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本期待翻译文章题目：Protein production and purification
发表时间：2008年2月
文章来源：nature methods
文章类型：综述（关于蛋白的表达、纯化、鉴定等）
文章结构：
1、Obtaining the cDNA and creating the expression clone
2、Cloning
3、Expressing the protein
4、Small-scale test expression
5、Protein purification
6、Protein characterization
7、Common ‘traps’ and ‘pitfalls’
8、Rescue strategies

后面的文章拆分并未全部与上面对应，故以下面为准。
请大家在后面跟贴说明自己要认领的部分。

[ 本帖最后由 hamye 于 08-6-16 13:43 编辑 ]

part2、3    已由蛋白丫头认领。
part4、5    已由稳稳飞认领。
part6         已由snoopyzxx同学认领。
part7         已由wwmhj认领。
part8、9   已由tieniu03认领。

请大家尽快完成，视本期效果，在适当时候将进行第二期的活动。

[ 本帖最后由 hamye 于 08-6-23 12:53 编辑 ]

part one

Protein production and purification

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 full-length protein or a fragment hereof? 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 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  esearcher 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.
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.

part two

Obtaining the cDNA and creating the expression clone cDNA

Recently, sequencing efforts and various cDNA consortia have made available large libraries of full-length, sequence-verified cDNAs. Although there are inevitably issues with clone contamination and mix-up, the resources are in general trustworthy. Among the most comprehensive and best annotated is the Mammalian Gene Collection, which maintains a repository of >19,000 human cDNAs, covering ~65% of all annotated genes. For genes or splice variants not easily obtained through more traditional routes, total gene synthesis can be used. Over the past few years, the cost of gene synthesis has dropped almost fivefold, and it will undoubtedly continue to decrease. One advantage of gene synthesis is the ability to change the codon bias of the gene to be more compatible with the recombinant host. However, for Escherichia coli, expression strains supplemented with additional tRNAs can often overcome the codon bias of the recombinant gene17. For example, in a study of 30 human genes by the Structural Genomics Consortium (SGC), there was no clear advantage in the use of codon-optimized genes compared with the natural sequence expressed in tRNA-supplemented strains (N.A. Burgess-Brown, S. Sharma, F. Sobott, C. Loenarz, U. Oppermann and O. Gileadi; submitted).

Selecting the N and C termini.
The objective of recombinant protein expression is usually to produce a sample that supports a certain biochemical or biological activity, such as enzyme catalysis or protein-ligand interactions. Frequently, the desired activity is supported by a discrete domain, and thus it is often not necessary to express the full-length protein to address a particular biological 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 structure is well-defined, it is not currently possible to predict which specific N- and C-terminal boundaries are most compatible with the expression of a soluble protein. Thus, pragmatism dictates testing many truncated forms of the protein to select one or more for scale-up production. For proteins of known or readily predicted three-dimensional structure, the borders should be engineered to encompass the domain of interest. As an example, ten constructs of the targeted domain might be made at the outset of every project, one corresponding to the full-length protein and nine representing the clones derived from amplifying a combination of three different 5′-end primers and three different 3′-end primers. Gräslund and colleagues have compared the success rate of the nested-primer approach with the predicted success rate if one had chosen only a single ‘optimal’ construct. In a sample set of 400 human protein domains, the use of multiple constructs increased the probability of generating a soluble protein twofold19.
To select the sets of PCR primers for proteins with a predictable three-dimensional structure, one should consider prior knowledge of the structure of a related protein, sequence conservation patterns, and predictions of secondary structure or unfolded/disordered regions20,21. Widely accepted guidelines are to: (i) remove predicted membrane-spanning regions; (ii) avoid disrupting predicted secondary structural elements; (iii) respect the boundaries of globular domains, if known; and (iv) avoid inclusion of low-complexity regions or hydrophobic residues at the termini22. The optimal 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 acids at each end19. For proteins without a predictable three-dimensional structure, the approximate boundaries of the region of interest might be identified using functional assays and scanning deletion mutagenesis, and then optimal boundaries for expression can be identified using nested sets of PCR primers, as above23. Boundaries of structured domains can also be determined experimentally by using limited proteolysis combined with mass spectrometry analysis24. Clearly, when using protein fragments, caution should be used in interpreting unexpected biological results.

part 3
Cloning
The most common methods now used in our groups to clone target genes into the requisite expression vector rely on homology-based approaches, using either recombination enzymes25 or ligation-independent cloning (LIC)26. Restriction enzyme–based 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.

part 4

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 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 site-specific 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.
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 maltose-binding 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.

[ 本帖最后由 hamye 于 08-5-22 15:59 编辑 ]

part 5

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 poly-merase 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 soluble 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 small-scale 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 µg 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.

part 6

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 ÄKTA 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 histidine-tagged 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 with lysozyme are equivalent in most cases.
The lysis buffer should contain a strong buffer (50–100 mM phosphate or HEPES) to overcome the contribution of the bacterial lysate, high ionic strength (equivalent to 300–500 mM NaCl) to enhance protein solubility and stability, protease inhibitors and a reducing agent such as Tris(2-carboxyethyl) phosphine hydrochloride (TCEP) to prevent oxidation of the protein. Loading large amounts of bacterial lysate (>1 l culture volume) on small (<1 ml) affinity columns may require prior removal of any particulate or viscous material. This can be accomplished by using enzymes that degrade nucleic acid and cell-wall material, such as DNase or Benzonase (Merck/EMD) and lysozyme, respectively. Some of the enzymes used in lysis are less active in the presence of reducing agents or high salt concentration; optimal lysis may require sequential addition of the components. Clarified lysates can also be filtered before loading on the affinity columns.
IMAC purification is performed in phosphate buffer, pH 8.0 and 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 (for example, 500 mM NaCl) maintain solubility and stability of the widest variety of proteins. Indeed, a substantial fraction of proteins precipitate if the salt concentration is reduced to physiological levels, particularly as the protein becomes more pure and concentrated. The choice of NaCl as the salt is mainly historical and, although not systematically explored, there is no reason to believe that sodium and chloride are optimal. Indeed, sodium and chloride levels in the cell are very low and are probably never the physiologically relevant counter-ions for intracellular proteins. A modest amount of imidazole (see resin manufacturer’s recommendations) should be included in the cell extraction buffer to reduce binding of 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 IMAC matrices. Finally, inclusion of glycerol (10%) during protein purification enhances the solubility and stability of many proteins.
Chromatography. After the lysate is loaded on the IMAC column, it should be washed with buffer including an intermediate concentration of imidazole (see manufacturer’s instructions), which will elute weakly bound contaminants without sacrificing large amounts of the recombinant protein. It is sometimes necessary to optimize the wash step with respect to the concentration of imidazole as well as the volume of the wash. Finally, the recombinant protein should be with a step gradient (for example, 300 mM imidazole). If EDTA and DTT are added after IMAC; add the EDTA first to sequester any nickel that has leached off and that could react with the DTT.
The choice of gel filtration as the next step may be surprising, considering its lower resolving power compared with ion exchange or other adsorption chromatography methods, but this step is often sufficient after IMAC if the protein was abundant in the lysate. Moreover, gel filtration is more generic, can be performed in any buffer condition, and can be used to resolve the oligomerization state of the target protein. In some cases, if the protein is judged insufficiently pure for the intended purpose, one can remove the tag with a histidine-tagged TEV protease and perform IMAC again as an additional ‘generic’ purification step, collecting the recombinant protein in the flowthrough. This step very efficiently removes histidine-rich proteins derived from the expression host, which may have copurified in the primary IMAC procedure, as well as the cleaved tag and the histidine-tagged protease.

part 7

Protein characterization
Characterizing the purified protein in some detail reduces the risk of wasting resources on protein material of inadequate quality. It also provides a means to ensure that different batches of the same protein have similar properties. Below, we outline a simple, generic protein characterization protocol that allows the experimentalist to judge whether the correct protein has been purified, whether additional molecular species are present and to estimate the approximate protein concentration. Other characterization methods that are very informative but not as widely applied, such as mass spectrometry, static or dynamic light scattering, and measuring protein thermal stability, are described in Supplementary Methods.
Inspection of gel filtration chromatogram. If size exclusion chromatography was used as the last purification step, a close look at the chromatogram is essential. Symmetric elution profiles are characteristic of homogeneous proteins, whereas asymmetric profiles reflect inhomogeneous, or partially aggregated, samples (Fig. 2), or whether the column itself is in poor condition. The elution profiles will also reveal the primary oligomerization state. The presence of additional oligomerization states may be of biological significance, or may be a sign of nonspecific aggregation. If the protein elutes in the void volume of the chromatogram, the protein is most likely forming large, nonspecific aggregates, which may be an indication of improper folding and compromised activity. It is also of value to analyze individual peaks by SDS-PAGE or mass spectrometry to analyze the protein in each peak.
SDS-PAGE analysis. After protein purification, samples should be resolved by denaturing SDS-PAGE. If stained with a dye such as Coomassie brilliant blue, the intensity of the bands will usually be proportional to the amount of protein53. This allows the purity of the sample to be estimated and whether the purified protein is of the expected size.
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.