part 8

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 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 can-not 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.
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.
‘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, wild-type 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 non-functional (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 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.

我要part2&3两部分，请版主批准

[ 本帖最后由 蛋白丫头 于 08-5-22 17:16 编辑 ]

大致看了下，是篇很好的综述
很期待大家把它翻译出来，

引用:

原帖由 蛋白丫头 于 08-5-22 17:11 发表
我要part2&3两部分，请版主批准

已确认。
辛苦了。

各位酷友如果对文章的划分及内容等各方面有不同的看法，请尽管提出来。

我认领part7,希望版主同意！

引用:

原帖由 wwmhj 于 08-5-23 19:29 发表
我认领part7,希望版主同意！

已确认!

大家积极参与！

我今天才看到个帖子 我的毕业论文就是翻译的这篇
你们能在3天内帮我修改修改吗？？？
拜托了