Labeling experiments for protein expression
Labeling experiments for protein expression
Most proteins are prepared using recombinant expression techniques. During the cloning process, additional residues or tags can be added to the N- or C-terminus of the target protein. These tags can range in length from just a few amino acid residues to full-length proteins or structural domains, and they can increase the yield of the target protein or give it new properties that can be used to characterize or study the target protein.
Operation method
Labeling experiments for protein expression Move I. Factors to consider when designing proteins with labels Selection of affinity and solubility Two challenges are faced when producing exogenous proteins in E. coli: one? is the low expression level of the protein expression system used; the second is that the expressed proteins are misfolded into insoluble aggregates-inclusion bodies. Expression problems caused by the presence of weak promoters, low transcriptional initiation or rare codons can be solved by introducing corrective sequences into the gene during assembly of the overexpression plasmid and by using E. coli supplemented with rare tRNAs. Fusing highly expressed endogenous proteins to the N-terminus of exogenous target proteins is not only a way to increase yield, but also to increase the solubility of the target proteins: this is the rationale for soluble labeling. In addition, affinity tags are essential for protein purification and provide a variety of strategies for binding target proteins to affinity substrates (Table I6.1). Some protein tags serve as both affinity and soluble tags. For example, glutathione-S-tamsferase (GST) enhances the solubility of some proteins, and the soluble tag maltosebindingprotein (MBP) can also be used for affinity purification of target proteins. Affinity tags and soluble tags are very different in molecular mass, and therefore the metabolic burden they impose on the host cell is also very different. For example, for a target protein with 100 amino acid residues fused with an MBP tag (43kDa), 5xng of the fusion protein must be expressed to obtain Img of the target protein. The purification costs required for affinity tags vary from the cost of the resin itself to the cost of the process (ease of regeneration and reuse, elution reagents, binding capacity, etc.) Lichty et al. (2005) compared eight affinity tags in terms of purification, throughput, and cost. Many similar studies have been carried out, and some reviews provide a good summary of affinity labels (Arnauetal., 2006b; Waugh, 2005) and soluble labels (EspositoandChatterjee, 2006) (Terpe, 2003), and list their advantages and disadvantages. Apart from the cost factor, the choice of affinity tag usually depends on the buffer suitable for the purification of the target protein. Histidine tags are not suitable for the purification of certain proteins that are sensitive to oxidation and protein hydrolysis damage because the medium for solid-phase metal affinity chromatography (IMAC) does not tolerate reducing agents or EDTA. similarly, great care should be taken when purifying proteins that are sensitive to metal ions using IMAC media. Similarly, great care should be taken when using IMAC media to purify proteins that are sensitive to metal ions. In contrast, if the target protein requires a denaturing environment or needs to be refolded, then histidine tagging and IMAC purification methods are a good choice. In some cases, the level of expression can also determine the choice of label - soluble tags such as mannan-binding protein (MBP), thioredoxin (Trx), N-utilizationsubstanceA (NusA) and glutathione-S-transferrin (GST) are also useful. NusA) and glutathione-S-transferase all have strong translation initiation signals that can drive high levels of expression, which is useful for structural studies. In addition, more specific epitope tags or tandem tags are more appropriate when low levels of expression are required, such as when studying complexes or physiological interactions. Protease cleavage sites for removal. Some proteins with histidine tags have been crystallized with little or no effect of the tag on the protein structure (Carsonetal., 20 0 7). For some proteins, histidine labeling actually contributes to protein crystal formation [e.g., Smits et al. (2008)]. Histidine tags can also be used for protein detection in conjunction with commercially available antibodies specific for the tag. There are also some non-standard 6X histidine forms of histidine tags. In order to reduce charge or increase stability, these tags have additional amino acid residues scattered throughout the histidine sequence, such as the HAT-tag and the 6XHN-tag, which are better able to bind Co2+-Talon resin (Clontech) and the MAT-tag (metalaffinitytag) (SigmaAldrich). GST is a highly expressed 26kDa eukaryotic protein. Studies have shown that when fused to the N-terminus of a target protein, GST cloned from Schistosoma japonicum (Sc/iistosomajaponicwm) is able to increase protein solubility and expression (SmithandJohnson,1988). When GST is located at the C-terminus (Fig. 16.1B), its solubilizing ability is relatively weak, but it still functions well as an affinity tag.GST binds to glutathione immobilized on the resin, which can be used for affinity purification of GST-tagged proteins. After binding to the resin, the fusion protein can be eluted by free reduced glutathione (10?40 mrnol/L) under neutral and mild conditions. Resins used for purification of GST fusion proteins, such as glutathione^sepharosebead, are relatively inexpensive and have a high loading capacity (5?10 mgGST/mL of resin), which can be used multiple times after regeneration. To bind glutathione, the GST tag must be present in the correctly folded form, so the fusion protein must be soluble and under nondenaturing conditions to be effectively purified.GST fusion proteins are usually expressed at high levels, so care must be taken to test the solubility of the protein. For some proteins, GST functions as a soluble tag [e.g., Kim and Lee (2008)].GST can be detected by colorimetricassay (c ○ l ○ rimetricassay) in the presence of its substrate, 1-gas-2,4-dinitroben-zene (l-chloro-2,4dinitroben-zene, CDNB) (Habigetal., 2004). Habigetal., 1974); this assay can also be used to monitor the correct folding of the GST in the fusion protein as well as its affinity when binding to glutathione on the resin is unsatisfactory. Again this tag can be detected with a commercial anti-GST antibody. The GST tag is relatively large, which makes it more susceptible to degradation by proteases, so purification of GST fusion proteins should be performed quickly to minimize its loss. Unlike proteins containing a histidine tag, samples of GST-tagged proteins can be prepared using a buffer containing EDTA to minimize protein degradation. Care must be taken when using reducing conditions because GST has 4 solvent-exposed cysteines, which are associated with oxidative aggregation of the protein.GST forms homodimers in solution, so the choice of a fusion GST tag is not a wise one for oligomeric proteins. GST binds to and elutes from glutathione relatively slowly, so GST fusion proteins need to be sampled and eluted at slower flow rates. Because glutathione absorbs strongly at 280nm, monitoring of the protein during elution should be done carefully. (1) Epitope tags A number of short amino acid sequences that can be recognized by commercially sourced antibodies can be used as tags for the detection and purification of proteins. The addition of epitope tags (epitopetag) has been widely used in molecular biology as a general practice for tracking recombinant proteins (FritzeandAnderson, 2000), and this method has a high degree of specificity, and the smaller tags minimize the impact on the structure and function of the target protein. These tags are usually added at the N-terminus or C-terminus, but can also be added to the target protein sequence (in the loop structure or in a solvent-exposed region between structural domains). Host cells usually do not have the same amino acid sequence as the epitope tag, which makes detection of the target protein easy. However, for purification, epitope-tag binding media are expensive (usually monoclonal antibodies immobilized on chromatographic chromatography resins) and are not suitable for large-scale protein preparation compared to other affinity purification media. Short peptides corresponding to tags, low PH, or other methods (e.g., chelating out the calcium ions required for tag binding, or using salts or polyols) can be used to elute the target proteins, but some of them can appear drastic compared to other methods of affinity purification (see Chapter 28 for details). FLAG tags, which are short hydrophilic peptide tags with only 8 amino acid residues (DYKDDDDK), can be used for the detection and purification of target proteins (EinhauerandJungbauer,2001;Prickettetal.,1989). In addition to using a number of highly specific anti-FLAG monoclonal antibodies, it is also possible to completely remove the tag after purification using the enterokinase digest site (DDDK) contained in the FLAG tag. A variant of the FLAG tag is the 3XFLAG tag (Sigma-Aldrich), which consists of three tandemly repeated FLAG-like sequences (Hernanet et al., 2001). sequences (Hernanetal., 2000). Other commonly used epitope tags include the influenza hemagglutinin epitope tag (HAtag) and the c~Myc tag (FritzeandAnderson, 2000). (2) S-tagging Raines et al. (2000) reviewed S-tagging, which utilizes the tight binding between the S-peptide segment (positions 1?20) at the N-terminus of the RNAase SCRNaseS) and the S-protein (residues 21?124) (RichardsandVithayarhil, 1959).The S-tagging system uses 1?15 amino acid residues at the N-terminus of the S-peptide segment. 15 amino acid residues at the N-terminus of the S-peptide, and S-proteins immobilized on agarose microspheres to purify the target protein. Typically, S-tagged vectors encode a site-specific protease cleavage site, and proteins can be eluted by cutting out the tag or by more drastic denaturing conditions to disrupt the interaction between the S-tag and the S-protein. (3) STREP-n tag The STREP-II tag (WSHPQFEK) takes advantage of the strong specific interaction between biotin and streptavidin (SchmidtandSkerm,1994). This peptide tag binds to the biotin pocket of the streptavidin.Strep-Tactin is a recombinant streptavidin optimized to bind the STREP-II tag and has been used in the preparation of affinity media. Bound target proteins can be eluted at low levels (2.5 mmol/L) of desthiobiotin, a biotin analog that competitively binds Strep-Tactin in a reversible manner (SkerraandSchmidt, 2000). The elution conditions are mild and the buffer used can contain high concentrations (up to 50 mmol/L) of reducing agents (e.g., DTT or i9-hydroxyethanol) as well as chelating agents (e.g., EDTA), making STREP-II labeling an excellent purification method for proteins sensitive to oxidizing agents.The resin of the Strep-Tactin chromatography method is capable of regeneration and can be reused several times. It can be reused several times. Although most cell extracts contain small amounts of naturally biotinylated host proteins (biotinylatedprotem), which usually do not interfere with purification, if necessary, affinity proteins can be added to the host proteins in order to remove the biotinylated host proteins (Schmidt and Skerra, 2007). There have also been some attempts to use streptavidin/avidin as a fusion tag, but these tags are difficult to use for affinity purification because the interactions between these proteins are so strong that they are difficult to disrupt. (4) CBP tag The calmodulin-binding peptide (CBP) is a short peptide with 26 amino acid residues. It is derived from the C-terminus of skeletalmusclemyosinUghtchainkinase and binds specifically to calmodulin [cf. Terpe (2003)]. Calmodulin immobilized on chromatographic media (e.g., calmodulin-agarose gels) can be used to purify target proteins with CBP tags. Although calmodulin binds very tightly to CBP (affinity on the nanomolar scale), this interaction is dependent on calcium ions, and bound proteins can be eluted off in one step with a mild buffer (e.g., EGTA)-step containing a calcium chelator. Because the CBP tag contains the target sequence for protein kinase A (proteinkinaseA), another use of the tag is 32P isotope labeling of fusion proteins (Vaillancourtetal., 2000). Because many endogenous proteins interact with calmodulin in eukaryotic cells, the CBP tag is not applicable to eukaryotic expression systems. In recombinant protein expression, especially when eukaryotic proteins are expressed in bacteria, the main bottleneck is the correct folding and solubility of the protein. Several soluble proteins have been used as tags to improve the folding of target proteins. These tags should be used in conjunction with other methods to promote protein folding, such as lowering the culture temperature after induction or co-expression of chaperone proteins (BaneyxandMujacic,2004;deMarcoetal.,2007;Sahdevetal.,2008).In order to obtain higher soluble target proteins, it is necessary to try multiple soluble tags (PelegandUnger, 2008). For proteins that are particularly difficult to solubilize, it is possible to try purifying the protein under denaturing conditions followed by refolding (CabritaandBottomley, 2004; JungbauerandKaar, 2007; Qoronflehetal., 2007; see Chapter 17 for details). Because of its ability to specifically bind maltose and atnylose, the MBP tag not only serves as a soluble tag (diGmmetal, 1988), but can also be used efficiently for affinity purification. MBP is a 43 kDa E. coli secretory protein that is expressed at high levels and enhances the solubility of proteins fused to its C-terminus (KapustandWaugh, 1999). MBP is a 43kDa E. coli secretory protein that is expressed at high levels and enhances the solubility of proteins fused to its C-terminus (KapustandWaugh, 1999). Recent studies have shown that MBP fused to the C-terminus of target proteins is also effective (Dysonetal., 2004). MBP is large, which places a large burden on cellular metabolism, but a high-throughput test has shown that the MBP tag is one of the best soluble tags (DysonetaU2004;Kataevaetal., 2005). However, about ¼ of MBP fusion proteins remain insoluble or the proteins become aggregation-prone after removal of the MBP tag ^ For example, we used an E. coli protein expressing the human calcitoningene-relatedpeptide- receptorcomponentprotein,^ a protein with the MBP tag. receptorcomponentprotein,CGRP-RCP), we found that aggregation of the target protein occurred after removal of the N-terminal MBP tag by enterokinase cleavage, although the protein had been successfully expressed and purified (Tolunetal., 2007). The solubilization-promoting effect of each soluble tag is different, and for those insoluble proteins, multiple tags need to be tried. The thioredoxin tag was ineffective for CGRP-RCP expression, and the target protein remained insoluble.Nallamsetty and Waugh (2006) suggested that soluble tags (such as MBP or NusA) provide assistance in the folding of target proteins, and that the solubility of those target proteins that are prone to aggregation after removal of the tags is determined by the nature of the protein itself, not by the tags used. Commercial vectors containing the MBP tag have a variety of tag removal sites (NewEnglandBiolabs) that can be used to express target proteins in the cytoplasm and periplasm. Cross-linked amylose resin can be used to bind MBP-tagged proteins, and the bound fusion protein can be readily eluted with elution buffer containing 10 mm01/1-maltose. This allows MBP-tagged proteins to be purified in one simple step in a mild environment. However, the starch affinity purification method cannot be performed in the presence of reducing agents or in a denaturing environment. The amylopectin resin is degraded to some extent by amylases in the crude extracts, especially extracts from cells grown in LB-rich medium, and this effect is minimized by the addition of glucose (∼0.2%) to the medium. The starch resin can be regenerated and reused many times. Thioredoxin (thi 〇 red 〇 xin, TrX) is a heat-stable, 12 kDa-sized intracellular protein of Escherichia coli, which is easily overexpressed and remains soluble even when overexpressed to more than 40% of the total cellular proteins (LaVallieetal., 1993), and therefore can be used as a soluble tag in recombinant protein preparation to avoid inclusion body formation (LaVallieetaL, 2000). The experimental results of Dyson et al. (2004) showed that the Trx tag could work better when added to the N-terminus of the target protein. Thioredoxin accumulates on adhesion sites in the cytoplasmic membrane (BayeretaL,W87), which allows Tnc fusion proteins to be released by simple osmotic pressure or freeze-thaw treatments, which provides a simple initial purification. An additional affinity tag, such as a histidine tag, is usually added in addition to the Tnc tag for further purification. The iV-utilizingsubstanceAtranscriptionantiterminationfactor (NusA) is a large protein with 495 amino acid residues that is the most soluble of the E. coli proteins when expressed in fusion with exogenous proteins, according to a statisticalsolubilitymodel ( statisticalsolubilitymodel), NusA is the most soluble of the E. coli proteins when expressed in fusion with exogenous proteins, which is the reason for choosing it as a solubility tag (Davisetal., 1999; DeMarcoetaL, 2004). In some high-throughput screening tests, NusA worked similarly or even better than MBP as a soluble tag. However, different results can be expected for different proteins.NusA tags are often used in conjunction with other affinity tags, such as histidine tags. A number of relatively small solubilityenhancementtag (SET) or solubilityenhancementpeptide (SEP) tags have been developed that utilize highly acidic sequences to enhance the solubility of certain target proteins ( KatoetaL, 2007; ZhangetaL, 2004). Some other minor tags are the GBl tag (56 amino acid residues), which is based on the IgG-binding BI structural domain of streptococcal protein G (ChengandPatel, 2004; Zhouetal., 2001) and the IgG-binding structural domain of protein A (ZZ structural domain, 116 amino acid residues; Inouyeandand, 2001). amino acid residues; InouyeandSahara,2009;Rondahletal.,1992). These relatively small tags are particularly useful for the preparation of protein samples for nuclear magnetic resonance (NMR) studies (KatoetaL, 20 0 7). On the other hand, some progress has been made in the creation of NMR-invisible soluble tags using the proteinligationmethod (Durstetal., 2008; Kobashigawaetal., 2009).10 Studies have shown that ubiquitin-like modifiers (smallubiquitin-type modifiers) fused to the N-terminal end of the target proteins can be used in the preparation of protein samples for NMR studies (KatoetaL, 20 ○ 7). smallubiquitin-likemodifi?er, SUMO) proteins (approximately 11 kDa) fused to the N-terminus of target proteins can greatly improve the stability and solubility of target proteins (Marblestoneetal., 2006). After purification of the target protein, the tag can be removed with a SUMO protease (the catalytic domain of Ulpl) that recognizes the SUMO structure (Leeetal., 2008; Panavasetal., 2009).The SUMO fusion system has also been modified for use in insect cells and other eukaryotic expression systems (Liuetal., 2008). The Halo tag is a recently constructed modulartaggingsystem with a 34kDa modified haloalkanedehalogenase protein that binds a variety of synthetic ligands (HaloTag ligand; Promega Corporation). ). The components of these ligands include a constant reactive junction that covalently binds to the Halo tag and a variable reporter end that confers many useful properties to the fusion protein. Thus, a single tag can be used for subcellular structural imaging of living cells, cell labeling and sorting, affinity purification, and immobilization on solid-phase supports (Losetal., 2008). Affinity purification was performed. Because the Halo tag binds covalently to the junction in a highly specific and irreversible manner, even very low expression proteins are efficiently bound to the chloroalkanelinker. The target protein can be eluted using tobacco etch virus protease (TEV), which cleaves the cleavage site between the Halo tag and the target protein. Surprisingly, tests using a set of recombinant proteins that are difficult to express in E. coli showed that this compact monomeric Halo tag significantly increased the solubility of the fusion protein (OhanaetaL, 2009). In these experiments, the Halo tag performed considerably better than MBP, indeed showing the function of a soluble tag. Since protein tags may interfere with the normal function of the target protein, tag removal, after it has fulfilled its role of promoting solubilization or affinity purification, is useful for biological and functional studies, especially for large tags such as GST or MBP, although there are some examples of proteins fused to tags that are more susceptible to crystallization (Smythetal., 2003). Most commercial expression vectors used to add tags to target proteins also introduce cleavage sites with specific sequences, and tags can be removed using recombinant protein endonucleases. After the affinity purification of the fusion protein is complete, the sample can be treated with protein endonuclease to cut out the tag, and after another pass through the affinity column the tag is separated from the recombinant protein, and the target protein is obtained by collecting the flow-through solution. Recombinant protein endonucleases usually carry an affinity tag as well, which makes it easy to remove it after the cleavage reaction is complete. Table 16.3 lists some commonly used protein endonucleases. Because enterokinase and Factor Xa cut at the C-terminus of the recognition site, which removes the tag and the recognition site sequence in its entirety, they are useful for removing tags from the N-terminus. However, these two enzymes are sometimes not very specific, cutting at secondary cleavage sites located at other basic residues. Thrombin is another protease used for tag removal that has a similar secondary cleavage site (JennyetaL, 2003; LiewetaL, 2005). The advantage of using a protease such as thrombin for label removal, especially for large-scale protein preparation, is that it is inexpensive and more efficient than other highly specific enzymes. PreScission protease is a more specific protease with a longer, more rigorous recognition sequence. It consists of protease 3C (3Cpro) from humanrhinovirus-14 and a GS Ding tag, the presence of which makes the enzyme useful for removing the GST tag. Another very specific and popular protease is the tobacco etch virus protease (Kapustetal., 2001), which has a preference for glycine as the first amino acid after the cleavage site (Table 16.3), but can tolerate other amino acid residues with a slight reduction in cleavage activity. This allows the enzyme to remove the N-terminal tag in its entirety in many cases, leaving no excess residues on the target protein (KapustetaL, 2002). Large quantities of tobacco etch virus protease can be easily prepared on their own, and the enzyme usually carries a histidine tag for easy purification and removal after the cleavage reaction is complete (Tropeaetal., 2009). There are many types of tobacco etch virus proteases sold by commercial companies, some with different affinity tags and some with higher activity and stability. A variant of the tobacco etch virus protease is the tobacco vein mottle virus protease, which has a different recognition sequence (Nallamsettyetal., 2004) and is able to cleave a cleavage site located between two different tags (Fig. 16.1C). Exonucleases can also be used to remove the N-terminal tag, as in the TAGzyme system (Arnauetal., 2006a, available from Qiagen). This method uses dipeptide aminopeptidase I (DAPase) to progressively digest the N-terminal tag up to the termination point of a dipeptide. Various similar systems have been designed for processing different sequences (Arnauetal., 2006b; 2008). Complete removal of the C-terminal tag is difficult because most protein endonucleases cut only at the C-terminal end of the recognized sequence. If complete tag removal is necessary, more specific cleavage sites can be designed that are recognized on a structural basis (e.g. SUM? protease; Malakhovetal., 2004). Alternatively, autocatalyticproteinself-splicingelement is utilized (Inteins; SalehandPerler, 2006). Both cleavage systems are coupled with the affinity-purified tag-SUM? fusion system (Buttetal., 2005; Leeetal., 2008): this consists of a protein intron-chitin-bindingdomain (CBD; commercialized by NewEnglandBiolabs, Inc. as the IBD). (commercialized by NewEnglandBiolabs as the IMPACT system; Chongetal.,1997) or protein intron-polyhydroxybutyratebinding and similar protein purification systems (Gilliesetal...?2008). In some cases, labeling can also be removed chemically. Although the reagents used in chemical cleavage methods are inexpensive and very effective, these reactions require vigorous solvents and conditions that lead to denaturation, so they are usually used only for small peptides. Cyanogen bromide (CNBr) cleaves methionine and can be used when fusion proteins can be engineered to have only one methionine located between the tag and the target peptide (D6belietal., 1998; FairlieetaL, 2002). Another chemical cleavage reagent, hydroxylamine, is able to cleave the peptide bond between asparagine and glycine (Huetal.,2008) 〇 Practical considerations: Although designing specific cleavage sites between tags and target proteins is relatively simple, effective cleavage of tags does not always occur and is difficult to predict. Each expression construct should be tested experimentally for cleavage efficiency. When less specific enzymes are used, it is also important to test for cleavage that may occur at secondary cleavage sites. Often the amount of enzyme and incubation time need to be worked out to optimize it. Maximizing cutting efficiency is especially important for oligomeric proteins, where tags must be removed from each monomer for high yields of the target protein (Kenigetal., 2006). Sequences to be cleaved also need to be spatially accessible to the protease and relatively free of complex structures; introducing spacer sequences or junction sequences of several residues between the recognition sequence and the target protein, or placing sequences that are unlikely to form a secondary structure close to the cleavage site, can sometimes overcome the inefficiency of cleavage. For proteases without affinity tags, on-column cleavage (injecting the protease into an affinity column bound to the fusion protein) is more efficient than batchreaction. Many kinds of protein tags can be used to facilitate the expression and purification of recombinant proteins. However, even with such a large library of tags, the success rate of structural genomics protein preparation facilities in obtaining soluble pure proteins is less than 50% (StructuralGenomicsConsortiumetal.,2008). Despite the many good strategies devised by these large-scale research programs, there is no universal set of soluble or affinity tags that can be applied to all proteins, given the diversity of protein folds and their different biochemical properties. The choice of tags is still very much dependent on the protein to be expressed and the purpose for which the protein was prepared. Here, we have reviewed most of the effective protein expression tags and the issues associated with their use. For more product details, please visit Aladdin Scientific website.