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(i) 5' Promoter Tag Gene of interest 3' Terminator Transcribe and translate Tag fused to the N-terminus of the protein of interest (ii) 5' Promoter Gene ot interest CS Tag 3' Terminator ^Transcribe and translate Tag fused to the C-terminus of the protein of interest 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. Purposes of fusion tags > 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. > 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. No single tag is ideally suited for all of these purposes. Fusion partner (tag) Size Tag placement Uses His-tag 6, 8, or 10 aa N- or C-terminus Purification, detection Thioredoxin 109 aa(11.7kDa) N- or C-terminus Purification, solubility enhancement Calmodulin-binding domain (CBD) 26 aa N- or C-terminus Purification Avidin/streptavidin Strep-tag 8aa 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 > 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. Combinations: Solubility-enhancing tag + purification tag: MBP + His6tag 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 Tag3 Advantages Disadvantages GST Efficient translation High metabolic burden initiation Inexpensive affinity resin Homodimeric protein Mild elution conditions Does not enhance solubility MBP Efficient translation High metabolic burden initiation Inexpensive affinity resin Enhances solubility Mild elution conditions NusA Efficient translation High metabolic burden initiation Enhances solubility Not an affinity tag Thioradoxin Efficient translation Not an affinity tagb initiation Enhances solubility Ubiquitin Efficient translation Not an affinity tag initiation Might enhance solubility FLAG Low metabolic burden Expensive affinity resin High specificity Harsh elution conditions BAP Low metabolic burden Expensive affinity resin Mild elution conditions Variable efficiency of enzymatic biotinylation Provides convenient means Co purification of E. coli of immobilizing proteins in biotin carboxyl carrier a directed orientation protein on affinity resin Does not enhance solubility Hiss Low metabolic burden Specificity of IMAC is not as high as other affinity HMtlMdl Inexpensive affinity resin Mild elution conditions Tag works under both Does not enhance native and denaturing solubility conditions STREP Low metabolic burden Expensive affinity resin High specificity Does not enhance solubility Mild elution conditions SET Enhances solubility Not an affinity tag CBP Low metabolic burden Expensive affinity resin High specificity Does not enhance solubility Mild elution conditions Stag Low metabolic burden Expensive affinity resin High specificity Harsh elution conditions (oron-column cleavage) Does not enhance solubility Advantages and disadvantages of used fusion tags > 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. X > Because affinity tags have the potential to interfere with structural and functional studies, provisions must also be made for removing them. "GST. glutathione S transferase: MBP. maltose binding protein: NusA. N utilization substance A: FLAG. FLAG tag peptide: BAP. biotin acceptor peptide: His,, hoxahistidinc 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 (His patch thiorodoxin) or avidin/streptavidin |38|. inns Otázka č. 1: Jaké jsou důvody pro využívaní tagů/kotev? Vyjmenujte 3. Increasing the yield of recombinant proteins using fusion technology Yield enhancing tags are proteins and peptides which can be involved in: > 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. > 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. > Helping to properly fold their partners leading to increased solubility of the target protein (in vivo and in vitro). Enhancing the solubility of recombinant proteins Solubility-enhancing tags - 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. - Advantage of N-terminal tags - Rather proteins (highly soluble proteins) than peptides - They are not universal - The mechanism by which partners exert their solubilising function is not fully understood. >PROTEINS Some commonly used solubility-enhancing fusion partners Tag Protein Source organism MBP Maltose-binding protein Escherichia coli GST Glut athione-S-transf erase Schistosoma japonicum Tra Thioredoxin Escherichia coli Nu s A N-Utilization substance Escherichia coli SU IVO Small ubiquitin modifier Homo sapiens SET Solubility-enhancing tag Synthetic DsbC Disulfide bond C Escherichia coli Skp Seventeen kilodalton protein Escherichia coli T7PK Phage T7 protein kinase Bacteriophage T7 GB1 Protein G B1 domain Streptococcus sp. ZZ Protein A IgG ZZ repeat domain Staphylococcus aureus Adopted from Esposito and Chatterjee, 2006 > PEPTIDES Poly-Arg Poly-Lys Generate parallel expression clones His6 (^St)—Target protein Dead end: insolubility His6 (NusA)—Target protein His6 ( MBP)—Target protein (a) Express in jfi E. coli x/' (gst]— Target protein ) His6 NusA Target protein I h^Targi 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 4 Target protein His6 (NusA Target protein Target protein His6 (NusA) Target protein Dead end: protein insoluble after cleavage of tag Dead end: difficult to separate cleaved protein from fusion Currenl Opinion in Biotechnology Schematic representation of the pathway from protein expression to purification using solubility tags (Esposito and Chatterjee, 2006). Enhancing the solubility of recombinant proteins 19, 84, 215 - human proteins involved in cancer produced in E.coli 19 84 215 27 28 29 34 27 28 29 34 27 28 29 34 sisisisi sisisisi sisisisi 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). Solubility-enhancing tags - the mechanism of action -The mechanism by which partners exert their solubilising function is not fully understood (they might act through a chaperone-like mechanism) - possibly differs between fusion proteins Examples of possible mechanisms Maltose binding protein (MBP) might bind reversibly to exposed hydrophobic regions of nascent target polypeptide, steering the polypeptides towards their native conformation by a chaperone like -mechanism NusA decreased translation rates by mediating transtriptional pausing, that might enable critical folding events to occur. Negative charged tags (highly acidic peptide) inhibit aggregation by increasing electrostatic repulsion between nascent polypepdides (Zhang et. 2004). Solubility-enhancing tag - mechanism of action Thioredoxin > Protein thiol-disulfide oxidoreductase. > E. coli thioredoxin is a compact, highly soluble, and thermally stable protein with robust folding characteristics. The active-site surface in thioredoxin is designed to fit many proteins. > Thioredoxin 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. > Fast reduction of intra - and inter-molecular disulfides in a hydrophobic environment. Cys 32 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. Holmgren, 1995; Berndt et al. 2006 In vitro 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 (in vitro solubility). 1 NH J Cht 1 Cht Cht 1 1 Cht Cht 1 J Cht Cht H;N+—C —CCfc-i HjN+—C—CQ£-I 1 H 1 H Arginine (R) Lysine (K) BPTI-22 = bovine pancreatic trypsin inhibitor variant containing 22 alanines 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. ->->->-> N- C- N- C- terminus The solubilization effect of poly-Lys tags is lower than that of poly-Arg tags (lysines are less hydrophilic than arginines). Kato et at., 2006 Biochemical properties of poly-Arg and poly- Lys tagged BPTI-22 protein Protein Solubility Cone |nijVÍ| Solubilization Re I. Trypsin Inhibitory Protein (Cone. |mg/ml| f Factor 7^ ("C) Artmty (%)' BPTI-22 I 7i' ■jüi:m;.. 3S.4 ■ NIK 1.70 (10.40) 1.00(1.04) 352 1.05 ■ N3K 2.66 (19.97) 136(2.00) 34.4 1.04 Nr>K 3.37 (35.60) 3.16(336) 34.3 1.05 -C1K 1.79 (10.95) 1.05(1.10) 34.6 1.05 . ..:k 2.41 (15.2«) 1.42(1.53) 362 1.05 ( 3R 7. IM J7.-J7I 4.21 (4.75) 35.0 1.02 -NiJL 1.69 (10.34) Ů.99U.03) 35i 1.02 -N3R 2.70 (1723) 139(1.72) 35.6 0.99 -N5R 620 (41.11) 3.65(4.11) 353 0.99 C1R LSI . 1 1.1.7. 1.06(1.11) 35.0 1.05 -C3R 3.02 (19.26) 1.7K(1.93) 34.4 1.05 ■ C5R 323 (54.56) 4.34(5.46) 34.3 1.08 ■ C6R 10.59 (73.41) 6.2: ..7..;4. 327 ].l BPTI-22" 5.63 (33.11) 3.31 (331) sir- 1.09 ' BPTT22f 2.01 Í11.32) Llňíl.lS) ND1 NAS _ 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. Prolan solubility wis determined as the maximum super rutin t concent ra Bo n rl .: supersaturated protein ujlLtinn llI i~C, in I OH m M acetate bu flkr p II 4.7. " Muiim.m Loncen rr ations lIlLU .:Ll.-.! ir: r ~. ■ i 11 ■ ■_: r ,ir:i ■■ per mi III hi 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. > Charged residues seem to act through repulsive electrostatic interaction and thus hamper intermolecular interaction arising from the hydrophobic cluster. 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. > Protein tags 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 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: > Chemical cleavage > Self - cleavage > Enzymatic cleavage Removal of fusion tags - chemical cleavage > Rarely used. Cyanogen bromide Met/X Hydroxylamine Asn-Gly 1 1 MRGSHHHHHH M12 M15 / / GMASMEKNNQ M28V / GNGQGHNVPN 40 1 DPNRNVDENA NANSAVKNNN NEEPSDKHIK EYLNKIQNSL STEWSPCSVT M105V CGNGIQVRIK PGSANKPKDE LDYANDIEKK ICKVEKCS Amino - acid sequence of the P. falciparum C-terminal segment of CSP (PfCSP C-ter) fused to a purification tag (Rais-Beghdadi et ah, 1998). Chemical cleavage is a harsh method, efficient, but rather non-specific and may lead to unnecesary denaturation or modification of the target protein. Removal of fusion tags - self - cleaving > Use of self-cleaving fusion tags 1. Inteins Intein Precursor (b) Protein splicing (intramolecular) Final protein Inteins (intervening proteins) are protein segments that can excise themselves from protein precursors in which the are inserted and rejoin the flanking regions. > 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. > Two categories of inteins: - inteins with pH-induced C-terminal cleaving activity - inteins with thiol-induced N- and C-terminal cleaving activity A pH intein N C Thiol intein A Tag pH 6.0-6.5 20-25°C, 16 h 15-30 mM thiol -1 4X, 16 h A pH intein N + 4m + C- Thiol intein A Target Protein H X intein Cysl Sacyl shift r 0 H2N Sv Thiocstcr 1 intein r iiii.il rcuecrit D-frSR + 0 C-terminal «-thioester intein Perler, (2005) Removal of fusion tags - self - cleaving fusion tag 2. hhhhhh—I sit.w-iofr~|— lpxtT:;— target protein 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). 3. N(pro) target protein 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. 6His or CBP"1 4. target protein —D P— SPM — 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). 5. target protein —VDAL ADGK— CPI) ]—hhhhhh 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. (Li, 2011) Removal of fusion tags - self - cleaving fusion tag Inteins (1) > Uncontrolled in vivo cleavage or in complete in vitro cleavage > Target protein modification - pH or thiols can modify the target protein > Protein compatibility with cleaving conditions - pH induced inteins > Compared to the traditional protease based method, the intein-based approach requires fewer steps and lower costs. Other system (2-5) > Tested on limited number of cases 'J'iihJu 3 General features of th e five self-clea\ age fusion systems discussed in the text Self HW:l Purification Cleavage Advantages Disadvantages cleaving (kDa) tag condition tag lutein 51: CBD, CBM, Thiols; pH Flexible fusion and cleavage options; Lack of solubility-enhancing capacity; in 22: phasin, and/or allowing generation of target protein vivo cleavage; incomplete cleavage: 17; 15b LLP temperature shift with native sequence miscleavagc SrtA P His-tag. biotin 5 mM Ca"' Potential of enhancing target protein expression and solubility In vivo cleavage; incomplete cleavage; introduction of an extra Gly residue to the /V-tcrininus of the target protein N,.n, 19 His-tag Kosniotropic conditions Allowing generation of target protein with native sequence Limited to proteins capable of refolding; in vivo cleavage; incomplete cleavage; long cleavage time FrpC 26 His-tag. CBD 10 mM Ca2+ Efficient and tightly controlled cleavage; insensitive to protease inhibitors Lack of solubility enhancing capacity: introduction of an extra Asp residue to the C-terininus of the target protein; single C-lerminal fusion option ("I'D His lag 50-HK) |iM lnsP6 Potential of enhancing target protein expression and solubility; efficient and tightly controlled cleavage; insensitive to protease inhibitors Introduction of up to four non-native residues to the ('-terminus of the target protein; single ('-terminal fusion option * Molecular weight of the self-cleaving tag h luteins with different sizes are available Li, 2011 Removal of fusion tags - enzymatic cleavage Cleavage site Protease Target 4-37°C, time varies Site-specific proteolytic cleavage: > Exopeptidases > Endopeptidases Exopeptidases (aminopeptidases and carboxypeptidases): DAPase (TAGZyme) Exo(di (peptidase Cleaves N-terminal. His-tag (C-terminal) for purification and removal A eromonus aminopeptida se Exopeptidase Cleaves N-terminal, effective on M. L. Requires Zn Aminopeptidase M Exopeptidase Cleaves N-terminal. does not cleave X-P Carboxypeptidase A Exopeptidase Cleaves C-terminal. No cleavage at X-R. P Carboxypeptidase B Exopeptidase Cleaves C-terminal R. K > APM, 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): > DAPase (dipeptidyl aminopeptidase I) TAGZyme stop points_ Amino acid DAPase Hop point (J-) sequence' Lysine (Lys, Kj Arginine (Arg. R) Proline (Pro. R) Proline (Pro. GlUamine (Gin. Q" IXaaXaa... Xaa Xaa I Arg-Xaa ... IXaa-Xaa... Xaa-Xaa i j(aa-Xaa fVoXaa. !Xaji-)Cjia... Xaa -Xnji I Xsa-Pro Xan-Xoa. Xaa-Xaa. ..Xaa-Xaa I Gln-Xaa... DAPase cleavage DAPase slop M K HQ HQ HQ HQ HHP SDK HT-Trx HHP-Trx M 123 4567 Removal of fusion tags - enzymatic cleavage Endopeptidases > The enzymatic cleavage site has to be placed between the fusion tag and the target protein. Enzyme Cleavage site Comments Enter ok inase DDDDK* Secondary sites at other basic aa Factor Xa IDGR* Secondary sites at GR Thrombin lvpr'cs Secondary sites. Bio tin labeled for removal of the protease PreScission LEVLFQ'GP GST tag for removal of the protease TEV protease EQLYFC/c His-tag for removal of the protease 3C protease ETLFQXP GST tag for removal of the protease Sortilse A LPET'G CaJ+-induction of cleavage, requires an additional affinity tag (e.g., his-tag) for on column tag removal Granzyme B D*X, N*X, M*N, S*X Serine protease. Risk for unspecific cleavage , Protease site His, MBP Target protein Enterokinase Asp-Asp-Asp-Asp-Lys/X Tuhle 4 Cleavage (%) of enterokinase through densitometry (Hosfield and Lu 19991 based on the amino acid residue X,. The SLUuence....-GSDYKDDDDK-Xi-ADQLTEEQIA-... of a GST-cat modulin fusion protein wi is tested LisiiiL' 5 inn protein digested with 0.2 Uof enterokinase tor 16 h at 37 °C Amino acid in position X ] Cleavage of enterokinase (%) Alanine 88 Methionine 86 Lysine 85 Leucine 85 Asparagine 85 Phenylalanine 85 Isoleucine 84 Aspartic aeid 84 Glutamic aeid sn Glutamine 79 \ aline 79 Arginine 78 Threonine 78 Tyrosine 78 Hislidine 76 Serine 76 Cysteine T4 Glycine 74 Tryptophan 67 Proline 61 Removal of fusion tags - enzymatic cleavage A critical review of the methods for cleavage of fusion proteins with thrombin and factor Xa Richard J. Jenny,3** Kenneth G. Mann,b and Roger L. Lundbladcd 3 Haettiatalagic Tec biologies, Inc., Essex Junction, VT, USA b Department oj' Bioiheinisny, Utthtrsity of Vermont, Burlington. IT, USA c Department of Pathology, University of North Carolina, Chape! Hill, NC. USA d Roger LLmdbtad, LLC. Chapel Hill, NC. USA Received 27 February 2003. and in revised form 7 May 2003 The purpose of this review was to demostrate that both thrombin and factor Xa can hydrolyze variety of peptide bonds in proteins. Sequences cleaved by thrombin in polypeptide hormones Polypeptide homones3 Sequence cleaved Secretin ELSLSRLRDSA Secretin ELSLSRLR (much slower than above) Vasoactive intestine polypeptide DM I'RI.RK Vasoactive intestine polypeptide YTRLRKQM Ch oleocystokinin APSGRVSM Choleocystokinin VSMIKNLQ Dynorphin A RJRPKLKW Somatostatin-28 AMAPRERK Somatostatin-28 NFFWKTFT Gastrin releasing peptide KM YPRCiNH Salmon calcitonin QIYPRIM aThe reaction mixtures contained 0.5 NIH units thrombin and I.Onmol peptide in 20 uL of 50 mM NH4CO3. pH 8.0, at 25 nC. The conditions were designed to obtain an enzyme/substrate ratio of 1:60 (w/w). Protein pExprcssion ^Purification Accuracy of cleavage has to be precisely verified! pRSETB:: AHP2 Enterokinase cleavage site N'MRGSHHHHHHGMASMTGGQQMGRDLYDDDDI^)PSSRSAAGTMEFMDALIA....................GIVPQVDIN C Theoretically: 3,4 kDa 18,9 kDa Intact mass spectrometry analysis a.i. 1000 800 600 400 200 4 0 4 1 3 2 2 1 Ilk 5 8 9 7 1 9 6 8 -1- 1 3 4 8 6 110 3 1 I 1117 5 1 1 □ 3 9 1116 7 AHP2 enterokinase 18 2 5 1 I _I_ AHP2 control A AHP2 standard 1 4 0 5 3 i...........Li. ... 1 1 0 I 3 4 I I 2000 4000 6000 8000 ' 10000 ' 12000 ' 14000 ' 16000 18000 20000 22000 m/z Removal of fusion tags - enzymatic cleavage > Unspecific cleavage (SOLUTION: optimization of protein cleavage conditions or using re-engineered proteases with increased specificity such as ProTEV and AcTEV proteases). > Optimization of protein cleavage conditions (mainly enzyme-to-substrate ratio, temperature, pH, salt concentration, length of exposure). > Precipitation of the target protein when the fusion partner is removed (so-called soluble aggregates; SOLUTION: another approach for protein solubilization has to be found). > 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). > High cost of proteases > Re-purification step > Failure to recover active or structurally intact protein > Target protein modification (some proteases like thrombin, TEV, Precision leave one or two amino-acids on the target protein near the cleavage site). The alternative is to leave the tag in place for structural analysis: The small tags are a better choice in structurural and functional analysis of proteins. Otázka 3: Jaký je rozdíl mezi inteinem a samo-vyštěpujícím tágem odvozeným od inteinu? Affinity chromatography (AC) > A 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). Affinity tag Protein of interest Sepharose Affinity tag binding partner Immoblized binding partner of Affinity tag fused to N- or C-terminus affinity tag of protein TPEG (substrate analogue of ß-galactosidase ß-galactosidase) Glutathione Glutathione-S-Transíerase Immunoglobulin G Protein A Cu II, Co II or Ni II poly His or poly Oys > AC has a concentrating effect, the high selectivity of separations derived from the natural specificities of the interacting molecules. > 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. > 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 Affinity tag Protein of interest Sepharose Affinity tag binding partner Immoblized binding partner of Affinity tag fused to N- or C-terminus affinity lag <>t pruti-in TPEG (substrate analogue of fl-galactoiiidase jl-galactosidase) Glutathione Glutathione-5-Transferase Immunoglobulin G Protein A Cu II, Co II or Ni II poly His or poly Cys Table 2 Sequence and size of affinity tags Tag Resi dues Sequence Size (kDa) Poly-Arg 5-6 RRRRR 0.80 (usually 5) Poly-His 2-10 HHHHHH 0.84 (usually 6) FLAG 8 DYKDDDDK 1.01 Strep-tag II 8 WSHPQFEK 1.06 c-myc 1 1 EQKL1SEEDL 1.20 S- 15 KETA AAKFERQHMDS 1.75 HAT- 19 KDHL1HNVHKEFHAHAHNK 2.31 3x FLAG 22 DYKDHDGDYKDHDIDYKDDDDK 2.73 Calmodulin-hinding peptide 25 KRRWKKNFIAVSAANRFKKISS SGA L 2.96 Cellulose-binding domains 27-189 Domains 3.00- 20.00 SBP 38 M D EKTTGW RGGH V V EGL AGEL EQ LR A RLEH H PQGQ REP 4.03 Chitin-binding domain 51 TN PC j V S A VV Q V NT A YT AG QL VT Y NGKT YKC LQ P HT S L AGWE PS N V PA LW Q LQ 5.59 Glutathione S-transferase 21 1 Protein 26.00 Maltose-binding protein 396 Protein 40.00 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 At't'inity tag Matrix Poly-Arg Poly-Hts FLAG Strep-tag II c-myc S HAT (natural histidine affinity tag) Calmodulin-binding peptide Cellulose-binding domain SBP Chi tin-binding domain Ghitathi o ne S -t r ans fe ra se Makosc-binding protein Cation-exchange resin Ni2+-NTA, Co*+-CMA (Talon) Anti-FLAG monoclonal antibody Strep-Tactin (modified streptavidin) Monoclonal antibody S-fragment of RNaseA Co2+-CMA (Talon) Calmodulin Cellulose Streptavidin Chi tin Glutathione Cross-linked amvlose Non - chromatographic tags Tag_Matrix ELP PHB annexin B1 None Intracellular PHA granules None > These tags can eliminate affinity resin. Proteins are isolated by other non-chromatographic methods (centrifugation, filtration) > typically combined with self-cleaving tags > 75 % - 95 % purity > Traditional purification tags > The tag binds strongly and selectively to an immobilized ligand on a solid support. > After optimization one could achieve > 90% purity. it 1 Additional separation step + DTT T Affinity tag-Inker-target prct™ % Protesá cleavage j MJd heattlg and/or s *\ 1 salt addition + Centrifugation % I Affinity tag - intein - target protein Pha&in - intein - target protein ELP - intein - target protein Purification tags Non - chromatographic tags + DTT 4, ^ Mild heating and/or ^ calt addition O 1 c Centrifugation The PHB system (c): > PHB (polyhydroxybutarate): subclass of biodegradable polymers produced in various organisms, use as storing excess carbon. > The system includes in vivo production of PHB small granules (from the plasmid carrying PHB-synthesis genes). > Target protein in fusion to self cleaving phasin tag. > Tagged protein binds to the PHB particles via phasin tag, which allows the granules and the tagged protein to be co-purified via centrifugation. Phasin - itiUtin - target protein ELP - intein - target protein > DTT induced cleaving activity of intein and thus elution of the target protein. The ELP system (d): > 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 Ni2+ NTA sepharose A ligand > The dissociation constant (Kd) for the ligand - target complex should ideally be in the range 10~4to 10~8 M in free solution to allow efficient elution under conditions which will maintain protein stability. > 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. Components of a matrix for affinity chromatography A matrix > 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. > 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. 34 Components of a matrix for affinity chromatography b) Inefficient binding target «lutes during binding and elution -1-1— 10 15 20 25 Elution volume, ml Efficient binding f target elutes in a single peak to 15 20 25 Elution volume, ml Fig. 56. Using spacer arms, a) Ligand attached directly to the matrix, b) Ligand attached tu the matrix via a spacer arm. Ni2+ NTA sepharose Spacer arm > 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. 35 Typical affinity purification steps adsorption of wash re-equilibration 1-2 -»- :■'lv -»- 1-2 cv cv 1-2 cv Column Volumes (cv) > 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 - 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 - Binding strength of His tag to metal ions: 93_ Cu2+ > Ni2+ > Zn2+ ~ Co2+ 27- 20 - Zn2+ Ni2+ Co2+ Cu2+ (Zouhar et al, 1999) > IMAC can be used under native and/or denatured conditions. > 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 a high salt concentration (0.5-1 M NaCl) reduce nonspecific electrostatic interaction. > Nonionic detergents or glycerol reduce nonspecific hydrophobic interactions. > Elution of contaminating proteins can be achieved by lowering the pH or using low concentrations of imidazole. > Elution of tagged protein is achieved at high imidazole concentrations (0-0.5 M), by strongly decreasing the pH, or by using EDTA. Immobilized metal affinity chromatography |PRQTErŇ~| His profease cleavage site + 4 charged metal chelate resin pi I I imidazole I [EDTA] t\ protease - + + |> His-tagged protein and IMAC under native conditions One-step purification of maize ß-glucosidase > Perfusion matrix: POROS MC/M > Functional group: iminodiacetate, metal ion Zn2+ > Removing contaminated proteins: linear gradient of imidazole (0-50 mM) and pH (pH 7-6.1) > Protein elution: 0.1 M EDTA > 80% recovery, 95 fold purification > Common production and isolation of the wild type protein and soluble mutant form for enzymatic measurements and crystallization. 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): > Binding to the column under strong denaturing conditions (8 M urea) > 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-OM urea). Identification of properly refolded (His)6Zm-p60.1 (maize P-glucosidase) using 10% native PAGE, followed by activity in gel staining: /(His)sZm-p60.r 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 A B (Zouhar et al, 1999) 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. His-tagged protein and IMAC under native conditions Two-step purification of Arabidopsis histidine phosphotransfer protein 5 > IMAC matrix: highly cross-linked spherical agarose (His)6AHP > Functional group: nitrilotriacetic acid, metal ion Ni2+ > Removing contaminated proteins: linear gradient of imidazole (20-500 mM) > Protein elution: 130 mM imidazol > Common production and isolation of the wild type protein for protein-protein interaction measurements and crystallization. 1st step - metal chelate affinity chromatography -► 2nd step - gel filtration 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 Ub- SGSG- •HisTaq- SA- FEV- AME-( :kii 3. Affinity purification after TEV cleavege CKI1 RD pETM-60::£KIlRD 200 mM imidazole mmm mssMmm \má uuuu m uuuu i,y pETM-60::CK] after cleavege is ird •t7" cki < - RD pETM-60 4. Size-exclusion chromatography 1 -10-20 mg for TB and M9 Pekařova B. Otázka č.4: Jakými metodami se izolují proteiny fúzované s nechromatografickými tagy/kotvami? Affinity purification for studying protein-protein interaction > Affinity purification provides a high-efficiency method for isolation of interacting proteins and protein complexes: > Co-immunoprecipitation > GST (or His) pull-down > Tandem affinity purification > Testing known protein-protein interaction. > Identification of novel protein-protein interactions. Co-immunoprecipitation (Co-IP) > 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. > An obvious advantage is that complexes are isolated in the state closest to the physiological condition. > 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. Cell lysis under mild conditions that do not disrupt protein-protein interactions (using low salt concentrations, non-ionic detergents, protease inhibitors, phosphatase inhibitors). 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). The antibody-protein(s) complex is then pelleted usually using protein-A or G sepharose, which binds most antibodies . Eluted immunoprecipitates are then fractionated by SDS-PAGE. 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 > 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. > They are used for purification of multiprotein complexes in vitro. > 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). > 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. Stepl, immobilize the Iusmn-lagged "baif fl*m the lysate, Gil JAIrmilrL-aiTi Co*3 ThHaH Ffljil Proltir-Conrimns ***** 4, Wash away unbound protein. Slsp 3, Wash away unbound protein. Slcp 5. Elule protein:protein interaction complex [.ieIjeh; ImtfiCling Cam pirn Slip 3, Bine1 "prey" protein to immobilized "bail protein, Prs/řreUinCcnlínlnj Slip 6. Analyze proTeininruten interaction comproíon SDS WYGE. 'ir, Markei Puiiltd Agfroiř Fvficd Tia\ Ud KiDlnr.h'io;™] ClfUnM Inlcr jcllor- > 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. > The proteins are resolved on SDS-PAGE and processed for further analysis. Tandem affinity purification (TAP) 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 (tandem affinity peptides) tags 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. Tap^agged polypeptide expressed near prryslalogkillpwfrs Olrwd^ftbiftdlngpeptkJeOPl —1 ""X. " i—1 ^ 11V protease dcavaqe she Proiein A IgG-bindlrig domain - PROTEIN PROTEIN INTERACTIONS C*l lyvsirt mildťrjnxíil lions / .ÖS PROTEIN DNA INTERACTIONS Cumlnk «vii h fun™ J úvh yde Chromatin fragmenlattari trrarrjtography * i AHfl rty chrarutog ra ptiy 5D5(jDl 1 RevprseciDüSnks Purify DNA PCB with specific přímen flehtet «fcsUriki PuiffyandLiWDNA Hybridlip Mass Spectrometry Single Locus PCR ISůguůiůryandwnscrlbédJ Location DNA Mitrcurray Colins (a) (b) (c) (d) Protein A Protein A— TEV CBP 20.7 kDa Protein G Protein G— TEV SBP 18.8 kDa FLAG -WWHI 1 »STOWT 4.6 kDa RGS-6xHis Biotin signal 6xHis 9.6 kDa ind Choudhary, 2008 Current Opinion in Biotechnology Tandem affinity purification Gene of Interest CBP TEN'Site — 1 Expression and purification r from a culture. ^ ^Affinity Column 1: IgG Beads IgGBead 6 _____J I Protein Cleavage with TEVProtease Column 2: Calmodulin Beads Ca2+ Ca 2+ 4 I Ca2+ Chelation, Elution 5 Sepai ation 61 Band Excision And Digestion *j I Peptide Separation ' I And MS Analysis ma 1. Protein 2. i 3. B 4. C 5. D (c) Lower background and higher complex yield with GS tag compared to TAP tag 5 mm 11 ♦ 12 (Chepelev et al. 2008) Affinity purification for studying protein-protein interaction > An affinity tags can influence protein-protein interactions (testing N- and C-terminal fusions). > Loss of weak or transient protein-protein interactions. > Non-specificity: controls, affinity tags with higher specificity > Verification of newly identified interactors by other methods and biologically relevant mutants. Comparison of a standard purification process with affinity purification > 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. > 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). > The yields obtained in purification of proteins using affinity chromatography can be over 90 % and include a reduced number of steps. A M I II M 1 Comparison of purification strategies for recombinant pGAP Purification step Total volume (nil) Activity (U/ml) Total activity (U) Yield (%) Standardpurification (untaggedpGAPi Cell extract 750 2.1) 1463 1 in; Phenyl-Sepharose HS 400 2.6 1040 71 Phenyl-Sepharose LS 160 5.6 888 61 Q Sepharose HP 57 10.3 587 4ii IM AC purification (h is- tagp GA P) Cell extract 120 19.0 2280 [ill; Ni-NTA Sepharose 84 26.0 2184 96 HT-pGAP Fig. 2. Comparison of purification strategies for recombinant pOAP. A B. amyloliqttefatiens pyroglutamyl aminopeptidase (pG.AP) was produced in E. co!i with ;md without an N-terminal his-tag {HT-pGAP, tag sequence: MEP(H)6LJ. For untagged pGAP, purification included ammonium sulfate precipitation and two consecutive separation steps using phenyl-Sepharose. Subsequently, a desalting step using a Sephadex G-25 F column and a final step using Q Sepharose HP were performed. For HT-pGAP, purification was performed with a single IMAC step. (A) Standard purification of pGAP. Lane M: MWM (Novex); lane 1: cell extract; lane 2: supernatant fraction of cell extract; lane 3: pool from first phenyl-Sepharose step: lane 4: pool from the second phenyl-Sepharose step; lane 5: pool after desalting; lanes 6-10: several fractions from Q Sepharose HP containing pGAP. (B) IMAC purification of HT-pGAP. Lane M: MWM; lane 1: cell extract; lane 2: supernatant fraction of the cell extract; lane 3: flow through fraction from the IMAC; lane 4: eluted HT-pGAP. See Table 2 for process yields. Arnau et at., 2006