St., Toronto, Ontario M5G 1L6, Canada. 4Architecture et Fonction des Macromolécules Biologiques, Centre National de la Recherche Scientifique, Case 932, 163 Avenue de Luminy, 13288 Marseille Cedex 09, France. 5Lawrence Berkeley National Laboratory and Department of Chemistry, University of California, 351A Donner Laboratory, Berkeley, California 94720, USA. 6Tsinghua University, Beijing 100084, China. 7University of Science and Technology of China, Hefei 230027, China. 8Los Alamos National Laboratory, Mailstop M888, Los Alamos, New Mexico 87507, USA. 9Weizmann Institute of Science, 2 Herzel Street, Rehovot 76100, Israel. 10The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, California 92037, USA. 11Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, California, 92121, USA. 12Biosciences Division, Argonne National Laboratory, 9700 South Cass Avenue, Argonne, Illinois 60439, USA. 13SGX Pharmaceuticals, Inc., 10505 Roselle Street, San Diego, California 92121, USA. 14Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA. 15Biology Department, 463, Brookhaven National Laboratory, Upton, New York 11973, USA. 16Case Western Reserve University, 10900 Euclid Ave., Cleveland, Ohio 44016, USA. 17Departments of Biopharmaceutical Sciences and Pharmaceutical Chemistry, University of California, San Francisco, 600 16th Street, San Francisco, California 94143, USA. 18Center for Advanced Biotechnology and Medicine, Rutgers University, 679 Hoes Lane, Piscataway, New Jersey 08854, USA. 19Department of Biological Sciences, Columbia University, 701 Fairchild Building, MC 2451, New York, New York 10027, USA. 20Wellcome Trust Centre for Human Genetics, Roosevelt Drive, Oxford OX37BN, UK. 21Max Delbrück Center for Molecular Medicine (MDC), Robert-R?ssle-Str. 10, 13092 Berlin, Germany, 22Protein Research Group, Genomic Sciences Center, Yokohama Institute, RIKEN, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan. 23Division of Structural Biology, Wellcome Trust Centre for Human Genetics, Roosevelt Drive, Oxford OX37BN, UK. 24Present addresses: Structural Biology, Helmoltz Center for Infection Research, Inhoffenstr. 7, 38124 Braunschweig, Germany (K.B.), and Department of Biology, and Center for Genomics and Systems Biology, New York University, 1009 Sliver Center, 100 Washington Square East, New York, NY 10003, USA (K.C.G.). 25A complete list of authors appears at the end of this paper. Correspondence should be addressed to A.E. (aled.edwards@utoronto.ca).
PUBLISHED ONLINE 30 JANUARY 2008; DOI:10.1038/NMETH.F.202

1Karolinska Institutet, Schéeles v?g 2, 171 77 Stockholm, Sweden. 2University of

NATURE METHODS | VOL.5 NO.2 | FEBRUARY 2008 | 135

REVIEW
Table 1 | Overview of targeted proteins
Organism Viruses Archaea Bacteria Eukarya Targets cloned 335 8,043 58,806 42,239 Targets purified Percentage purified 118 2,917 17,350 8,008 35 36 30 19

These data were obtained from TargetDB (21 December 2007) and include data from all structural genomics centers listed, plus updated information from the SGC. Source, http://targetdb.pdb.org/ statistics/TargetStatistics.html and http://www.thesgc.com/structures/target_progress.php.

? 2008 Nature Publishing Group http://www.nature.com/naturemethods

guided by evidence-based trends, probabilities and cautionary notes that have emerged from large-scale structural genomics studies. In this review, which is targeted to the researcher with limited experience in protein expression and purification, we draw on our collective experiences to suggest a ‘consensus’ starting point for soluble protein expression and purification. Over the past decade, our laboratories have collectively targeted and purified tens of thousands of different proteins from the Eubacteria and Archaea, and thousands from the Eukarya, including fungal, nematode, parasite, plant and human proteins (Table 1). These proteins belong to many different classes, including proteins with no predictable structure, human proteins of therapeutic relevance, proteins from parasites and viruses, integral membrane proteins and multiprotein complexes. A near-complete list of these proteins is available in a database (TargetDB) maintained by the Protein Data Bank (PDB; http://targetdb.pdb.org/) under the auspices of the US National Institute of General Medical Sciences (NIGMS)-funded Protein Structure Initiative (http://www.nigms.nih.gov/Initiatives/ PSI/). The European research network Structural Proteomics in Europe (SPINE) also provides detailed target lists online (http:// www.spineurope.org/).

In efforts to identify an optimal approach(es) for the initial production and purification of a ‘typical’ protein, our groups have explored many different technologies and strategies. Our common objective has been to balance success rates with ease and breadth of use, speed, cost and versatility1–16. By comparing our independently optimized approaches, it is apparent that our preferred methods have, in many instances, evolved to be quite similar, but by no means identical (Table 2). Accordingly, in an effort to provide guidance to scientists interested in generating purified recombinant proteins, representatives from our research groups collaborated to articulate our ‘consensus’ advice (Box 1), along with a brief rationale for each choice. In essence, we tried to answer the question “what would you try first?”, understanding that several choices are often possible or even desirable. We also provide guidance for those cases in which the initial attempt fails or problems are encountered, in other words, “What next?”. In Supplementary Methods online, we provide links to online protocols offered by several structural genomics groups as well as detailed experimental protocols for the methods described here. It is important to emphasize three aspects of this review. First, it is meant to serve as a guide to those members of the research community who are interested in expressing recombinant proteins, but who feel that they may not have the breadth of experience to decide among the various possible approaches. Second, we selected this consensus strategy because it is simple and has the widest use. There are other methods that are perhaps equivalent, but space limitations preclude an in-depth discussion of all possible cloning, expression and purification strategies. Third, the methods described here were developed with the intention to produce purified, soluble protein in close-to-milligram quantities; there are many applications for purified protein (biochemical assays, antibody production) that may not have such requirements.

Table 2 | Summary of approaches used by SG centers
Main target sources Human and human pathogens Cloning method LIC Expression Affinity promoter tags T7 6His Small-scale expression method 96-well plates 96-well plates Scale-up cultivation method 1–2 l in Tunair shake flasks 1–5 l in shake flasks Purification strategy IMAC and gel filtration using ?KTA systems IMAC and gel filtration using ?KTA systems Protein characterization ESI-MS

Center Structural Genomics Consortium Architecture et Fonction des Macromolécules Biologiques Berkeley Structural Genomics Center

References 16,19

Gateway Mammalian viruses, higher eukaryotes, bacteria, phages Bacteria LIC

T7

6His

MALDI-TOF

32,51

T7

6His

96-well plates

1 l cultures in IMAC and gel Fernbach flasks filtration using ?KTA systems IMAC, and ion exchange chromatography and gel filtration

DLS, MALDI, ANSEC 85

China Structural Human Genomics Consortium Integrated Center for Structure and Function Innovation

LIC, restriction enzyme– based

T7

6His

3 ml culture 2 l in in test tubes Erlenmeyer flasks 96-well plates

SDS-PAGE, DLS and 8,9 mass spectrometry

Mycobacterium Restriction T7 and tuberculosis, enzymes and arabinose Bacillus LIC subtilis, Thermotoga maritima

6His

0.5–1 l in soda IMAC and gel bottles or filtration baffled shake flasks

SDS-PAGE, densitometry, DLS and MALDI

136 | VOL.5 NO.2 | FEBRUARY 2008 | NATURE METHODS

REVIEW
Table 2 | (continued)
Main target sources Cloning method Expression Affinity promoter tags N-6His Small-scale expression method Scale-up cultivation method Purification strategy Protein characterization

Center

References

Israel Structural Higher Proteomics eukaryotes, Center human pathogens

Restriction T7 enzyme– based or LIC

? 2008 Nature Publishing Group http://www.nature.com/naturemethods

4 ml culture 0.5 l culture in in test tubes 2-l shake flasks or 1.25 l culture in 5-l flasks (total 5–6 l per large-scale production) 96-well Parallel 12–96 plates, ANSEC cultures in and mass GNFermentor spectromerty

IMAC, gel filtration, MALDI and ESI-MS 10 ion exchange chromatography and TEV cleavage

Joint Center for Structural Genomics

Bacteria

Polymerase incomplete primer extension

Arabinose

6His

IMAC, TEV cleavage, ANSEC, LC-MS, IMAC subtraction, SDS-PAGE ion exchange chromatography and gel filtration if necessary IMAC, TEV cleavage, SDS-PAGE gel filtration ?KTAxpress and Ni-NTA column purification and gel filtration

18,86–88

Midwest Center Bacteria for Structural Genomics New York Structural GenomiX Research Center for Structural Genomics Northeast Structural Genomics Consortium Human and >130 other species (ATCC and gene synthesis) Prokaryotes and eukaryotes, including human

LIC

T7

6His

96-well plates 96-well plates

Plastic bottles

89

Topo (blunt)

C-6His and N-6HisSmt3

1–3 l shake flasks SeMet high yield

MALDI and 1 ESI-MS; protein identification by mass spectrometry and/or DNA sequencing Caliper 13 microfluidics, MALDI-TOF mass spectrometry, light scattering, NMR SDS-PAGE, ESI-MS, 5,14,51,90 MALDI-TOF MS; LC-ESI-MS followed by ZIC-HILIC for glycosylated proteins

LIC

T7

6His

96-well plates

1–2 l in baffled IMAC, gel filtration shake flasks using ?KTAxpress, ion exchange chromatography if required 1–2 l cultures IMAC and gel filtration using ?KTA systems

Oxford Protein Bacteria, Production human, viral Group (SPINE) pathogens

LIC

T7; N- or C-6His β-actin or hCMV for mammalian cells T5 and T7

96-well plates; 25-cm2 dishes for mammalian cells

Protein Sample Human Production and higher Facility, Max eukaryotes Delbrück Center for Molecular Medicine RIKEN Structural Genomics/ Proteomics Initiative Human, mouse, bacteria, and archaea

Restriction enzyme– based, Gateway

Mainly 1–10 ml N-7His, culture occasionally N-GST or N-MBP

1–8 l in shake flasks

IMAC and TEV Mass spectrometry, 2,3,7 cleavage, IMAC DLS and gel filtration, and ion exchange chromatography using ?KTA systems if necessary IMAC and TEV cleavage, IMAC subtraction, ion exchange chromatography, gel filtration using ?KTA systems if necessary IMAC and gel filtration using ?KTA systems DLS, NMR, MALDI-TOF and quadrupole-TOF tandem mass spectrometry 72–76

Two-step PCR and TA cloning

T7

Histidine 30 ?l in cell- 9–27 ml dialysis affinity tag free synthesis cell-free (HAT) synthesis in 96-well plates

SPINE2Complexes

Human, viral proteins involved in subversion of human signaling pathways

LIC

T7; N- or Cβ-actin or 6His tag hCMV for mammalian cells

96-well plates; 25-cm2 dishes for mammalian cells

1–2 l cultures

SDS-PAGE, ESI-MS, 51,71 MALDI-TOF mass spectrometry; LC–ESI-MS followed by ZIC-HILIC for glycosylated proteins

ESI-MS, electrospray ionization–mass spectrometry; MALDI-TOF, matrix-assisted laser desorption/ionization–time of flight; DLS, dynamic light scattering; ANSEC, analytical size-exclusion chromatography; ZIC-HILIC, zwitterionic chromatography–hydrophilic interaction liquid chromatography; LC-MS, liquid chromatography–mass spectrometry; SeMet, selenomethionine.

NATURE METHODS | VOL.5 NO.2 | FEBRUARY 2008 | 137

REVIEW
question. In expressing a protein domain, the choice of the N- and C-terminal boundaries represents an important consideration because even small differences can dramatically influence both solubility and expression. For example, Klock and colleagues18 evaluated a nested set of 2,143 N- and C-terminal truncations from 96 targets and found considerable variation in both solubility and aggregation behavior by altering the protein length by just a few amino acids. Despite the best efforts, and even for proteins whose domain Obtaining the cDNA and creating the expression clone structure is well-defined, it is not currently possible to predict cDNA. Recently, sequencing efforts and various cDNA consortia which specific N- and C-terminal boundaries are most compatible have made available large libraries of full-length, sequence-verified cDNAs. Although there are inevitably issues with clone contamina- with the expression of a soluble protein. Thus, pragmatism dictates tion and mix-up, the resources are in general trustworthy. Among testing many truncated forms of the protein to select one or more the most comprehensive and best annotated is the Mammalian for scale-up production. For proteins of known or readily predicted three-dimensional structure, the borders should be engineered Gene Collection, which maintains a repository of >19,000 human cDNAs, covering ~65% of all annotated genes. For genes or splice to encompass the domain of interest. As an example, ten constructs variants not easily obtained through more traditional routes, total of the targeted domain might be made at the outset of every project, one corresponding to the full-length protein and nine repgene synthesis can be used. Over the past few years, the cost of gene synthesis has dropped almost fivefold, and it will undoubt- resenting the clones derived from amplifying a combination of edly continue to decrease. One advantage of gene synthesis is the three different 5′-end primers and three different 3′-end primers. ability to change the codon bias of the gene to be more compatible Gr?slund and colleagues have compared the success rate of the with the recombinant host. However, for Escherichia coli, expres- nested-primer approach with the predicted success rate if one had sion strains supplemented with additional tRNAs can often over- chosen only a single ‘optimal’ construct. In a sample set of 400 come the codon bias of the recombinant gene17. For example, in a human protein domains, the use of multiple constructs increased study of 30 human genes by the Structural Genomics Consortium the probability of generating a soluble protein twofold19. To select the sets of PCR primers for proteins with a predictable (SGC), there was no clear advantage in the use of codon-optimized genes compared with the natural sequence expressed in tRNA- three-dimensional structure, one should consider prior knowledge supplemented strains (N.A. Burgess-Brown, S. Sharma, F. Sobott, of the structure of a related protein, sequence conservation patterns, C. Loenarz, U. Oppermann and O. Gileadi; submitted). and predictions of secondary structure or unfolded/disordered Selecting the N and C termini. The objective of recombinant regions20,21. Widely accepted guidelines are to: (i) remove predictprotein expression is usually to produce a sample that supports a ed membrane-spanning regions; (ii) avoid disrupting predicted certain biochemical or biological activity, such as enzyme catalysis secondary structural elements; (iii) respect the boundaries of globor protein-ligand interactions. Frequently, the desired activity is ular domains, if known; and (iv) avoid inclusion of low-complexsupported by a discrete domain, and thus it is often not necessary ity regions or hydrophobic residues at the termini22. The optimal to express the full-length protein to address a particular biological step size between the nested primers is not yet fully understood; we commonly make constructs to encode proteins that vary in length by 2–10 amino BOX 1 SUMMARY OF CONSENSUS PROTOCOL acids at each end19. For proteins without a predictable three-dimensional structure, ? Obtain the cDNA by amplifying either genomic DNA (prokaryotic genes, or eukaryotic the approximate boundaries of the region genes with no introns) or full-length, sequence-verified cDNAs (eukaryotes) or by total of interest might be identified using funcgene synthesis. tional assays and scanning deletion muta? Use ligation-independent cloning (LIC) to clone the full-length cDNA (or the fragment genesis, and then optimal boundaries for of interest) into an E. coli expression vector. expression can be identified using nested ? Use T7 RNA polymerase–driven expression and an N-terminal oligohistidine tag sets of PCR primers, as above23. Boundaries (include a cleavage site for a sequence-specific protease to enable removal of the tag). of structured domains can also be deter? Express the protein in a derivative of the E. coli BL21(DE3) strain, with induction at low mined experimentally by using limited temperature (15–25 °C) in rich medium and with good aeration. If expressing proteins proteolysis combined with mass spectromfrom organisms that have codon biases differing from those used by E. coli, use a strain etry analysis24. Clearly, when using protein supplemented with the appropriate tRNA genes. fragments, caution should be used in inter? Solubilize and purify the protein in a well-buffered solution containing an ionic preting unexpected biological results. strength equivalent to 300–500 mM of a monovalent salt, such as NaCl.
? Use immobilized metal affinity chromatography (IMAC) as the initial purification step. ? If additional purification is required, use size-exclusion chromatography (gel filtration). If necessary, use ion exchange chromatography as a final ‘polishing’ step. ? The affinity tag may be removed to minimize non-native sequences in the recombinant protein and to achieve further purification. Use a recombinant, hexahistidine-tagged protease and reapply the sample to IMAC column to remove the protease and any cellular proteins that bound to the metal affinity resin.

There are two important provisos to the methods and strategies described in this review. First, our experience is dominated by studies with nonmembrane cytosolic and/or fragments of proteins that comprise soluble domains. Second, although the protocols for the ‘first attempt’ described here have proven to be optimal for the broadest range of proteins, in any individual case, the methods will fail more often than they succeed.

? 2008 Nature Publishing Group http://www.nature.com/naturemethods

Cloning The most common methods now used in our groups to clone target genes into the requisite expression vector rely on homologybased approaches, using either recombination enzymes25 or ligation-independent cloning (LIC)26. Restriction enzyme–based

138 | VOL.5 NO.2 | FEBRUARY 2008 | NATURE METHODS

REVIEW
approaches are used less frequently. A comparison of the methods is shown in Supplementary Table 1 online. Recombination-based methods include, for example, the bacteriophage lambda integrase system27 and the Cre-lox recombination system28. These methods are rapid, easy and produce few false positives. However, the requirement for special cloning sites imposes constraints: either additional amino acid codons are inserted at either end of the gene, making the PCR primers quite long, or the work-around cloning strategies are more complicated. The unique feature of these methods is the ability to transfer the cloned sequence among a series of compatible vectors that can be used to express the gene in different hosts or with different tags. For bacterial expression, however, the probability of identifying a clone that expresses a soluble protein is increased by making different variants of a single protein in the same E. coli host rather than by cloning a single variant into vectors with different tags and expression hosts19,29. Ligation-independent cloning, which is used by most of our groups, has the disadvantage compared with recombination-based approaches in that one needs to clone sequences independently into each vector (if this is required). However, the method is inexpensive and simple. One scientist can routinely generate two 96-well plates of distinct clones in a week without the benefit of automation. Expressing the protein E. coli as the recombinant host for initial studies. The stably folded, globular domains of prokaryotic and eukaryotic proteins (for example, catalytic domains or protein interaction domains) are a major focus both of the biomedical research community and of our laboratories. These proteins are generally suitable for expression in E. coli. Over the years, much effort has been put into optimizing E. coli as an expression host for proteins from higher organisms30. This strategy has generated a wide arsenal of tools that can be used to increase the yield of soluble protein. A surprising variety of other classes of proteins, from full-length bacterial and human proteins, to protein complexes, and even some human integral membrane proteins can also be produced in E. coli. In terms of full-length proteins, analysis of large-scale protein expression trials shows that up to 50% of proteins from the Eubacteria or Archaea and 10% of proteins from the Eukarya can be expressed in E. coli in soluble form31 (http://targetdb.pdb.org/). Overall, the probability of successfully expressing a soluble protein decreases considerably at molecular weights above ~60 kDa (Fig. 1). Proteins that do not express in soluble form may not be modified or folded properly, or may precipitate within E. coli through formation of inclusion bodies. Remarkably, expression in a heterologous host does not solely account for the poor success rates; even after extensive screens of expression conditions, 30% of proteins from E. coli itself cannot be produced in soluble form when overexpressed in E. coli32. On the basis of these studies, our view is that the first attempt for the recombinant production of any protein—whatever the source—is to try E. coli as the expression host. It is fast and inexpensive to test a wide variety of possible strategies in E. coli, and one can complete a fairly comprehensive analysis within a relatively short period of time. Alternative systems should be used only after the E. coli system has been reasonably explored. This view balances the fact that there is definitely a lower probability of expressing some classes of proteins in E. coli (full-length eukaryotic proteins, integral membrane proteins) compared with
0.7 0.6 Soluble clone to purified protein

0.5 Fraction successful 0.4 Clone to purified protein

? 2008 Nature Publishing Group http://www.nature.com/naturemethods

0.3 Purified protein to structure 0.2

0.1 0 0

100 200 300 400 500 600 700 800 900 1,000 Construct length

Figure 1 | Solubility as a function of construct length. Fraction of successful purifications and structure determinations as a function of protein length (data from New York Structural GenomiX Research Center). Dotted line, fraction of cloned targets resulting in successful large-scale purifications. Dashed line, fraction of soluble clones (those that express soluble protein at a 1-ml scale) that yield pure protein at large scale. Solid line, fraction of purified proteins resulting in successful crystal structure determinations. There are relatively few targets with lengths greater than 800 amino acids, so these fractions have been extrapolated and are shown in gray.

other systems (human or insect cells), with the fact that the E. coli system is useful in many cases, and also is far more cost-effective and convenient. Strain of E. coli. For high-level protein production purposes, BL21(DE3) is an appropriate E. coli strain. It has the advantage of being deficient in both lon and ompT proteases and it is compatible with the T7 lacO promoter system33. For eukaryotic proteins, it is often important to use BL21(DE3) derivatives carrying additional tRNAs to overcome the effects of codon bias. Historically, ampicillin has been the most commonly used antibiotic-selection marker, but it is being replaced by carbenicillin, which is more stable. Vectors encoding resistance to kanamycin or chloramphenicol are now widely used as well. Fusion to oligohistidine tags. We suggest that the protein should be produced as a fusion to an affinity tag because tags dramatically aid in protein purification and rarely adversely affect biological or biochemical activity34. However, in selecting which tag to use, one is faced with a daunting number of choices. Our groups have explored most of the available options, and we observed that no affinity tag emerged as significantly more efficacious in successfully producing soluble, active recombinant proteins35. Despite the lack of a clear winner based on success rate, most of our research groups selected an N-terminal hexahistidine tag that can be removed by a sitespecific protease, such as the tobacco etch virus (TEV) protease36. However, many other instances can be found in which proteins can be expressed in soluble form only as fusions to other affinity tags29.
NATURE METHODS | VOL.5 NO.2 | FEBRUARY 2008 | 139

REVIEW
The rationale for the choice of an N-terminal hexahistidine is manifold. First, an N-terminal tag ensures that the bacterial transcription and translation machineries always encounter 5′ and N-terminal sequences that are compatible with robust RNA synthesis and protein expression, respectively. Second, oligohistidine-tagged proteins can be purified using a relatively simple protocol using immobilized metal affinity chromatography (IMAC)37. Third, histidine tags rarely affect the characteristics of the protein, which distinguishes it, for example, from glutathione S-transferase (GST), which itself is a dimer that then imposes dimerization on the recombinant protein. Fourth, the hexahistidine tag is relatively small and usually does not dramatically alter the solubility properties of the target protein. By contrast, larger tags, such as the maltosebinding protein (MBP), can often increase the apparent solubility of the recombinant moiety, even when the protein is either insoluble by nature, or unstable or unfolded and, therefore, less likely to be active38–40. Fifth, for the specific application of protein crystallography, short histidine tags appear to be neutral actors; in most of our projects, we routinely attempt crystallization and NMR structure determination with both cleaved and uncleaved proteins, and their relative representation among the resulting three-dimensional structures is roughly equivalent. A recent PDB-wide survey41 also indicates that hexahistidine tags do not have a consistent impact on the N-terminal structure of the target protein. T7 RNA polymerase–based expression vectors. The most commonly used expression systems are based on pET vectors (Merck/ EMD; the pET System manual, 2006), which drive expression of a recombinant gene under the control of the T7 RNA polymerase promoter and lac operator33,42. The vectors are designed for use in λDE3 lysogen strains of E. coli, which harbor a genomic copy of the gene for T7 RNA polymerase under the control of the lac repressor. Under repressive conditions, T7 RNA polymerase is not produced, and transcription of the target gene is negligible. After induction, when the T7 RNA polymerase is produced, most of the cellular protein synthesis machinery will be devoted to producing the target protein. On occasion, low-level expression of T7 polymerase within these strains leads to expression of the recombinant protein and may slow or prevent growth of the transformed bacteria. The expression of such highly toxic proteins can be effected by using T7 lysozyme-expressing strains42, strains in which the T7 RNA polymerase is under the control of the arabinose promoter43, by producing the protein in a cell-free system44 or by driving expression of the recombinant protein directly by the more tightly regulated arabinose promoter system45. Expression conditions. Using T7 systems, protein expression can be induced either with the chemical inducer isopropyl-β-D-thiogalactoside (IPTG) or by manipulating the carbon sources during E. coli growth (auto-induction; ref. 46 and the pET System manual; Merck/EMD, 2006). In both cases, the cells can, and should, be grown to high densities (OD600 of 4–20) 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). One feature of the T7 system is that many recombinant proteins often precipitate when expressed at 37 °C, but are sol140 | VOL.5 NO.2 | FEBRUARY 2008 | NATURE METHODS

uble when the temperature during induction is 15–25 °C, presumably because slower rates of protein production allow newly transcribed recombinant proteins time to fold properly50. Thus, lower temperatures during induction should be used as the default. Small-scale test expression Small-scale test expression is widely used as a predictive tool to determine which of the derivative clones actually produces soluble protein and to establish the optimal scale for the large-scale growth. A major concern is that the expression level and solubility of a recombinant protein is influenced by the culture conditions and the degree of aeration, and these parameters do not always scale with culture volume. The results from small and large-scale growth also vary owing to differences in sample preparation and protein purification methods that are used for each scale of growth. Therefore, whereas positive smallscale experiments are often predictive of the results from large-scale growth, there will inevitably be a substantial proportion of false negatives in which an apparently nonexpressed or insoluble protein can be in fact, expressed in soluble form when grown on a larger scale. If the total number of constructs to be tested is small (for example, <20 constructs), it may be wiser to proceed immediately to larger-scale cultures to avoid any potential complications. For analysis of large numbers of constructs, parallel small-scale protein purification can be performed efficiently in volumes of 1–20 ml, in 96-well format. This scale typically produces 10–200 ?g of protein, which is sufficient for many analytical tests. The results can be used to optimize the construct design and experimental conditions before embarking on larger scale purifications49,51,52. Protein purification As a chromatographic procedure, IMAC has the advantages of having strong, specific binding, mild elution conditions and the ability to control selectivity by including low concentrations of imidazole in chromatography buffers. There is a broad array of common resins with slightly different binding capacities and binding strengths, but all tolerate harsh cleaning procedures (TALON Metal Affinity Resins User Manual, Clontech, 2007; the QIAexpressionist, Qiagen, 2003; and HisTrap HP, 1 ml and 5 ml (instructions), Amersham Biosciences, GE Healthcare, 2003). Most purification steps can be integrated by high-performance liquid chromatography; the most commonly used devices are the ?KTA systems from GE Healthcare. The final purity of the protein can be optimized by controlling the ratio of recombinant protein to the column size; lower-affinity contaminants can be competed with a relative excess of the histidinetagged recombinant protein. Accordingly, it is beneficial to determine the amount of the soluble target protein to be loaded on the column, and this can be estimated from small-scale expression trials. As a general rule, to maximize purity, one should load the column with a slight excess over the predicted binding capacity. Although not necessary, it is relatively straightforward to implement these protein purification protocols on automated chromatography systems, which have proven reliable, effective and simple to use. Preparation of the bacterial lysate. Preparation of the bacterial lysate is a critical step. Optimal conditions maximize cell lysis and the fraction of the recombinant protein that is extracted while minimizing protein oxidation, unwanted proteolysis and sample contamination with genomic DNA. Mechanical lysis by high-pressure homogenization or sonication, or lysis by freeze-thaw procedures

? 2008 Nature Publishing Group http://www.nature.com/naturemethods

REVIEW
with lysozyme are equivalent in most cases. 600 250 The lysis buffer should contain a strong 500 buffer (50–100 mM phosphate or HEPES) 200 400 to overcome the contribution of the bacte150 rial lysate, high ionic strength (equivalent 300 to 300–500 mM NaCl) to enhance protein 200 100 solubility and stability, protease inhibi100 50 tors and a reducing agent such as Tris(20 carboxyethyl) phosphine hydrochloride 0 45.0 50.0 55.0 60.0 65.0 70.0 75.0 40.0 50.0 60.0 70.0 80.0 90.0 (TCEP) to prevent oxidation of the protein. Retention volume (ml) Retention volume (ml) Loading large amounts of bacterial lysate (>1 l culture volume) on small (<1 ml) Figure 2 | Gel filtration profiles. Representative good (left) and bad (right) gel-filtration profiles of affinity columns may require prior removal two different proteins purified on an ?KTAxpress system using a HiLoad Superdex 200 column (GE of any particulate or viscous material. This Healthcare). can be accomplished by using enzymes that degrade nucleic acid and cell-wall material, such as DNase or Benzonase (Merck/EMD) and lysozyme, respec- insufficiently pure for the intended purpose, one can remove the tively. Some of the enzymes used in lysis are less active in the pres- tag with a histidine-tagged TEV protease and perform IMAC again ence of reducing agents or high salt concentration; optimal lysis may as an additional ‘generic’ purification step, collecting the recombirequire sequential addition of the components. Clarified lysates can nant protein in the flowthrough. This step very efficiently removes histidine-rich proteins derived from the expression host, which also be filtered before loading on the affinity columns. IMAC purification is performed in phosphate buffer, pH 8.0 and may have copurified in the primary IMAC procedure, as well as the cleaved tag and the histidine-tagged protease. an ionic strength equivalent to 300–500 mM NaCl. HEPES buffer (and, to a lesser extent, Tris buffer) at pH 7.5–8.0 can also be used. It has been consistently observed that conditions of high ionic strength Protein characterization (for example, 500 mM NaCl) maintain solubility and stability of the Characterizing the purified protein in some detail reduces the widest variety of proteins. Indeed, a substantial fraction of proteins risk of wasting resources on protein material of inadequate qualprecipitate if the salt concentration is reduced to physiological levels, ity. It also provides a means to ensure that different batches of particularly as the protein becomes more pure and concentrated. the same protein have similar properties. Below, we outline a The choice of NaCl as the salt is mainly historical and, although not simple, generic protein characterization protocol that allows the experimentalist to judge whether the correct protein has been systematically explored, there is no reason to believe that sodium and chloride are optimal. Indeed, sodium and chloride levels in purified, whether additional molecular species are present and the cell are very low and are probably never the physiologically rel- to estimate the approximate protein concentration. Other charevant counter-ions for intracellular proteins. A modest amount of acterization methods that are very informative but not as widely imidazole (see resin manufacturer’s recommendations) should applied, such as mass spectrometry, static or dynamic light scattering, and measuring protein thermal stability, are described in be included in the cell extraction buffer to reduce binding of Supplementary Methods. less histidine-rich proteins to the IMAC column. For intracellular proteins, care should be taken to maintain a reducing environment. TCEP, unlike dithiothreitol (DDT), is compatible with all known Inspection of gel filtration chromatogram. If size exclusion chroIMAC matrices. Finally, inclusion of glycerol (10%) during protein matography was used as the last purification step, a close look at the chromatogram is essential. Symmetric elution profiles are charpurification enhances the solubility and stability of many proteins. acteristic of homogeneous proteins, whereas asymmetric profiles reflect inhomogeneous, or partially aggregated, samples (Fig. 2), or Chromatography. After the lysate is loaded on the IMAC column, it should be washed with buffer including an intermediate concentra- whether the column itself is in poor condition. The elution profiles tion of imidazole (see manufacturer’s instructions), which will elute will also reveal the primary oligomerization state. The presence of weakly bound contaminants without sacrificing large amounts of additional oligomerization states may be of biological significance, the recombinant protein. It is sometimes necessary to optimize the or may be a sign of nonspecific aggregation. If the protein elutes in wash step with respect to the concentration of imidazole as well as the void volume of the chromatogram, the protein is most likely the volume of the wash. Finally, the recombinant protein should forming large, nonspecific aggregates, which may be an indication be with a step gradient (for example, 300 mM imidazole). If EDTA of improper folding and compromised activity. It is also of value and DTT are added after IMAC; add the EDTA first to sequester any to analyze individual peaks by SDS-PAGE or mass spectrometry to nickel that has leached off and that could react with the DTT. analyze the protein in each peak. The choice of gel filtration as the next step may be surprising, considering its lower resolving power compared with ion exchange SDS-PAGE analysis. After protein purification, samples should be or other adsorption chromatography methods, but this step is often resolved by denaturing SDS-PAGE. If stained with a dye such as sufficient after IMAC if the protein was abundant in the lysate. Coomassie brilliant blue, the intensity of the bands will usually be Moreover, gel filtration is more generic, can be performed in any proportional to the amount of protein53. This allows the purity of buffer condition, and can be used to resolve the oligomerization the sample to be estimated and whether the purified protein is of state of the target protein. In some cases, if the protein is judged the expected size.
Absorbance units (mAU)

? 2008 Nature Publishing Group http://www.nature.com/naturemethods

NATURE METHODS | VOL.5 NO.2 | FEBRUARY 2008 | 141

REVIEW
Table 3 | Common E. coli proteins that copurify by IMAC
Protein GroES Fur SlyD CA RplB DnaJ GroEL DnaK Accession number NP_418566 NP_415209 NP_755987 NP_414668 P60422 NP_414556 AAS75782 NP_414555 Monomer mass (kDa) 10.39 16.79 20.85 25.10 29.86 41.10 57.35 69.11

fractions using SDS gel electrophoresis. Some cellular proteins characteristically resolve into the soluble and insoluble fractions and these serve as excellent internal controls (Supplementary Fig. 1 and 2 online). The recombinant protein fails to bind the IMAC column. The pH of the lysate should be 7.5–8.0 for efficient binding, and the buffer should not contain chelators (EDTA or citrate), high imidazole concentrations (for example, >30 mM for Ni-NTA resins) or DTT. In some instances, it is necessary to reduce the amount of imidazole in the loading buffer to <5 mM. The column must be properly charged with metal ions and, when charging columns, make sure the concentrated NiSO4 solution is buffered and set to pH 7.5. It is also important to remember that imidazole is a base; the final solutions must be adjusted to the correct pH. In some cases the target protein may bind weakly to the IMAC column, so the concentration of imidazole in the wash step should be reduced (for example, 20 mM). The wrong or a mutant protein was expressed or purified. An incorrect protein may occasionally be expressed and purified, which most commonly results from a simple clone mix-up. In that instance the problem will be detected either by gel electrophoresis or mass spectrometry of the purified protein. If the recombinant protein is expressed at low levels, it is also relatively common to purify an endogenous E. coli protein that binds to, and elutes from, the IMAC column and that also adventitiously migrates with the predicted mobility of the target protein54. In some cases, this E. coli protein may even appear to be induced after the expression of T7 RNA polymerase. Determining whether you have purified your recombinant protein or an endogenous bacterial protein can readily be accomplished with mass spectrometry, but is more difficult by denaturing gel electrophoresis. A western blot to the affinity tag can sometimes be useful to track the recombinant protein. If the expression construct is sequenced before the experiment, errors introduced in primer synthesis or PCR will be detected. In practice, PCR-generated sequence errors are so rare that it is often more practical to do the expression trials first, and to sequence the successful expression constructs later. Of course, if none of the constructs express a protein, it is essential to sequence the expression clones and, ultimately, to sequence the clones selected for scale-up and purification. Bacterial proteins copurify with the recombinant protein. Copurification of E. coli proteins with the histidine-tagged recombinant protein is very common, especially when the expression level of the recombinant protein is low. Contaminants include proteins that contain multiple histidine residues (for example, SlyD; Table 3), and molecular chaperones that may bind to the resin directly or to the recombinant protein54,55. The affinity resin has limited capacity, so loading near-saturating amounts of the recombinant protein on a column improves purity. Tag cleavage followed by affinity purification is also effective in removing contaminants, as these proteins are unaffected by the protease and bind to the column after reapplication of the cleavage reaction. Samples copurifying with chaperones should be regarded with suspicion because this indicates that the protein may have some unfolded character. In cases where the target protein cannot be separated from the chaperones by additional chromatography, use an alternative expression system, process a different construct of the protein or try working with a closely related ortholog.

? 2008 Nature Publishing Group http://www.nature.com/naturemethods

Based on information in references 54 and 55, and on unpublished observations.

UV absorption spectroscopy. To quantify the amount and concentration of purified protein, the simplest and most common method is the Bradford assay53, which measures the binding of Coomassie brilliant blue to the protein. As some proteins bind the dye anomalously, it is also useful to measure the UV absorption at A280 and calculate the concentration of the protein by using the predicted molar extinction coefficient at A280 (http://www.expasy.org/tools/ protparam.html). By taking a UV absorption spectrum, it is also possible to uncover contamination with DNA or RNA, or reveal common copurifying cofactors (for example, NAD, FAD, heme). Storing purified protein. Aliquots of the protein to be stored should be placed in thin-walled PCR plastic tubes, frozen in liquid nitrogen and stored at –80 °C. Small aliquots should be frozen to avoid damaging freeze-thaw cycles, and aliquots should be thawed on ice. Concentrated proteins (for example, >1 mg/ml) tend to be more stable to freeze-thaw cycles. Proteins are usually concentrated using centrifuge-driven filter devices with adequate molecular weight size cutoffs. Care should be taken during centrifugation to avoid local over-concentration and irreversible precipitation or aggregation of the protein on the filtration membrane. It is advisable to explore the stability of the protein to concentration and freeze-thaw cycles before processing the entire batch. The frozen and thawed sample should be compared with protein that was not frozen for biochemical activity, visible precipitation, changes in physical properties (for example, dynamic light scattering or gel filtration profile) or crystallization characteristics. In our collective experience, relatively few proteins are irreversibly inactivated by one freeze-thaw cycle. In those rare instances, the protein can be stored at 4 °C for short periods of time, at –20 °C in high concentrations of glycerol, or as an ammonium sulfate suspension. Common ‘traps’ and ‘pitfalls’ Poor lysis. In small-scale test expression and solubility trials designed to assess the extent to which a protein partitions to the soluble or insoluble fractions, it is important to ensure that the cells are lysed and fractionated properly. Although this is not technically challenging, we have found that it is very common to fail to achieve complete bacterial lysis, which leads to an underestimation of the proportion of recombinant protein in the soluble fraction. Care should also be taken when removing the soluble fraction after centrifugation; it is relatively easy to contaminate the soluble fraction with insoluble material, which can lead to an overestimate of the amount of recombinant protein in the soluble fraction. As a quality control, it is advisable to inspect the protein profiles of the
142 | VOL.5 NO.2 | FEBRUARY 2008 | NATURE METHODS

REVIEW
Samples contain additional proteins or multiple protein species or states. If the protein target is contaminated with other proteins, one can perform additional purification steps such as ion-exchange chromatography. Purifying samples contaminated with different post-translationally modified species or proteolytic fragments of the same protein is more challenging, but not necessarily intractable. For example, different phosphorylated states of a protein can sometimes be resolved using ion-exchange chromatography56.
? 2008 Nature Publishing Group http://www.nature.com/naturemethods

‘Pure’ samples precipitate or fail to concentrate. Pure proteins often precipitate out of solution, even at relatively low (<1 mg/ml) concentrations. This behavior is sometimes coupled with sample inhomogeneity, either in the form of contaminating protein or alternate folded states. Precipitation can also occur by aggregation owing to the presence of hydrophobic or hydrophilic patches on the surface of the target protein. In either case, the problem worsens as the protein concentration increases. There are no generic solutions but some potential solutions, which must be explored for each protein, are to: find a more stabilizing buffer through screening using analytical gel filtration or thermal denaturation (see Supplementary Methods), maintain the protein at lower concentration (<0.1–0.5 mg/ml), maintain an adequate reduced state to prevent protein oxidation (>5 mM DTT, refreshed as required), maintain the salt concentration at high levels (ionic strength >500 mM of a monovalent salt), add glycerol to 10%, add arginine in the range of 50–500 mM, add a mild nondenaturing detergent (0.1% β-octylglucoside) or keep the protein at its optimal temperature (determined empirically). Rescue strategies In even the best of circumstances, it is unusual to generate a soluble version of any given protein on the first attempt. As such, it is important to have a series of alternative approaches. Here we provide various suggestions in the order in which we would usually apply them. Changing expression conditions. Adjustment of the expression conditions seldom results in radical changes but, as some optimization can be done quite easily, it is worth the effort. The first step is to lower the temperature to slow down protein production. Different types of media can also be tested; rich media, such as Terrific Broth, 2×YT or ZYP5052 (auto-induction), often support good expression. Changing the E. coli strain can also improve expression of a soluble protein51. Expression of more variants of the protein sequence. As described above, it is important to test the expression of a range of constructs to identify those that express a soluble derivative. We suggest expressing as many as 10 constructs in the initial attempts. If this proves unsuccessful, then it may be advisable to explore additional constructs, particularly if one has knowledge that a structurally related protein can be expressed in soluble form. Alternate tags. Our consensus strategy is to append an N-terminal histidine tag to each construct. If the histidine-tagged recombinant protein does not express or is insoluble, then the probability that it will be expressed in an active form with another N-terminal fusion partner is reduced considerably. Our advice, therefore, is not to iteratively append different N-terminal fusions but to first explore a C-terminal fusion to the histidine tag instead. Some proteins that are completely insoluble with an N-terminal histidine tag can be expressed in soluble form with a C-terminal histidine tag57.

Although we do not advise extensive sampling of other N-terminal fusions, this strategy can sometimes lead to production of soluble, stable fusion protein. If the aim is to study the function of the target protein, and the fusion protein is an acceptable reagent, then it may be an appropriate strategy. However, this approach has its caveats. In the absence of a robust and quantitative functional assay, one reasonably uses solubility as a proxy for function. However, proteins that are soluble only with a larger tag can be ‘dragged’ into solution by the tag, and revert to an insoluble form if the fusion partner is removed38–40.This indicates that the integrity of the recombinant protein as a fusion protein may be suspect. For example, wildtype GFP is mostly insoluble when expressed in E. coli at 37 °C but is largely expressed in the soluble fraction as an MBP fusion58. Nonetheless, bacterial colonies expressing the MBP-GFP fusions display only weak fluorescence, suggesting that the GFP is nonfunctional (G.S. Waldo; unpublished data). Accordingly, before any functional studies, considerable attention should be paid to whether a target protein appears to be soluble only because it is a passenger on a larger tag. Coexpression of interacting proteins. Many proteins are obligate components of multiprotein assemblies and these often require an interacting protein for correct folding and stability21,59,60. Such proteins, and those with unstructured polypeptide chain segments, often cannot be expressed in E. coli in soluble form, but it has proven possible to improve the properties of these proteins by coexpressing the cognate interacting protein61–63. This strategy is only starting to be used in the large-scale projects, in those cases when entire families of interacting proteins are being studied. Ligand supplementation. Many proteins can be stabilized by the binding of a small molecule—a principle that has found widespread application in generic screening for protein ligands64,65. This property can be exploited to increase the proportion of recombinant protein expressed in soluble form or to stabilize a protein during purification. If a sufficiently soluble, cell-permeable and avid ligand is available, one can use it to stabilize newly synthesized proteins and promote solubility66,67. This concept has also not yet been explored sufficiently in a systematic way. Other expression hosts. If bacterial expression is unsuccessful to this point, other hosts should be considered. Common eukaryotic alternatives are the baculovirus expression system in insect cells68, the yeasts Pichia pastoris69 and Saccharomyces cerevisiae70, human cells71, or cell-free systems using prokaryotic or eukaryotic extracts72–76. These cell-free systems, which have been used extensively to generate thousands of purified proteins for structural studies77–79, can be used to produce proteins that are toxic to E. coli79 and can use PCR-amplified linear DNA fragments, without cloning into a vector, for screening and optimization. All these other expression systems are reasonably simple to use, but they are somewhat more time-consuming to work with than are bacteria and require equipment less commonly found in a typical laboratory. Coexpression of chaperones. Proper in vivo folding of a recombinant protein can be promoted by coexpression of molecular chaperones, which are typically produced from cotransformed plasmids carrying several chaperones with synergistic effects, such
NATURE METHODS | VOL.5 NO.2 | FEBRUARY 2008 | 143

REVIEW
as the pG-Tf2 vector80—a combination of GroEL-GroES81 and trigger factor82. In our hands, chaperones have been used successfully only in isolated cases, and we know of no study of considerable size that has demonstrated broad efficacy. Refolding. A commonly tried but only episodically successful protocol to rescue insoluble protein is to denature the protein and try to refold it in vitro. The method can be successful83,84, particularly for extracellular proteins. However, even the most robust protocols only refold a small fraction of the input protein, and it is difficult to purify the refolded fraction. The best procedures use an activity assay to monitor refolding, and affinity reagents that select any refolded, active protein. We would advise using refolding as a last resort for intracellular proteins. Summary The methods and strategies for protein expression and purification have been reviewed for the expert many times in excellent, comprehensive ways. Here we attempted to provide a resource for those entering the field, reflecting the experiences of our groups in the application of the various methods to large numbers of proteins. We understand there are many possible routes to obtain high-quality protein and acknowledge that the methods described above should be considered as a starting point that can be embellished once sufficient expertise has been obtained. Detailed protocols for the methods described in this review can be found in the Supplementary Methods.
Note: Supplementary information is available on the Nature Methods website. ACKNOWLEDGMENTS The Structural Genomics Consortium is a registered charity (number 1097737) that receives funds from the Canadian Institutes for Health Research, the Canadian Foundation for Innovation, Genome Canada through the Ontario Genomics Institute, GlaxoSmithKline, Karolinska Institutet, the Knut and Alice Wallenberg Foundation, the Ontario Innovation Trust, the Ontario Ministry for Research and Innovation, Merck & Co., Inc., the Novartis Research Foundation, the Swedish Agency for Innovation Systems, the Swedish Foundation for Strategic Research and the Wellcome Trust. The New York Structural GenomiX Research Center for Structural Genomics is supported by the US National Institute of General Medical Sciences (U54 GM074945). Work at the MDC was supported by the German Federal Ministry for Education and Research (BMBF) through the Leitprojektverbund Proteinstrukturfabrik and the German National Genome Network (NGFN; FKZ 01GR0471, 01GR0472), and by the Fonds der Chemischen Industrie. The Protein Sample Production Facility is funded by the Helmholtz Association of German Research Centres. The China Structural Genomics Consortium is supported by the National 863 Hi-Tech Research and Development Program of China. The Israel Structural Proteomics Center is supported by The Israel Ministry of Science, Culture and Sport, the Divadol Foundation, the Neuman Foundation, the European Commission Sixth Framework Research and Technological Development Programme ‘SPINE2-Complexes’ Project under contract 031220. The RIKEN Structural Genomics/Proteomics Initiative was supported by the National Project on Protein Structural and Functional Analyses, Ministry of Education, Culture, Sports, Science and Technology of Japan. The Joint Center for Structural Genomics is supported by the US National Institutes of Health (NIH) Protein Structure Initiative grant U54 GM074898 from the NIGMS. The Northeast Structural Genomics Consortium is supported by the NIH NIGMS (U54-GM074958). The Midwest Center for Structural Genomics is supported by the NIH (GM074942) and by the US Department of Energy, Office of Biological and Environmental Research (DE-AC02-06CH11357). The Oxford Protein Production Facility is funded by the UK Medical Research Council and Biotechnology and Biological Sciences Research Council. SPINE2-Complexes is funded by the European Commission (contract 031220) under the Framework 6 RTD Programme and is coordinated from the Division of Structural Biology, Wellcome Trust Centre for Human Genetics, Oxford, UK . The Berkeley Structural Genomics Center is supported by the NIH (GM62412). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIGMS or the NIH. Published online at http://www.nature.com/naturemethods/ Reprints and permissions information is available online at http://npg. nature.com/reprintsandpermissions 1. Sauder, J.M. et al. High throughput protein production and crystallization at NYSGXRC. in Structural Proteomics: High-Throughput Methods Vol. 426 (eds. B. Kobe, M.Guss & H. Thomas) 561–575 (Humana Press, Totowa, New Jersey, USA, 2008). Büssow, K. et al. Structural genomics of human proteins-target selection and generation of a public catalogue of expression clones. Microb. Cell Fact. 4, 21 (2005). Heinemann, U., Büssow, K., Mueller, U. & Umbach, P. Facilities and methods for the high-throughput crystal structural analysis of human proteins. Accounts Chem. Res. 36, 157–163 (2003). Banci, L. et al. First steps towards effective methods in exploiting highthroughput technologies for the determination of human protein structures of high biomedical value. Acta Crystallogr. D62, 1208–1217 (2006). Aricescu, A.R. et al. Eukaryotic expression: developments for structural proteomics. Acta Crystallogr. D62, 1114–1124 (2006). Heinemann, U. Establishing a structural genomics platform: The Berlin-based Protein Structure Factory. Gene Funct. Dis. 3, 25–32 (2002). Scheich, C., Kummel, D., Soumailakakis, D., Heinemann, U. & Büssow, K. Vectors for co-expression of an unrestricted number of proteins. Nucleic Acids Res. 35, e43 (2007). Bartlam, M., Xu, Y. & Rao, Z. Structural proteomics of the SARS coronavirus: a model response to emerging infectious diseases. J. Struct. Funct. Genomics 8, 85–97 (2007). Gong, W.M. et al. Structural genomics efforts at the Chinese Academy of Sciences and Peking University. J. Struct. Funct. Genomics 4, 137–139 (2003). Albeck, S. et al. Three-dimensional structure determination of proteins related to human health in their functional context at The Israel Structural Proteomics Center (ISPC). Acta Crystallogr. D61, 1364–1372 (2005). Lesley, S.A. et al. Structural genomics of the Thermotoga maritima proteome implemented in a high-throughput structure determination pipeline. Proc. Natl. Acad. Sci. USA 99, 11664–11669 (2002). Montelione, G.T., Zheng, D., Huang, Y.J., Gunsalus, K.C. & Szyperski, T. Protein NMR spectroscopy in structural genomics. Nat. Struct. Biol. 7 (Suppl.), 982–985 (2000). Acton, T.B. et al. Robotic cloning and Protein Production Platform of the Northeast Structural Genomics Consortium. Methods Enzymol. 394, 210–243 (2005). Alzari, P.M. et al. Implementation of semi-automated cloning and prokaryotic expression screening: the impact of SPINE. Acta Crystallogr. D62, 1103–1113 (2006). Gileadi, O. The scientific impact of the Structural Genomics Consortium: a protein family and ligand-centered approach to medically-relevant human proteins. J. Struct. Funct. Genomics 8, 107–119 (2007). Gileadi, O. et al. Methods in Molecular Biology. in Structural Proteomics: HighThroughput Methods. Vol. 426 (eds., B. Kobe, M. Guss & T. Huber) 222–246 (Humana Press, Totowa, New Jersey, USA, 2008). You, J., Cohen, R.E. & Pickart, C.M. Construct for high-level expression and low misincorporation of lysine for arginine during expression of pET-encoded eukaryotic proteins in Escherichia coli. Biotechniques 27, 950–954 (1999). Klock, H.E., Koesema, E.J., Knuth, M.W. & Lesley, S.A. Combining the polymerase incomplete primer extension method for cloning and mutagenesis with microscreening to accelerate structural genomics efforts. Proteins published online, doi: 10.1002/prot.21786 (14 November 2007). Gr?slund, S. et al. The use of systematic N- and C-terminal deletions to promote production and structural studies of recombinant proteins. Protein Expr. Purif. (in the press). Ginalski, K., Elofsson, A., Fischer, D. & Rychlewski, L. 3D-Jury: a simple approach to improve protein structure predictions. Bioinformatics 19, 1015–1018 (2003). Ward, J.J., McGuffin, L.J., Bryson, K., Buxton, B.F. & Jones, D.T. The DISOPRED server for the prediction of protein disorder. Bioinformatics 20, 2138–2139 (2004). Yang, Z.R., Thomson, R., McNeil, P. & Esnouf, R.M. RONN: the bio-basis function neural network technique applied to the detection of natively disordered regions in proteins. Bioinformatics 21, 3369–3376 (2005). Cornvik, T. et al. An efficient and generic strategy for producing soluble human proteins and domains in E. coli by screening construct libraries. Proteins 65, 266–273 (2006). Gao, X. et al. High-throughput limited proteolysis/mass spectrometry for protein domain elucidation. J. Struct. Funct. Genomics 6, 129–134 (2005). Hartley, J.L., Temple, G.F. & Brasch, M.A. DNA cloning using in vitro site-

2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

? 2008 Nature Publishing Group http://www.nature.com/naturemethods

19. 20. 21. 22. 23. 24. 25.

144 | VOL.5 NO.2 | FEBRUARY 2008 | NATURE METHODS

REVIEW
specific recombination. Genome Res. 10, 1788–1795 (2000). 26. Aslanidis, C. & de Jong, P.J. Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Res. 18, 6069–6074 (1990). 27. Landy, A. Dynamic, structural, and regulatory aspects of lambda site-specific recombination. Annu. Rev. Biochem. 58, 913–949 (1989). 28. Guo, F., Gopaul, D.N. & Van Duyne, G.D. Asymmetric DNA bending in the Cre-loxP site-specific recombination synapse. Proc. Natl. Acad. Sci. USA 96, 7143–7148 (1999). 29. Hammarstr?m, M., Hellgren, N., van Den Berg, S., Berglund, H. & H?rd, T. Rapid screening for improved solubility of small human proteins produced as fusion proteins in Escherichia coli. Protein Sci. 11, 313–321 (2002). 30. Peti, W. & Page, R. Strategies to maximize heterologous protein expression in Escherichia coli with minimal cost. Protein Expr. Purif. 51, 1–10 (2007). 31. Braun, P. & LaBaer, J. High throughput protein production for functional proteomics. Trends Biotechnol. 21, 383–388 (2003). 32. Vincentelli, R. et al. Medium-scale structural genomics: strategies for protein expression and crystallization. Accounts Chem. Res. 36, 165–172 (2003). 33. Studier, F.W., Rosenberg, A.H., Dunn, J.J. & Dubendorff, J.W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 185, 60–89 (1990). 34. Uhlén, M., Forsberg, G., Moks, T., Hartmanis, M. & Nilsson, B. Fusion proteins in biotechnology. Curr. Opin. Biotechnol. 3, 363–369 (1992). 35. Arnau, J., Lauritzen, C., Petersen, G.E. & Pedersen, J. Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins. Protein Expr. Purif. 48, 1–13 (2006). 36. Carrington, J.C. & Dougherty, W.G. A viral cleavage site cassette: identification of amino acid sequences required for tobacco etch virus polyprotein processing. Proc. Natl. Acad. Sci. USA 85, 3391–3395 (1988). 37. Porath, J. Immobilized metal ion affinity chromatography. Protein Expr. Purif. 3, 263–281 (1992). 38. Nallamsetty, S. & Waugh, D. Solubility-enhancing proteins MBP and NusA play a passive role in the folding of their fusion partners. Protein Expr. Purif. 45, 175–182 (2006). 39. Nallamsetty, S. & Waugh, D.S. A generic protocol for the expression and purification of recombinant proteins in Escherichia coli using a combinatorial His6-maltose binding protein fusion tag. Nat. Protoc. 2, 383–391 (2007). 40. Waugh, D.S. Making the most of affinity tags. Trends Biotechnol. 23, 316–320 (2005). 41. Carson, M., Johnson, D.H., McDonald, H., Brouillette, C. & Delucas, L.J. Histag impact on structure. Acta Crystallogr. 63, 295–301 (2007). 42. Dubendorff, J.W. & Studier, F.W. Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor. J. Mol. Biol. 219, 45–59 (1991). 43. Wycuff, D.R. & Matthews, K.S. Generation of an AraC-araBAD promoterregulated T7 expression system. Anal. Biochem. 277, 67–73 (2000). 44. Shimizu, Y. et al. Cell-free translation reconstituted with purified components. Nat. Biotechnol. 19, 751–755 (2001). 45. Guzman, L.M., Belin, D., Carson, M.J. & Beckwith, J. Tight regulation, modulation, and high-level expression by vectors containing the arabinose pBAD promoter. J. Bacteriol. 177, 4121–4130 (1995). 46. Studier, F.W. Protein production by auto-induction in high density shaking cultures. Protein Expr. Purif. 41, 207–234 (2005). 47. Lesley, S.A. High-throughput proteomics: protein expression and purification in the postgenomic world. Protein Expr. Purif. 22, 159–164 (2001). 48. Tunac, J. A new high-aeration capacity shake-flask system. J. Ferm. Bioeng. 68, 15–159 (1989). 49. Brodsky, O. & Cronin, C.N. Economical parallel protein expression screening and scale-up in Escherichia coli. J. Struct. Funct. Genomics 7, 101–108 (2006). 50. Vera, A., Gonzalez-Montalban, N., Aris, A. & Villaverde, A. The conformational quality of insoluble recombinant proteins is enhanced at low growth temperatures. Biotechnol. Bioeng. 96, 1101–1106 (2007). 51. Berrow, N.S. et al. Recombinant protein expression and solubility screening in Escherichia coli: a comparative study. Acta Crystallogr. D62, 1218–1226 (2006). 52. Page, R. et al. Scalable high-throughput micro-expression device for recombinant proteins. Biotechniques 37, 364–370 (2004). 53. Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254 (1976). 54. Bolanos-Garcia, V.M. & Davies, O.R. Structural analysis and classification of native proteins from E. coli commonly co-purified by immobilised metal affinity chromatography. Biochim. Biophys. Acta 1760, 1304–1313 (2006). 55. Howell, J.M., Winstone, T.L., Coorssen, J.R. & Turner, R.J. An evaluation of in vitro protein-protein interaction techniques: assessing contaminating background proteins. Proteomics 6, 2050–2069 (2006). 56. Bullock, A.N., Debreczeni, J., Amos, A.L., Knapp, S. & Turk, B.E. Structure and substrate specificity of the Pim-1 kinase. J. Biol. Chem. 280, 41675–41682 (2005). 57. Alam, M., Ho, S., Vance, D.E. & Lehner, R. Heterologous expression, purification, and characterization of human triacylglycerol hydrolase. Protein Expr. Purif. 24, 33–42 (2002). 58. Kapust, R.B. & Waugh, D.S. Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused. Protein Sci. 8, 1668–1674 (1999). 59. Iakoucheva, L.M., Brown, C.J., Lawson, J.D., Obradovic, Z. & Dunker, A.K. Intrinsic disorder in cell-signaling and cancer-associated proteins. J. Mol. Biol. 323, 573–584 (2002). 60. Frenkiel-Krispin, D. et al. Plant transformation by Agrobacterium tumefaciens: modulation of single-stranded DNA-VirE2 complex assembly by VirE1. J. Biol. Chem. 282, 3458–3464 (2007). 61. Tolia, N.H. & Joshua-Tor, L. Strategies for protein coexpression in Escherichia coli. Nat. Methods 3, 55–64 (2006). 62. Romier, C. et al. Co-expression of protein complexes in prokaryotic and eukaryotic hosts: experimental procedures, database tracking and case studies. Acta Crystallogr. 62, 1232–1242 (2006). 63. Bullock, A.N., Debreczeni, J.E., Edwards, A.M., Sundstr?m, M. & Knapp, S. Crystal structure of the SOCS2-elongin C-elongin B complex defines a prototypical SOCS box ubiquitin ligase. Proc. Natl. Acad. Sci. USA 103, 7637–7642 (2006). 64. Vedadi, M. et al. Chemical screening methods to identify ligands that promote protein stability, protein crystallization, and structure determination. Proc. Natl. Acad. Sci. USA 103, 15835–15840 (2006). 65. Niesen, F.H., Berglund, H. & Vedadi, M. The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nat. Protoc. 2, 2212–2221 (2007). 66. Elleby, B. et al. High-level production and optimization of monodispersity of 11beta-hydroxysteroid dehydrogenase type 1. Biochim. Biophys. Acta 1700, 199–207 (2004). 67. Strauss, A. et al. Improved expression of kinases in Baculovirus-infected insect cells upon addition of specific kinase inhibitors to the culture helpful for structural studies. Protein Expr. Purif. 56, 167–176 (2007). 68. Smith, G.E., Summers, M.D. & Fraser, M.J. Production of human beta interferon in insect cells infected with a baculovirus expression vector. Mol. Cell. Biol. 3, 2156–2165 (1983). 69. Boettner, M., Prinz, B., Holz, C., Stahl, U. & Lang, C. High-throughput screening for expression of heterologous proteins in the yeast Pichia pastoris. J. Biotechnol. 99, 51–62 (2002). 70. Holz, C., Hesse, O., Bolotina, N., Stahl, U. & Lang, C. A micro-scale process for high-throughput expression of cDNAs in the yeast Saccharomyces cerevisiae. Protein Expr. Purif. 25, 372–378 (2002). 71. Aricescu, A.R., Lu, W. & Jones, E.Y. A time- and cost-efficient system for highlevel protein production in mammalian cells. Acta Crystallogr. D62, 1243–1250 (2006). 72. Yokoyama, S. Protein expression systems for structural genomics and proteomics. Curr. Opin. Chem. Biol. 7, 39–43 (2003). 73. Kigawa, T. et al. Preparation of Escherichia coli cell extract for highly productive cell-free protein expression. J. Struct. Funct. Genomics 5, 63–68 (2004). 74. Matsuda, T. et al. Cell-free synthesis of zinc-binding proteins. J. Struct. Funct. Genomics 7, 93–100 (2006). 75. Endo, Y. & Sawasaki, T. Cell-free expression systems for eukaryotic protein production. Curr. Opin. Biotechnol. 17, 373–380 (2006). 76. Mikami, S., Masutani, M., Sonenberg, N., Yokoyama, S. & Imataka, H. An efficient mammalian cell-free translation system supplemented with translation factors. Protein Expr. Purif. 46, 348–357 (2006). 77. Yokoyama, S., Terwilliger, T.C., Kuramitsu, S., Moras, D. & Sussman, J.L. RIKEN aids international structural genomics efforts. Nature 445, 21 (2007). 78. Murayama, K. et al. Crystal structure of the rac activator, Asef, reveals its autoinhibitory mechanism. J. Biol. Chem. 282, 4238–4242 (2007). 79. Miyazono, K. et al. Novel protein fold discovered in the PabI family of restriction enzymes. Nucleic Acids Res. 35, 1908–1918 (2007). 80. Nishihara, K., Kanemori, M., Yanagi, H. & Yura, T. Overexpression of trigger factor prevents aggregation of recombinant proteins in Escherichia coli. Appl. Environ. Microbiol. 66, 884–889 (2000). 81. Wynn, R.M., Song, J.L. & Chuang, D.T. GroEL/GroES promote dissociation/reassociation cycles of a heterodimeric intermediate during alpha(2)beta(2) protein assembly. Iterative annealing at the quaternary structure level. J. Biol. Chem. 275, 2786–2794 (2000). 82. Kaiser, C.M. et al. Real-time observation of trigger factor function on translating ribosomes. Nature 444, 455–460 (2006).

? 2008 Nature Publishing Group http://www.nature.com/naturemethods

NATURE METHODS | VOL.5 NO.2 | FEBRUARY 2008 | 145

REVIEW
83. Willis, M.S. et al. Investigation of protein refolding using a fractional factorial screen: a study of reagent effects and interactions. Protein Sci. 14, 1818–1826 (2005). 84. Vincentelli, R. et al. High-throughput automated refolding screening of inclusion bodies. Protein Sci. 13, 2782–2792 (2004). 85. Kim, S.H. et al. Structural genomics of minimal organisms and protein fold space. J. Struct. Funct. Genomics 6, 63–70 (2005). 86. Lesley, S.A. & Wilson, I.A. Protein production and crystallization at the joint center for structural genomics. J. Struct. Funct. Genomics 6, 71–79 (2005). 87. Kreusch, A. & Lesley, S.A. High throughput cloning, expression and purification technologies. in Genomics, Proteomics, and Vaccines (ed., G. Grandi) 171–184 (Wiley Press, Chichester, UK, 2004). 88. McMullan, D. et al. High-throughput protein production for X-ray crystallography and use of size exclusion chromatography to validate or refute computational biological unit predictions. J. Struct. Funct. Genomics 6, 135–141 (2005). 89. Stols, L., Millard, C.S., Dementieva, I. & Donnelly, M.I. Production of selenomethionine-labeled proteins in two-liter plastic bottles for structure determination. J. Struct. Funct. Genomics 5, 95–102 (2004). 90. Geerlof, A. et al. The impact of protein characterization in structural proteomics. Acta Crystallogr. D62, 1125–1136 (2006).

? 2008 Nature Publishing Group http://www.nature.com/naturemethods

The authors are: Susanne Gr?slund1, P?r Nordlund1, Johan Weigelt1, James Bray2, Opher Gileadi2, Stefan Knapp2, Udo Oppermann2, Cheryl Arrowsmith3, Raymond Hui3, Jinrong Ming3, Sirano dhe-Paganon3, Hee-won Park3, Alexei Savchenko3, Adelinda Yee3, Aled Edwards3, Renaud Vincentelli4, Christian Cambillau4, Rosalind Kim5, Sung-Hou Kim5, Zihe Rao6, Yunyu Shi7, Thomas C Terwilliger8, Chang-Yub Kim8, Li-Wei Hung8, Geoffrey S Waldo8, Yoav Peleg9, Shira Albeck9, Tamar Unger9, Orly Dym9, Jaime Prilusky9, Joel L Sussman9, Ray C Stevens10, Scott A Lesley10,11, Ian A Wilson10,11, Andrzej Joachimiak12, Frank Collart12, Irina Dementieva12, Mark I Donnelly12, William H Eschenfeldt12, Youngchang Kim12, Lucy Stols12, Ruying Wu12, Min Zhou12, Stephen K Burley13, J Spencer Emtage13, J Michael Sauder13, Devon Thompson13, Kevin Bain13, John Luz13, Tarun Gheyi13, Fred Zhang13, Shane Atwell13, Steven C Almo14, Jeffrey B Bonanno14, Andras Fiser14, Sivasubramanian Swaminathan15, F William Studier15, Mark R Chance16, Andrej Sali17, Thomas B Acton18, Rong Xiao18, Li Zhao18, Li Chung Ma18, John F Hunt19, Liang Tong19, Kellie Cunningham18, Masayori Inouye18, Stephen Anderson18, Heleema Janjua18, Ritu Shastry18, Chi Kent Ho18, Dongyan Wang18, Huang Wang18, Mei Jiang18, Gaetano T Montelione18, David I Stuart20,23, Raymond J Owens20,23, Susan Daenke20,23, Anja Schütz21, Udo Heinemann21, Shigeyuki Yokoyama22, Konrad Büssow21,24, Kristin C Gunsalus18,24

146 | VOL.5 NO.2 | FEBRUARY 2008 | NATURE METHODS


赞助商链接
相关文章:
实验十蛋白质的表达、分离纯化和鉴定
实验十 蛋白质的表达、分离纯化和鉴定第一部分 蛋白质的表达、分离纯化 目的要求(1)了解重组蛋白表达的方法和意义。 (2)了解重组蛋白亲和层析分离纯化的方法。 ...
蛋白的表达纯化
蛋白的表达纯化一、溶液配制 凝胶上样缓冲溶液配方: 1×SDS 凝胶上样缓冲溶液配方: Tris-HCl pH6.8 50mmol/L 巯基乙醇 SDS 溴酚蓝 甘油 蛋白纯化 配制以下...
蛋白质表达及纯化策略
蛋白表达纯化策略 一.目录: 含组氨酸标签的蛋白诱导表达及纯化 GST-融合蛋白的纯白化策略 DEAE 纯化蛋白策略 分子筛纯化蛋白策略 二.试剂: 所用试剂均购自上海...
抗体降解蛋白的表达纯化及特性分析_图文
抗体降解蛋白的表达纯化及特性分析 - 抗体降解蛋白的表达纯化及特性分析 马喆,华承薇 * # 5 (南京农业大学动物医学院,南京 210095) 摘要:检测体外反应体系中 Id...
蛋白表达纯化实验步骤
蛋白表达纯化实验步骤_生物学_自然科学_专业资料。蛋白表达纯化实验步骤(待改进) 1、 取适当相应蛋白高表达的动物组织提 total-RNA。 2、 设计蛋白表达引物。引物...
蛋白表达和纯化
蛋白表达纯化 材料和方法 细胞生长深红红螺菌培养物于 摄氏度下在如前所述改良的 摄氏度下在如前所述改良的Ormerod’ s培养基 深红红螺菌培养物于30摄氏度下...
HIS-TAG蛋白质的表达纯化
蛋白质的表达、分离、纯化实验的实验原理 和操作步骤携带有目标蛋白基因的质粒在大肠杆菌 BL21 中,在 37℃,IPTG 诱 导下, 超量表达携带有 6 个连续组氨酸残基...
大肠杆菌表达系统与蛋白表达纯化
8.大肠杆菌表达系统与蛋白表达纯化大肠杆菌表达系统遗传背景清楚,目的基因表达水平高,培养周期短,抗污染能力强等特点, 是分子生物学研究和生物技术产 业化发展进程中...
目的蛋白表达与纯化protocol
目的蛋白表达纯化protocol_基础医学_医药卫生_专业资料。目的蛋白表达纯化 protocol 小量表达测试: 1、挑一单克隆到 3ml LB(含抗生素)中,370C,220rpm 振荡培...
GST蛋白表达、纯化步骤
GST蛋白表达纯化步骤_生物学_自然科学_专业资料。Protein Expression Protocol: 1. Transform control construct and tag-fusion-constructs into E.coli BL ...
更多相关标签: