Adobe Systems Adobe Systems 1 Protein expression and purification Radka Dopitová Kód předmětu: C8980 VII. Fusion proteins and affinity purification Fusion proteins (tagged proteins) Translation fusion of sequences coding a recombinant protein and a) short peptides [ex. (His)n, (Asp)n, (Arg)n ... ]. b) protein domains, entire proteins [ex. MBP, GST, thioredoxin …]. Engineering a tagged protein requires adding the DNA encoding the tag to either the 5’ or 3’ end of the gene encoding the protein of interest to generate a single, recombinant protein with a tag at the N- or C-terminus. The stretch of amino acids containing a target cleavage sequence (CS) is included to allow selective removal of the tag. Expression plasmids containing various tags are commercially available. Ø Increasing the yield of recombinant proteins – Fusion of the N-terminus of the target protein to the C-terminus of a highly expressed fusion partner results in high level expression of the target protein. Ø Enhancing the solubility of recombinant proteins – Fusion of the N-terminus of the target protein to the C-terminus of a soluble fusion partner often improves the solubility of the target protein. Ø Improving detection – Fusion of the target protein to either terminus of a short peptide (epitope tag) or protein which is recognized by an antibody (Western blot analysis) or by biophysical methods (e.g. GFP by fluorescence) facilitates the detection of the resulting protein during expression or purification. Ø Localization – A tag, usually located on the N-terminus of the target protein, which acts as an address for sending a protein to a specific cellular compartment. Ø Facilitating the purification of recombinant proteins – Simple purification schemes have been developed for proteins used at either terminus which bind specifically to affinity resins. Purposes of fusion tags No single tag is ideally suited for all of these purposes. Self splicing tags? Fusion partner (tag) Size Tag placement Uses His-tag 6, 8, or 10 aa N- or C-terminus Purification, detection Thioredoxin 109 aa (11.7 kDa) N- or C-terminus Purification, solubility enhancement Calmodulin-binding domain (CBD) 26 aa N- or C-terminus Purification Avidin/streptavidin Strep-tag 8 aa N- or C-terminus Purification, secretion Glutathione S-transferase (GST) 26 kDa N-terminus Purification, solubility enhancement Maltose binding protein (MBP) 396 aa (40 kDa) N- or C-terminus Purification, solubility enhancement Green fluorescent protein (GFP) 220 aa (27 kDa) N- or C-terminus Localization, detection, purification Poly-Arg 5-16 aa N- or C-terminus Purification, solubility enhancement N-utilization substance A (NusA) 495 aa (54.8 kDa) N-terminus Solubility enhancement Combinatorial tagging Combinations: Solubility-enhancing tag + purification tag: MBP + His6 tag 2x purification tag: IgG-binding domain + streptavidin-binding domain Localization tag + purification tag: GFP + His6 tag Localization tag + 2x purification tag + immunodetection: GFP + SBP domain + His8 tag + c-Myc Ø No single tag is ideally suited for all purposes. Therefore, combinatorial tagging might be the only way to harness the full potential of tags in a high-throughput setting. Loclization tag !!!!! Ø Proteins do not naturally lend themselves to high-throughput analysis because of their diverse physiological properties. Affinity tags have become indispensable tools for structural and functional proteomics. Ø Because affinity tags have the potential to interfere with structural and functional studies, provisions must also be made for removing them. Advantages and disadvantages of used fusion tags Waugh, 2005 x vysokokapacitni Otázka č. 1: Jaké jsou důvody pro využívaní tagů/kotev? Vyjmenujte 3. Ø Increasing the efficiency of translation initiation (e.g. GST, MBP, NusA…) - Advantage of N-terminal tags - Providing a reliable context for efficient translation initiation - Ribosome efficiently initiates translation at the N-terminal methionin of the tag - Deleterious secondary structures are more likely to occur in conjunction with short N-terminal tags because short RNA-RNA interactions tend to be more stable than long-range interactions. Ø Protection against proteolytic degradation - Several studies have shown that the nature of terminal residues in a protein can play a role in recognition and subsequent action by proteases and in some cases affinity tags might improve the yield of recombinant proteins by rendering them more resistant to intracellular proteolysis. Ø Helping to properly fold their partners leading to increased solubility of the target protein (in vivo and in vitro). Increasing the yield of recombinant proteins using fusion technology Yield enhancing tags are proteins and peptides which can be involved in: Solubility-enhancing tags - Advantage of N-terminal tags - Rather proteins (highly soluble proteins) than peptides -Fusion with a soluble fusion partner often helps to properly fold their fusion partners leading to improved solubility (in vivo and in vitro) of the target protein. -The choice of a fusion partner is still a trial-and-error experience. - Fusion partners do not perform equally with all target proteins, and each target protein can be differentially affected by several fusion tags (Esposito and Chatterjee, 2006) - Schematic representation of the pathway from protein expression to purification using solubility tags (Esposito and Chatterjee, 2006). Enhancing the solubility of recombinant proteins Ø PEPTIDES Poly-Arg Poly-Lys Adopted from Esposito and Chatterjee, 2006 ØPROTEINS Enhancing the solubility of recombinant proteins 19, 84, 215 – human proteins involved in cancer produced in E.coli Example of SDS PAGE with soluble (s) and insoluble (i) fractions following lysis. The results produced from the four different expression vectors (27: His tag only; 28: thioredoxin + His tag; 29: GST + His tag; 34: GB1 + His tag) are shown for three different target proteins (Hammarstrom et al., 2006). -The mechanism by which partners exert their solubilising function is not fully understood and possibly differs between fusion proteins Solubility-enhancing tags - the mechanism of action Maltose binding protein (MBP) has an intrinsic chaperone-like activity. MBP might bind reversibly to exposed hydrophobic regions of nascent target polypeptide, steering the polypeptides towards their native conformation by a chaperone like –mechanism. Examples of possible mechanisms N-utilization substance (NusA) decreased translation rates by mediating transtriptional pausing, that might enable critical folding events to occur. MBP and N-utilization substance (NusA) attract chaperones. The fusion tag drives its partner protein into a chaperone-mediated folding pathway. MBP and N-utilization substance (NusA) interact with GroEL in E. coli (Huang and Chuang, 1999). Small ubiquitin related modifier (SUMO) promotes the proper folding and solubility of its target proteins possibly by exerting chaperoning effects in a similar mechanism to the described for its structural homolog Ubiquitin (Ub; Khorasanizadeh et al., 1996). Negative charged tags (highly acidic peptide) inhibit aggregation by increasing electrostatic repulsion between nascent polypepdides (Zhang et. 2004) . Steer smerovat, mirit, vest Doplnit-Tom Brom Thioredoxin ØIts intrinsic oxido-reductase activity responsible for the reduction of disulfide bonds through thio-disulfide exchange ØThioredoxin serves as a covalently joined molecular chaperone independently of redox activity. Thioredoxin may, thus, act to prevent the aggregation and precipitation of fused nascent proteins, giving them an extended opportunity to adopt their correct tertiary folds. Solubility-enhancing tag – mechanism of action Holmgren, 1995; Berndt et al. 2006 Proposed mechanism of thioredoxin-catalyzed protein disulfide reduction. Reduced thioredoxin [Trx-(SH)2] binds to a target protein via its hydrophobic surface area. Nucleophilic attack by the thiolate of Cys32 results in formation of a transient mixed disulfide, which is followed by nucleophilic attack of the deprotonated Cys35 generating Trx-S2 and the reduced protein. Conformation changes in thioredoxin and the target protein occur during the reaction. Thioredoxin fusions have proved to be especially useful in avoiding inclusion body formation, particularly for the production of small, normally secreted, mammalian cytokines in an active form in the E. coli cytoplasm. E. coli thioredoxin is a compact, highly soluble, and thermally stable protein with robust folding characteristics. These properties perhaps allow the molecule, when fused to a protein of interest, to serve as a covalently joined molecular chaperon. Thioredoxin may, thus, act to prevent the aggregation and precipitation of fused nascent proteins, giving them an extended opportunity to adopt their correct tertiary folds. But Trxs and Grxs do not necessarily act as disulfide reductases, under certain conditions these redoxins promote disulfide bond formation and synergistically work with PDI and/or chaperones. Besides their disulfide formation and isomerization activity,thioredoxins and related oxidoreductases have been shown to promote the folding of proteins independent of their redox activity both by directly promoting protein folding and by enhancing the refolding activity of other molecular chaperones. The solubilization factor is defined as the molar ratio between the solubility of tagged BPTI-22 variants and that of the reference BPT-22 molecule. Lys tag Arg tag N- C- N- C- terminus Short peptide tags Poly-Lys tag, poly-Arg tag = one, three and five lysine or arginine residues fused to the C- or N-terminus of the target protein The solubilization effect of poly-Lys tags is lower than that of poly-Arg tags (lysines are less hydrophilic than arginines). In vitro solubility-enhancing tags Arginine (R) Lysine (K) Solubility as defined here is the maximum protein concentration of the supernatant after centrifugation of the supersaturated protein sample (in vitro solubility). BPTI-22 = bovine pancreatic trypsin inhibitor variant containing 22 alanines Kato et al., 2006 Biochemical properties of poly-Arg and poly- Lys tagged BPTI-22 protein The addition of 0.5 M Arg barely increased its solubility, and trypsin activity was inhibited by the high arginine concentration. On the other hand, addition of 50 mM Arg+Glu was more effective and increased protein solubility more than threefold. Kato et al., 2006 barely- bidne Ø The solubilization factor of all C-terminal tags was slightly higher than that of the respective N-terminal tags. Ø The C-terminus of BPTI-22 is close to a large hydrophobic patch, whereas the N-terminus is located on the opposite side of the molecule, away from the hydrophobic patch. Kato et al, 2006 Ø Charged residues seem to act through repulsive electrostatic interaction and thus hamper intermolecular interaction arising from the hydrophobic cluster. Solubility-enhancing tags – comparison of peptide and protein tags, conclusions Ø Protein tags are inherently large and need to be correctly folded in order to enhance solubility. Ø Protein tags are often natural affinity tags. Ø Peptide tags are small, and, importantly, they do not need to be folded, which provides a significant advantage over protein tags. Ø The use of small tags (< 30 amino acids long) does not increase protein size substantially and reduces steric hindrance, which simplifies downstream structural and functional applications without the need to remove the tag. Ø The solubilization enhancement effect depends on the size of the target protein. Solubility enhancement of fusion partners such as thioredoxin, GB1 is less pronounced for larger target proteins (above 25 kDa). MANY TAGS SUFFER FROM THE SAME PROBLEM – THEY DO NOT FUNCTION EQUALLY WELL WITH ALL TARGET PROTEINS. Otázka 2: Který tag/kotvu by jste využily pro zvýšení rozpustnosti proteinu bohatého na cysteiny? Removal of fusion tags- the Achilles' heel of the fusion approach All tags, whether small or large, have the potential to interfere with the biological activity of a protein, impede its crystallization (presumably due to the conformational heterogeneity allowed by the flexible linker region), be too large for NMR analysis, cause a therapeutic protein to become immunogenic or otherwise influence the target protein`s behavior. The fusion tags can be removed by: Ø Chemical cleavage Ø Self - cleavage Ø Enzymatic cleavage Ø Rarely used. Cyanogen bromide Met/X Hydroxylamine Asn-Gly Removal of fusion tags – chemical cleavage Amino – acid sequence of the P. falciparum C-terminal segment of CSP (PfCSP C-ter) fused to a purification tag (Rais-Beghdadi et al., 1998). M28V M105V M12 M15 Chemical cleavage is a harsh method, efficient, but rather non-specific and may lead to unnecesary denaturation or modification of the target protein. Bromkyanid nesetrna Removal of fusion tags - self - cleaving Ø Use of self-cleaving fusion tags Inteins (intervening proteins) are protein segments that can excise themselves from protein precursors in which the are inserted and rejoin the flanking regions. 1. Inteins Perler, (2005) Ø Self - splicing inteins can be mutated at the N- or C- terminal splice junction to yield self cleaving inteins, which can be used to mediate self cleaving of various tags. Výsledek obrázku pro inteins intein tag difference Stepeni vazby pomoci inteinu vyvolane thiolem (DTT, merkaptak).. Thioesterova vazba mezi cilovym proteinem a N koncovym cysteinem inteinu muze byt vytvorena pomoci posunu acylove skupiny z N na S . Thiol katalyzuje transesterifikacni reakci, jejiz vysledkem je uvolneny cilovy protein. Nove vytvoreny thioester mezi thiolem a C koncovou AK ciloveho prteinu není stabilni a hydrolyzuje za mirnych podminek a vznika volny C konec ciloveho proteinu. Intervening – lezici mezi Ø Two categories of inteins: - inteins with pH-induced C-terminal cleaving activity - inteins with thiol-induced N- and C-terminal cleaving activity 1. Inteins Trans-esterification reaction – formation of thioester between thiol and C- term amino acid of target protein. This product is not stable and easily hydrolyze to release target protein and tag. Stepeni vazby pomoci inteinu vyvolane thiolem (DTT, merkaptak).. Thioesterova vazba mezi cilovym proteinem a N koncovym cysteinem inteinu muze byt vytvorena pomoci posunu acylove skupiny z N na S . Thiol katalyzuje transesterifikacni reakci, jejiz vysledkem je uvolneny cilovy protein. Nove vytvoreny thioester mezi thiolem a C koncovou AK ciloveho prteinu není stabilni a hydrolyzuje za mirnych podminek a vznika volny C konec ciloveho proteinu. Intervening – lezici mezi Removal of fusion tags - self – cleaving fusion tag System based on the catalytic domain of Staphylococcus aureus sortase A (SrtA). SrtA cleaves the Thr-Gly bond at the conserved LPXTG motif in the substrates. Cleavage is inducible by adding calcium (cofactor of SrtA). (Li, 2011) N-terminal protease (Npro) is the first protein of the pestivirus polyprotein. It posesses autoproteolytic activity and catalyzes the cleavage by switching from chaotropic to cosmotropic conditions. 2. 3. 4. 5. FrpC modul (from G+ bacteria Neisseria meningitides): FrpC protein undergoes calcium – inducible autocatalytic proccesing at the peptide bond between residues Asp and Pro. Cleavage reaction is catalyzed by a self proccessing modul (SPM). Vibrio cholerae secretes a large multifunctional autoprocessing repeats-in-toxin (MARTX) toxin that undergoes proteolytic cleavage during translocation into host cells. Proteolysis of the toxin is mediated by a conserved internal cystein protease domain (CPD), which is activated upon binding of inositol hexakisphosphate. chaotropic agent is a molecule in water solution that can disrupt the hydrogen bonding network between water molecules (i.e. exerts chaotropic activity). This has an effect on the stability of the native state of other molecules in the solution, mainly macromolecules(proteins, nucleic acids) by weakening the hydrophobic effect. For example, a chaotropic agent reduces the amount of order in the structure of a protein formed by water molecules, both in the bulk and the hydration shells around hydrophobic amino acids, and may cause its denaturation. Conversely, an antichaotropic agent (kosmotropic) is a molecule in an aqueous solution that will increase the hydrophobic effects within the solution.^[^citation needed^] Antichaotropic salts like ammonium sulphate can be used to precipitate substances from the impure mixture. This is used in protein purification processes, to remove undesired proteins from solution Removal of fusion tags - self – cleaving fusion tag Ø Uncontrolled in vivo cleavage or in complete in vitro cleavage Ø Target protein modification – pH or thiols can modify the target protein Ø Protein compatibility with cleaving conditions – pH induced inteins Ø Compared to the traditional protease based method, the intein-based approach requires fewer steps and lower costs. Inteins (1) Other system (2-5) Ø Tested on limited number of cases Li, 2011 Removal of fusion tags – enzymatic cleavage Site-specific proteolytic cleavage: Ø Exopeptidases Ø Endopeptidases Exopeptidases (aminopeptidases and carboxypeptidases): ØAPM, CPA and CPB release sequentially a single amino-acid from the N- or C- terminus of a protein until the stop site is reached. TAGZyme system (Qiagen): Ø DAPase (dipeptidyl aminopeptidase I) TAGZyme stop points Arnau et al., 2006 Removal of fusion tags - enzymatic cleavage Endopeptidases Ø The enzymatic cleavage site has to be placed between the fusion tag and the target protein. Enterokinase Asp-Asp-Asp-Asp-Lys/X Removal of fusion tags - enzymatic cleavage The purpose of this review was to demostrate that both thrombin and factor Xa can hydrolyze a variety of peptide bonds within the fused proteins of interest. Accuracy of cleavage has to be precisely verified! 2000 4000 6000 8000 10000 12000 14000 16000 18000 20000 22000 m/z 0 200 400 600 800 1000 a.i. AHP2_standard AHP2_control AHP2_enterokinase Enterokinase cleavage site 3,4 kDa 18,9 kDa N´MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDPSSRSAAGTMEFMDALIA….…………….GIVPQVDIN C´ Theoretically: pRSETB::AHP2 Intact mass spectrometry analysis SUMO gene fusion system SUMO protease recognizes the tertiary structure of SUMO rather than an amino acid sequence. As a result, SUMO protease never cleaves within the fused protein of interest! SUMO protease recognizes the tertiary sequence of SUMO. As a result, SUMO protease never cleaves within the fused protein of interest. SUMO protease recognizes the tertiary sequence of SUMO. As a result, SUMO protease never cleaves within the fused protein of interest. SUMO protease recognizes the tertiary sequence of SUMO. As a result, SUMO protease never cleaves within the fused protein of interest. SUMO protease recognizes the tertiary sequence of SUMO. As a result, SUMO protease never cleaves within the fused protein of interest. ØOptimization of protein cleavage conditions (mainly enzyme-to-substrate ratio, temperature, pH, salt concentration, length of exposure). ØCleavage efficiency (Optimization is needed. The efficiency varies with each fusion protein in an unpredictable manner, probably due to aggregation or steric issues; the problem can be solved by introducing short linkers between the protease site and the fusion tag). ØUnspecific cleavage (SOLUTION: optimization of protein cleavage conditions or using re-engineered proteases with increased specificity such as ProTEV and AcTEV proteases). ØPrecipitation of the target protein when the fusion partner is removed (so-called soluble aggregates; SOLUTION: another approach for protein solubilization has to be found). ØFailure to recover active or structurally intact protein ØTarget protein modification (some proteases like thrombin, TEV, Precision leave one or two amino-acids on the target protein near the cleavage site). ØRe-purification step is needed to separate the protease from target protein. ØHigh cost of proteases Ø The alternative is to leave the tag in place for structural analysis: The small tags are a better choice in structural and functional analysis of proteins. Removal of fusion tags - enzymatic cleavage Otázka 3: Jaký je rozdíl mezi inteinem a samo-vyštěpujícím tagem odvozeným od inteinu? Affinity chromatography (AC) ØA type of adsorption chromatography, in which the molecule to be purified is specifically and reversibly adsorbed to a complementary binding substance (ligand, L) immobilized on an insoluble support (matrix, M). Ø AC has a concentrating effect, the high selectivity of separations derived from the natural specificities of the interacting molecules. Ø AC can be used (1) to purify substances from complex biological mixtures, (2) to separate native forms from denatured forms of the same substance, and (3) to remove small amounts of biological material from large amounts of contaminating substances, (4) and to isolate protein complexes from the native source. Ø the first application was in 1910 (adsorption of amylase onto insoluble starch) but it developed during the 1960s and 1970s. Affinity tags and affinity purification A tag is fused to the N- or C-terminus of the protein of interest to facilitate purification, which relies on a specific interaction between the affinity tag and its immobilized binding partner. Genetically engineered fusion tags allow the purification of virtually any protein without any prior knowledge of its biochemical properties. Purification tags Affinity tags Non - chromatographic tags Tag Matrix Ø Traditional purification tags Ø The tag binds strongly and selectively to an immobilized ligand on a solid support. Ø After optimization one could achieve > 90% purity. Ø These tags can eliminate affinity resin. Proteins are isolated by other non-chromatographic methods (centrifugation, filtration) Ø typically combined with self-cleaving tags Ø 75 % - 95 % purity Purification tags Non - chromatographic tags The PHB system (c): Ø PHB (polyhydroxybutarate): subclass of biodegradable polymers produced in various organisms, use as storing excess carbon. Ø The system includes in vivo production of PHB small granules (from the plasmid carrying PHB-synthesis genes). Ø Target protein in fusion to self cleaving phasin tag. Ø Tagged protein binds to the PHB particles via phasin tag, which allows the granules and the tagged protein to be co-purified via centrifugation. Ø DTT induced cleaving activity of intein and thus elution of the target protein. The ELP system (d): Ø ELP (elastin-like polypeptide) selectively and reversibly precipitates in response to changes in temperature and buffer salts. This allows soluble and insoluble contaminants to be removed by filtration or centrifugation. Components of a matrix for affinity chromatography A ligand Ø The dissociation constant (Kd) for the ligand - target complex should ideally be in the range 10-4 to 10-8 M in free solution to allow efficient elution under conditions which will maintain protein stability. Ø A ligand has to be attached to the matrix with a suitable chemically reactive group. The mode of attachment must not compromise the reversible interaction between the ligand and protein. Ni2+ NTA sepharose 36 Components of a matrix for affinity chromatography One of the most common methods for immobilizing ligands involves cyanogen bromide activation of agarose to produce imidocarbonate derivatives, which react with amino groups to generate isourea linkages. A matrix Ø Typically, a macroporous polysaccharide bead such as agarose, that provides a porous structure so that there is an increased surface area to which the target molecule can bind. Ø A matrix has a suitable attachment site for the ligand. Typically matrices are chemically activated to permit the coupling of the ligand. A number of activation methods are available which depend on the nature of the matrix and the availability of compatible reactive groups on the ligand. 37 Ø A spacer arm will be required in cases where direct coupling of the ligand to the matrix results in steric hindrance and subsequently the target protein will fail to bind to the immobilized ligand efficiently. The introduction of a spacer arm between the ligand and the matrix minimizes this steric effect and promotes optimal adsorption of the target protein to the immobilized ligand. Components of a matrix for affinity chromatography Ni2+ NTA sepharose Spacer arm Ø In the equilibration phase, buffer conditions are optimized to ensure that the target molecules interact effectively with the ligand and are retained by the affinity medium as all other molecules wash through the column. Ø During the washing step, buffer conditions are created that wash unbound substances from the column without eluting the target molecules or that re-equilibrate the column back to the starting conditions (in most cases the binding buffer is used as a wash buffer). Ø In the elution step, buffer conditions are changed to reverse (weaken) the interaction between the target molecules and the ligand so that the target molecules can be eluted from the column. Typical affinity purification steps Affinity chromatography - Immobilized metal ion affinity chromatography (IMAC) Ø The most common purification tag is typically composed of six consecutive histidine residues. Ø Histidine, cysteine, and tryptophan residues are known to interact specifically with divalent transient metal ions such as Ni2+, Cu2+, Co2+, and Zn2+. Ø Histidine is the amino acid that exhibits the strongest interaction with immobilized metal ion matrices as the electron donor groups on the histidine imidazole ring readily form coordination bonds with an immobilized transition metal. Binding strength of His tag to metal ions: Cu2+ > Ni2+ > Zn2+ ~ Co2+ Ø IMAC can be used under native and/or denatured conditions. Ø A highly purified protein can often be obtained in one or, at most, two purification steps. Zn2+ Ni2+ Co2+ Cu2+ (Zouhar et al., 1999) •Interakce proteinu s matricí je zprostředkovaná neobsazenými d-orbitaly iontů přechodných kovů, které vážou volné elektronové páry převážně z dusíkového atomu imidazolových skupin histidinových zbytků v proteinu. at Ø Optimal binding of recombinant protein with metal ion is achieved at pH 7–8. Ø Buffers with a high salt concentration (0.5–1 M NaCl) reduce nonspecific electrostatic interaction. Ø Nonionic detergents or glycerol reduce nonspecific hydrophobic interactions. Ø Elution of contaminating proteins can be achieved by lowering the pH or using low concentrations of imidazole. Ø Elution of tagged protein is achieved at high imidazole concentrations (0–0.5 M), by strongly decreasing the pH, or by using EDTA. His-tagged protein and IMAC under native conditions One-step purification of maize -glucosidase Ø Perfusion matrix: POROS MC/M Ø Functional group: iminodiacetate, metal ion Zn2+ Ø Removing contaminated proteins: linear gradient of imidazole (0–50 mM) and pH (pH 7-6.1) Ø Protein elution: 0.1 M EDTA Ø 80% recovery, 95 fold purification Ø Common production and isolation of the wild type protein and soluble mutant form for enzymatic measurements and crystallization. His-tagged protein and IMAC under native conditions (Zouhar et al., 1999) His-tagged protein and IMAC under denatured conditions – Purification of proteins expressed in inclusion bodies. – Purification in a high concentration of urea or guanidine chloride. – Result is a pure protein, but in a denatured form (sufficient for immunization). Recovery of native conformers (necessary for functional and structural analysis): Ø Binding to the column under strong denaturing conditions (8 M urea) Ø Two possibilities of renaturation: 1. The protein is eluted from the column and renatured by dialysis or rapid dilution in renaturing buffers. 2. Renaturation of the protein bounded to the column (matrix assisted refolding procedure): gradient from denatured to renatured buffers or pulsion renaturation (8-0M urea). Identification of properly refolded (His)6Zm-p60.1 (maize -glucosidase) using 10% native PAGE, followed by activity in gel staining: A = crude protein extract prepared from maize seedlings containing the native enzyme B = (His)6Zm-p60.1, renatured product (matrix assisted refolding procedure – 23 renaturing cycles) C = (His)6Zm-p60.1 purified by native IMAC KM (His)6Zm-p60.1 purified by native IMAC: 0.64 ± 0.06 mM KM (His)6Zm-p60.1 renatured product: 0.6 ± 0.08 mM Determination of vmax and kcat was hampered by the fact that the refolding process yielded a number of improperly folded polypeptides. (Zouhar et al., 1999) 1st step - metal chelate affinity chromatography 2nd step - gel filtration Purity: 96% Concentration of the protein: 22 mg/ml After ultrafiltration His-tagged protein and IMAC under native conditions Two-step purification of Arabidopsis histidine phosphotransfer protein 5 Ø IMAC matrix: highly cross-linked spherical agarose Ø Functional group: nitrilotriacetic acid, metal ion Ni2+ Ø Removing contaminated proteins: linear gradient of imidazole (20–500 mM) Ø Protein elution: 130 mM imidazol Ø Common production and isolation of the wild type protein for protein-protein interaction measurements and crystallization. Crystallization (His)6AHP5 3. Affinity purification after TEV cleavege CKI1RD pETM-60::CKI1RD pETM-60 200 mM imidazole 20 mM imidaz. 10 CV pETM-60 CKI1RD pETM-60::CKI1RD after cleavege 4. Size-exclusion chromatography 1 L→ ~10-20 mg for TB and M9 His-tagged protein and IMAC under native conditions Four-step purification of Arabidopsis CKI1RD 1.Affinity purification (IMAC) 2.Tag removal (TEV protease) 3.Affinity purification (IMAC) 4.Size exclusion chromatography Pekárová B. Ub-SGSG-HisTag-SA-TEV-AME-CKI1 Otázka č.4: Jakými metodami se izolují proteiny fúzované s nechromatografickými tagy/kotvami? Affinity purification for studying protein-protein interaction Ø Co-immunoprecipitation Ø GST (or His) pull-down Ø Tandem affinity purification Ø Affinity purification provides a high-efficiency method for isolation of interacting proteins and protein complexes: Ø Testing known protein-protein interaction. Ø Identification of novel protein-protein interactions. Ø The principle: If protein X is immunoprecipitated with an antibody of X, then protein Y, which is stably associated with X in vivo, may also be precipitated. This precipitation of protein Y, based on a physical interaction with X, is referred to as co-immunoprecipitation. Ø An obvious advantage is that complexes are isolated in the state closest to the physiological condition. Ø When a good quality antibody of X is available, Co-IP is a fast method and there is no need to clone and express the component(s) of the complex. Co-immunoprecipitation (Co-IP) 1.Cell lysis under mild conditions that do not disrupt protein-protein interactions (using low salt concentrations, non-ionic detergents, protease inhibitors, phosphatase inhibitors). 2.The protein of interest (X) is specifically immunoprecipitated from the cell extracts (using an antibody specific to the protein of interest or to its fusion tag). 3.The antibody-protein(s) complex is then pelleted usually using protein-A or G sepharose, which binds most antibodies . 4.Eluted immunoprecipitates are then fractionated by SDS-PAGE. 5.A protein of known identity is most commonly detected by performing a western blot .Identification of novel interaction is carried out by mass spectrometry analysis. Pull-down assay Ø Pull-down assays are a common variation of co-immunoprecipitation and are used in the same way, but pull down does not involve using an antibody specific to the target protein being studied. Ø They are used for purification of multiprotein complexes in vitro. Ø The target protein is expressed in E. coli as GST fusion and immobilized on glutathione-sepharose beads (GST alone is often used as a control). Ø Cellular lysate is applied to the beads or column, and the target protein competes with the endogenous protein for interacting proteins, forming complexes in vitro. Ø Centrifugation is used to collect the GST fusion probe protein and adhering proteins. Ø The complexes are washed to remove nonspecifically adhering proteins. Ø Free glutathione is used to elute the complexes from the beads, or alternatively the beads with attached complexes are boiled directly in an SDS-PAGE sample buffer. Ø The proteins are resolved on SDS-PAGE and processed for further analysis. Tandem affinity purification (TAP) TAP tag: a double affinity tag (highly specific) which is fused to a protein of interest as an efficient tool for purification of native protein complexes. Examples of TAP (tandem affinity peptides) tags Two-step purification strategy in order to achieve higher purity of isolated multiprotein complexes under near physiological conditions. This method was originally developed for use in yeast and quickly adapted to higher eukaryotes such as insect cells, human cells and plant cells. Colins and Choudhary, 2008 Tandem affinity purification vLeene et al., 2007 (Chepelev et al. 2008) Ø An affinity tags can influence protein-protein interactions (testing N- and C-terminal fusions). Ø Loss of weak or transient protein-protein interactions. Ø Non-specificity: controls, affinity tags with higher specificity Ø Verification of newly identified interactors by other methods and biologically relevant mutants. Affinity purification for studying protein-protein interaction Comparison of a standard purification process with affinity purification Ø Generally, the yield and efficiency of any specific purification procedure depends on the level of optimization developed for individual proteins and the method. It is therefore recommended to use the data presented in different comparisons as indicative rather than definitive, which it is not e.g., identical elution conditions are optimal for different proteins. Ø Standard chromatographic methods include several steps to obtain a relative pure protein. This results in a time-consuming procedure and a relatively low yield of recovery (typically 50 % of the starting material for optimized processes). Ø The yields obtained in purification of proteins using affinity chromatography can be over 90 % and include a reduced number of steps. Arnau et al., 2006