dodododododod ododododododo □ odododododod ododododododo dodododododod ododododododo dodododododod JNVAViaaZA 3fOAZOU Oa 3DI1S3ANI ododododododo dodododododod ^.ö-VMw »soudoqoseauejny|uo>iaid aaohoaactbi v azaaviAi M ^^k^^ 3IND V>iSdOdA3 /flSh\ ľWWAdO \IA±S10»S OA1SH31SINIW | RM s flAII % "TL i a i ■ dodododododod ododododododo dodododododod ododododododo dodododododod ododododododo dodododododod ?jfy£% juBABiapzAdO JAlSnO>IS OA1SH31 Á>|!|qnd9J 9>|S93 wsjDodzoj luju;b;s b wapuoj luju|BDOs iuÁ>|sdojA3 uBAODUBu^n|ods si :pp[ojd 0}U9j_ n n n n n o BAOJldOQ B^pF^ pWS BAOJB5[3J B5[UBjg 'BpiTBf JTUIOqtlf|° °0 uo|iiíoijLincl o d o d o d o d o d o d o d o d o d o d o d o d o d o d ododododododo VlIZÜBAINn VAO>IAWSVW 086819 :rH90ip9Jd po» ododododododo dodododododod ododon^nododo < □ o ^^/s-^jsSé'o □ ododol.^.jOdodo dodododododod ododododododo dodododododod ododododododo Fusion proteins (tagged proteins) Translation fusion of sequences coding a recombinant protein and a) short peptide [ex. (His)n, (Asp)n, (Arg)n ... ] b) oligopeptide [ex. MBP, GST, thioredoxin ...] tu 5' Promoter Tag CS Gene of interest 3' Terminator Transcribe and translate _I_ 11 I c Tag fused to the N-terminus of the protein of interest 5' Promoter Gene of interest CS Tag 3' Terminator 1 Transcribe and translate n I c Tag fused to the C-terminus of the protein of interest Engineering a tagged protein requires adding DNA encoding the tag to either the 5' or 3' end of the gene encoding the protein of interest to generate 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. Uses of fusion tags Mncreasing 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. > 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. > 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. ^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. ^Facilitating the purification of recombinant proteins - Simple purification schemes have been developed for proteins used at either terminus which bind specifically to affinity resins. 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 Advantages and disadvantages of used fusion partners 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. No single tag is ideally suited for all purposes. Therefore, combinatorial tagging might be the only way to harness the full potential of affinity tags in a high-throughput setting. Because affinity tags have potential to interfere with structural and functional studies, provisions must also be made for removing them. Tag^ GST IV 3 P Kus A Thioredoxin Ubiujuitin FLAG BAP Advantages Disadvantages Efficient translation initiati on Inexpensive affinity resin Mild elution conditions Efficient translation initiati on Inexpensive affinity resin Enhances solubility Mild elution conditions Efficient translation initiati on Enhances solubility Not an affinity tag Efficient translation initiati on Enhances solubility Efficient translation initiati on Might enhance solubility Low metabolic burden High specificity Low metabolic burden Mild elution conditions Provides convenient means of immobilizing proteins in a directed orientation High metabolic burden Homodimeric protein Does not enhance solu bil ity High metabolic burden High metabolic burden Not an affinity tag Not an affinity tag Expensive affinity resin Harsh elution conditions Expensive affinity resin Variable efficiency of enzymatic biotinylation Co-purification of E. coti biotin esrboxyl carrier protein on affinity resin Does not enhance solubility Hise STREP SET CBP S-tag Low metabolic burden Inexpensive affinity resin Mild elution conditions Tag works under both native and denaturing conditions Low metabolic burden High specificity Mild elution conditions Enhances solubility Low metabolic burden High specificity Mild elution conditions Low metabolic burden High Specificity Specificity of IMAC is not as high as other affinity methods Does not enhance solubility Expensive affinity resin Does not enhance solubility Not an affinity tag Expensive affinity resin Dd6s not enhance solubility Expensive affinity resin Harsh elution conditions (oron-column cleavage) Does not enhance solubility JGST, glutathione S-transferase: MBP, maltose binding protein: NusA, N utilization substancí; A; FLAG, FLAG tag peptide: BAP, biotin acceptor peptide: HÍSjj, hexahistidine tag: STREP, streptavidin-binding peptide: SET, solubility enhancing tag: CBP, calmodulin binding peptide. derivatives of thioredoxin have been engineered to have affinity for immobilized metal ions (Hispatch thioredoxin) or avidi n/streptavidin [361. Combinatorial tagging The aim is to get the maximum possible benefit from affinity tags. 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 Increasing the yield of recombinant proteins using fusion protein technology Yield enhancing tags are proteins and peptides which can be involved in: > increasing the efficiency of translation initiation, > protection against proteolytic degradation, and > 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 > 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 initiate translation at the N-terminal methionine 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. > Protection against proteolytic degradation - Several studies have shown that the nature of terminal residues in 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. Solubility-enhancing tags - Are generally proteins or peptides that enhance solubility and even promote the proper folding of the target proteins. PROTEINS GST (glutathione S-transferase), MBP (maltose binding protein) - Also act as affinity tags for protein purification NusA (N-utilization substance A), TRX (thioredoxin) - Require additional affinity tags for use in protein purification PEPTIDES Poly-Arg (also acts as affinity tag for protein purification) Poly-Lys Generate parallel expression clones Dead end: insolubility His6 GST Target protein His6 NusA Target protein (a) $ Express in E. coli N y His6 GST Target protein His6 MBP His6 Trx Target protein Target protein Target protein Protease cleavage site His6(^MBP His6 ( Trx Target protein Target protein His6 MBP (e) Target protein IMAC FT SUCCESS Target protein Good cleavage His6 Trx (b) Purify by IMAC Cleave with protease Poor cleavage Target protein (d) Target protein (c) His6 (NusA Target protein His6 (^JusA) Target protein Dead end: protein insoluble after cleavage of tag Dead end: difficult to separate cleaved protein from fusion Current Opinion in Biotechnology Schematic representation of the pathway from protein expression to purification using solubility tags. The mechanism by which partners exert their solubilising function is not clear (they might act through a chaperone-like mechanism) and possibly differs between fusion proteins. Solubility-enhancing tag - Thioredoxin - Serves as a general protein disulfide oxidoreductase. - Is present in all species from Archaebacteria to humans. Folding of thioredoxin. The redox-active disulfide in the active site (Cys32-Cys35) is located on a protrusion between the strand p2 and the helix a2 . Only the sulphur of Cys32 is exposed to the solvent. 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. Trx-(SH)2 reduces insulin disulfides at pH 7 with a rate constant of 105 M-1 s-1, which is about five orders of magnitude faster than insulin reduction by dithiothreitol (DTT), a well-known dithiol reductant. Solubility-enhancing tag - Thioredoxin SUMMARY > The active-site surface in thioredoxin is designed to fit many proteins. Thioredoxin thus uses a chaperone-like mechanism of conformational changes to bind a diverse group of proteins and fast thiol-disulfide exchange chemistry in a hydrophobic environment to promote high rates of disulfide reduction. Example of SDS PAGE gels with soluble (s) and insoluble (i) fraction following lysis. The results when 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., 2005). > Increasing the solubility of the target proteins by overproduction of thioredoxin strongly suggests that the redox state affects the solubility of target proteins. 19 84 215 27 28 29 34 27 28 29 34 27 28 29 34 sisisisi t Is Is I i I s I s i s i s i K - if - - -____ 19, 84, 215 - human proteins involved in cancer Solubility-enhancing tags - 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 Solubility as defined here is the maximum protein concentration of the supernatant after centrifugation of the supersaturated protein sample. Nhfe ^2 NH Cht 1 Cht Cht 1 Cht Cht 1 Cht Cht HjN+—C1 —CCfc- HjN+—C—CCfc-1 H 1 H Arginine (R) Lysine (K) —>->->-> 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. BPTI-22 = bovine pancreatic trypsin inhibitor variant containing 22 alanines The solubilization effect of poly-Lys tags is lower than that of poly-Arg tags (lysines are less hydrophilic than arginines). N- C- N- C-terminus Charged residues seem to act through repulsive electrostatic interaction and thus hamper intermolecular interaction arising from the hydrophobic cluster. Kato et al., 2006 Biochemical properties of poly-Arg and poly- Lys tagged BPTI-22 protein Prot-e in Solubility Protein Cone |mAÍ| (Cone. I mg/ml I )J Solubilization Factor35 ReL Trypan Inhibitory Activity (%f IJTTl JJ L7C1 tio.nuj 33.4 \ i \: N3K i. Ik i ./k Ok 1.70 (10.40) 2.66 537 (35.60) 1.79 ((0,93) 141 (15.2ÍÍ) 7.16 (47.4?) 1.00(1.04) 136(2.00) 3.16(336) 1.05(1.10) 1.42(1.53) 4.21 (4.75) 35.2 34.4 34.3 34.6 36.2 35.0 1.05 1.04 1.05 1.05 1.05 1.02 -zrn— -N3R ■ NSR CAR ■ C3R ■ C5R ■ C6R JUH'J 21" BPTI-22f l.*y i 10.3 J i 2.70 (17:23) 6.20 (41.J 1) LSI (11.07) 3.02 (19.26) «323 (54.56) 10.59 (73.41) 5.63(33.11) 2.01 0 1.32) 139(1.72) 3.65(4.11) 1.06(1.11) ].7H(L93) 4.84(5.46) 6.22 (734) 3.31(3.31) 1.18(1.lít) "KT 35.6 353 35.0 34.4 34.a 32.7 TT" TöT 0.99 0.99 1.05 1.05 LOK 1.1 TÖT NA£ I 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 over threefold. Prolan solubility was determined as the maximum supernatant concento tio a nt a siipersat lit alt J protein solution M 4~C In 10(1 mM Acetate b j (far pll 4.7. * iVtaJiiminn concentrations calculated in milligrams per null r I iter are inilkated in parentliesis. The Mw of BPTI-22, -NIK and -CiK, -N3K and -C3K, -NSKand -C5K, -NIR and -OR, -N3R and -C3R.-N5R and -OR, and -C6R arc, respectively: 5880,6123,6579,6636,6151, 6463,6776, and6932 Da. Calculated aii the ratio bet weert the molar protein solubility of BPTI-22 and [It at uf LaLjried BPTI-22. Values En parent tits lh indicate thi.- ratio .= !_■. i "■. -. . in milligrams per milliliters. 11 Relative trypsin inhibitory activity calculated is the ratio between the activity o( BPTI-22 and that ot Iaj"r^ed EPTI-22. BPTI-22, which Lacks R39, an areinine residue invoked in two hydrogen hooding interactitrcis with the trypsin residue backbone, ** has a reduced trypsin inhibitor activity corresponding to ■~6Cftitot the wt-BPTIand BPT1-:S,55] at stoic hiinnetry and a protein concentration of 280nAt™ * Solubility in the .same buffer as alfovebut with the addition of 50 mM L-Arg. 1- L-Glu. "The CD thermal meltirtjj curve could not KL il el er mi r ;e d ice to the strong absorption of arginine and glutamic acid. f Protein solubility with 500 m M Arg-HCl added to the above buffer. * The trypsin activity could not be deter mEned because the high arpuine coEicentratiou inhibited trypsin activity, The addition of a poly-Arg or poly-Lys tag to the N- or C-terminus of BPTI-22 can increase its solubility without significantly affecting its structure, stability or activity. Kato et al., 2006 A) BFTI-23: RPAFCLEPPYAGPAKAEIIRYFYNA.^aAAQAFVYG0AAAKRNNFA8AADALAACAAA FIGURE 3 Hydrophobic residues in BPTI-22. A: One letter amino acid sequence of BPTI-22 with the hydrophobic residues (AtVJ5L,HI1) shown in green letters. B: Left, BPTI-22 ribbon model with ft-helices colored red and 8- strands colored blue. Right, surface representation of BPTI-22 with the hydrophobic area determined as low electrostatic potential regions according to MOL-MOL,35 colored green. The molecule is oriented with the 8-sheet pointing to the back in (a) and to the front in (b). "I'he N- and tl-termini are labeled "N" and 11C" respectively. 'Hie t> terminal end is located on the same face as a large hydrophobic patch shown in green, whereas the N-terminal end is on the opposite side of the molecule and is shown with a light gray letter "N" in panel (b). The solubilization factor of all C-terminal tags was slightly higher than that of the respective N-terminal tags. 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 Solubility-enhancing tags - Comparison of peptide and protein tags, conclusions > Protein tags are inherently large and need to be correctly folded in order to enhance solubility. > Proteins tag are often natural affinity tags > Peptide tags are small, and, importantly, they do not need to be folded, which provides a significant advantage over protein tags. > The use of small tags (< 30 amino acids long) does not increase protein size substantially and diminishes steric hindrance, which simplifies downstream structural and functional applications without the need to remove the tag. > The solubilization enhancement effect depends on the size of the target protein. Solubility enhancement of fusion partners 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. Removal of fusion tags All tags, whether small or large, have the potential to interfere with biological activity of a protein, impede its crystallization, or otherwise influence its behavior. 1. Site-specific proteolytic cleavage Enterokinase Asp-Asp-Asp-Asp-Lys/X A min a icid in jimdan K| <:il enwrciuiLLie Alwin« SS M« Han in« 1 BS Leucine B5 as F'h-iTT.bJjJETK B5 holend ne A.spjjric icid (jhiajTuc acid fj hi amine TO 79 ^ [MBP Alanine Threnirine 11 Tjrn.dne n 1 ll-;G'J]Tt 76 Serine rt i\.*eine 7* T: TnqiKijihaji 67 Praline *l TEV protease optimal cleavage sequence: Glu-Asn-Leu-Tyr-Phe-Gln/Ser Thrombin X4-X3-Pro-Arg[Lys]/X1-X2 (X4, X3-hydrophobic residues; X1, X2-non-acidic residues) Some frequently used recognition sites: Leu-Val-Pro-Arg/Gly-Ser; Leu-Val-Pro-Arg/Gly-Phe; Met-Tyr-Pro-Arg/Gly-Asn. Factor Xa Ile-Glu[Asp]-Gly-Arg/X1 X1 can be any amino acid except arginine and proline. 2. Use of self-processing fusion partners derived from self-splicing inteins -Disulfide (analogy to RNA splicing) Inteins are selfish DNA elements inserted in-frame and translated together with their host proteins. This precursor protein undergoes an autocatalytic protein splicing reaction. The process of protein splicing removes inteins and splices the exteins together to make a mature protein. The principle disadvantages of the intein approach: y the large size of the catalytic machinery that must be incorporated into the fusion protein, which increases the metabolic burden on the cells y the dependence of processing efficiency on the sequence context at the fusion junction y the slow rate of auto-processing bond Precursor Precursor Precursors 7^3 (a) Protein cleavage Intein (b) Protein spiking (intramolecular) Inteins Final protein Final protein c) Protein splicing ntermolecular) Copyright © 2009 Pearson Education. Inc. Final protein y the fact that inteins neither enhance the solubility nor facilitate the purification of their fusion partners 3. Chemical cleavage - Rarely used Cyanogen bromide Met/X Hydroxylamine Asn-Gly Chemical cleavage is a harsh method leading to non-specific cleavage, whereas enzymatic cleavage can be specific but inefficient. Removal of fusion tags - Achilles' heel of the fusion approach 1. Unspecific cleavage (SOLUTION: optimization of protein cleavage conditions or using re-engineered proteases with increased specificity such as ProTEV and AcTEV) 2. Optimization of protein cleavage conditions (mainly enzyme-to-substrate ratio, temperature, pH, salt concentration, length of exposure) 3. Precipitation of target protein when the fusion partner is removed (so-called soluble aggregates; SOLUTION: other approach for protein solubilization has to be found) 4. Cleavage 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) 5. High cost of proteases 6. Re-purification step 7. Failure to recover active or structurally intact protein The alternative is to leave the affinity tag in place for structural analysis: These multi-domain proteins are usually > less conducive to forming well-ordered diffraction crystals, presumably due to the conformational heterogeneity allowed by the flexible linker region > too large for NMR analysis Small tags are better choice in structural biology Unspecific His-tag cleavage pRSETB: :AHP2 Enterokinase cleavage site N'MRGSHHHHHHGMASMTGGQQMGRDLYDDDDK DPSSRSAAGTMEFMDALIA....................GIVPQVDIN C' ___J ' Theoretically: ^3.4 kDa LC/MS-MS analysis 18.9 kDa a.i. 1,000 800 600 400 200 4 0 4 1 3 2 2 1 llk ilřJll 4 9 2 3 19 6 8 3 8 9 7 3 B 9 4 4 0 5 3 \ \ 1 3 4 S 6 110 3 1 1117 5 1 1 D 3 9 1116 7 I 110 3 4 AHP2_enterokinase \ 8 2 5 8 AHP2_control 3 5 8 11 AHP2_standard 2 2 3 3 7 2000 4000 6000 8000 10000 12000 14000 16000 18000 20000 22000 m/z 0 Affinity chromatography > Affinity chromatography exploits the natural specific recognition between biological molecules. Ligand Affinity to Enzyme substrate analogue, inhibitor, cofactor Antibody antigen, virus, cell Lectin polysaccharide, glycoprotein, cell surface receptor, cell Nucleic acid complementary base sequence, histones, nucleic acid polymerase, nucleic acid binding protein. Hormone, vitamin receptor, carrier protein Glutathione glutathione-S-transferase or GST fusion proteins Metal ions poly (His) fusion proteins, native proteins with histidine, cysteine and/or tryptophan residues on their surfaces. > Essential tool for protein purification (protein preparation for structural genomics, antibody generation, and biochemical analysis) and protein complex isolation. > The essential element of affinity chromatography is the affinity ligand immobilized onto an inert, hydrophilic solid support (or matrix), which is used in a column to purify the desired target molecule. 21 Affinity purification of tagged proteins 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. Components of a matrix for affinity chromatography > The ligand must bind strongly (e.g., Kd < 1 nM) to the target molecule to facilitate its capture from a complex protein mixture. > Higher affinity produces better specificity, and thus better purification. > When the affinity is high enough (e.g., Kd < 1 nM) , small amounts of affinity gel matrices can be used to purify proteins from large volumes of crude extracts. > When the affinity is not high enough and the protein is of low abundance, partial purification using other methods to enrich the protein of interest may be required. > Affinity tag procedures are particularly useful when target proteins must be isolated from complex protein mixtures. 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 supports (typically, a macroporous polysaccharide bead such as agarose) tether the active ligands and provide a porous structure so that there is an increased surface area to which the target molecule can bind. A ligand can be covalently affixed to substituent groups within the matrix (e.g., amino, hydroxyl, carbonyl, and thio groups) that are easily activated using conventional chemical methods. 24 Components of a matrix for affinity chromatography Ni2+ NTA sepharose 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. 25 Overview of tags using in affinity chromatography Table 2 Sequence and size of affinity tags Tag Residues Sequence Size (kDa) Poly-Arg 5-6 RRRRR 0.80 (usually 5) Poly-His 2-10 HHHHHH 0.84 (usual lv 6) FLAG 8 DYKDDDDK 1.01 Strep-tag II 8 WSHPQFEK 1.06 omyc 1 1 EQKL1SEEDL 1.20 S- 15 KETAAAKFERQHMDS 1.75 HAT- 19 KDHLIHNVHKEFHAHAHNK 2.3] 3x FLAG 22 DYKDHDGDYKDHDIDYKDDDDK 2.73 Calmodulin-binding peptide 26 KRRWKKNF1AVSAANRFKKISSSGAL 2.96 Cellulose-binding domains 27-189 Domains 3.00- 20.00 SBP 38 M D EKTTG W R G G H V V EG LAG EL EQ LR A R LEH H PQ G Q R EP 4.03 Chi tin-binding domain 51 TNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNVPALWQLQ 5.59 Glutathione S-trans terase 211 Protein 26.00 Ma kose-bin ding protein 396 Protein 40.00 Typical affinity purification steps > 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. > 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). > 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. Affinity chromatography Glutathione S-transferase > Enzymatically active fusion partner. GSTs perform a protective role in the cell by detoxifying endogenous compounds during oxidative stress, chemical carcinogens, environmental pollutants and a range of pharmaceutical compounds, leading to drug resistance. > GST ensures a high concentration of GST in the cell extract - this acts effectively as a purification step, i.e. GST may form 10% of total cell protein. > One-step affinity chromatography is used. > This chromatographic approach relies on the specificity of the interaction between GST and its substrate - glutathione. > GST affinity column, which contains immobilized glutathione, binds GST whereas most contaminating proteins fail to bind to the column. > Glutathione interacts with GST and promotes the specific elution of the enzyme. Schematic representation of GST purification: Optimal binding of GST to immobilized glutathione requires a low flow rate 1 mL min-1) due to the relative weak affinity of the enzyme for the substrate. Specific elution of GST is achieved by applying a solution of reduced glutathione. Affinity chromatography - Immobilized metal ion affinity chromatography (IMAC) > The most common purification tag is typically composed of six consecutive histidine residues. > Histidine, cysteine, and tryptophan residues are known to interact specifically with divalent transient metal ions such as Ni2+, Cu2+, Co2+, and Zn2+. > 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. m (kDa) 170 — 116 — 86 - 56 - Binding strength of His tag to metal ions: 27- Cu2+ > Ni2+ > Zn2+ ~ Co2+ 20 - Zn2+ Ni2+ Co2+ Cu2+ > IMAC can be used under native and/or denatured conditions. > Immobilized Fe3+, Ga3+, Al3 = metal ions that have been used for selective enrichment of phosphopeptides and phosphoproteins. > A highly purified protein can often be obtained in one or, at most, two purification steps. His-tagged protein and IMAC under native conditions > Optimal binding of recombinant protein with metal ion is achieved at pH 7-8. > Buffers with high salt concentration (0.51 M NaCl-) reduce nonspecific electrostatic interaction. > Nonionic detergents or glycerol reduce nonspecific hydrophobic interactions. > Elution of contaminating proteins can be achieved by lowering pH or using low concentrations of imidazole. > Elution of tagged protein is achieved at high imidazole concentrations (0-0.5 M), by strongly decreasing pH, or by using EDTA. Immobilized metal affinity chromatography I PROTEIN ] His protease cleavage site + -4 -4 charged metal chelate resin pi I [imidazole] || [EDTA] [\ protease + + + + + r 1> -* His-tagged protein and IMAC under native conditions One-step purification - Perfusion matrix: POROS MC/M - Functional group: iminodiacetate, metal ion Zn2+ - Removing contaminated proteins: linear gradient of imidazole (0-50 mM) and pH (pH 6.1-7) - Protein elution: 0.1 M EDTA - 80% recovery, 95 fold purification - Common production and isolation of wild type and soluble mutant form for enzymatic measurements and crystallization His-tagged protein purification under denatured conditions Denaturing IMAC - purification of proteins expressed in inclusion bodies - purification in high concentration of urea or guanidine chloride - result is pure protein, but in denatured form (sufficient for immunization) Recovery of native conformers (necessary for functional and structural analysis): > binding to the column under strong denaturing conditions (8 M urea) >Two renaturation possibilities: 1. Protein is eluted from column and renatured by dialysis or rapid dilution in renaturing buffers (8-0M urea). 2. Renaturation of protein bounded to the column (matrix assisted refolding procedure): gradient from denatured to renatured buffers or pulsion renaturation. Identification of properly refolded (His)6Zm-p60.1 (maize p-glucosidase) using 10% native PAGE, followed by activity in gel staining: 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 kcatwas hampered by the fact that the refolding process yielded a number of improperly folded polypeptides. A = crude protein extract prepared from maize seedlings containing 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 Affinity purification for isolation of protein complexes Many protein-protein associations that exist within the intact cell are conserved during purification. This property can be exploited to facilitate the detection and identification of physiologically relevant protein-protein interactions. Affinity based method used for detection and identification: > Co-immunoprecipitation > Tandem affinity purification > GST pull-down > Testing an interaction between two known proteins > Identification of novel protein-protein interactions Co-immunoprecipitation 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. 1. Cell lysis under mild conditions that do not disrupt proteinprotein interactions (using low salt concentrations, nonionic 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 or autoradiography when the interaction partner is labeled with S35 methionine. Identification of novel interaction is carried out by mass spectrometry analysis. Pull-down assay y Pull-down assays are a common variation of immunoprecipitation and are used in the same way, although this approach is more suited to an initial screening for interacting proteins. y They are used for purification of multiprotein complexes in vitro. y The protein of interest is expressed in E. coli as GST (or His) fusion and immobilized on glutathione-sepharose beads (GST alone is often used as a control). y 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. > Centrifugation is used to collect the GST fusion probe protein and adhering proteins. > The complexes are washed to remove nonspecifically adhering proteins. y 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. y The proteins are resolved on SDS-PAGE and processed for further analysis. Tandem affinity purification (TAP) 'op "agotd MltfSeptlde expressed near physiological iete s Calmodulin blfidlng peptide (CÍP) -TEV protease doaraqc site Protein A IgG-bindlng domain - PROTEIN-PROTEIN INTERACTIONS CeltohlnmidCTndlllw» / Tandem afilnTty rhramamgraphy SD5 gel 1 i PROTEIN'DNA INTERACTIONS \ CrossJInkwIth rcnTSfdehyde V Chromatin fragmeniaiJon Affinity chromaipgraphy Reverse crosslinks =uriyDNA PCR with specific prirren; flewse crosslinks Purify and label DNA Hybridize Mast Spectrometry Single Lotus PCR Genom k: sequences IRegu latoiy and i i; ní ex;-.!: Location DNA Microarray 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. Examples of TAP tags (a) Protein A Protein A TEV CBP 20.7 kDa (b) Protein G Protein G TEV SBP 18.8 kDa (c) FLAG ^ÜCTTP- 3TT 4.6 kDa m RGS-6xHis Biotin signal ! 6xHis 9.6 kDa Current Opinion in Biotechnology 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. Tandem affinity purification A new (and so far the best) TAP tag for plants: The GS tag (b) Higher oait expression with GS tag fusions TAP tag 5Q 10 2 GS tag 59 IP. .2.. ^ — — CK31 tsg CKS1 MO co 10 a SO to 2 CDKA; 1 tag MO eo. 10 i SO 1Q 2 GFP-iag van Leene et al., 2008 Affinity purification for isolation of protein complexes - Tags can influence protein-protein interactions (testing N- and C-terminal fusion). - Loss of weak or transient protein-protein interactions (in vivo chemical cross-linking, e.g. using formaldehyde). - Verification of newly identified interactors by other methods and biologically relevant mutants - Non-specificity: controls, affinity tags with higher specificity