INVAVT3QZA 3ľOAZOÍI OO 3 D 11 S 3 A N I l«iiidIS OA±SU31ŠINII/M QMS OA1SU31S.IMIW ■ ^^^2 ^^_|^^_ ^ mm X)|!iqndsj 3>|S3-) wsBOdzoj luiujfis B Luspuoj wiu|Bpos lu^sdojAj uBAO0ireuijn|ods af }>p!ojd ojuai uopeoijund □ o □ o □ o o □ o □ o □ □ o □ o □ o o □ o □ o □ □ o □ o □ o o □ o □ o □ □ o □ o □ o £jiuyjB puB supjoad uoisnj □ o o □ o □ o □ □ o □ o □ o uoi^oiiund pire uoisssidxa uisjojfT □ ODO O □ O □ ' o □ o □ □ ODO o □ o □ □ o □ o vnzyaAiNn vaoiawsvw 0868O :ni§mp9Jd po» 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 u ]. 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. (i) 5' Promoter Tag CS Gene of interest 3' Terminator Transcribe and translate T Tag fused to the N-terminus of the protein of interest (ii) 5' Promoter Gene of interest CS Tag 3' Terminator Transcribe and translate _T_ N I | c Tag fused to the C-terminus of the protein of interest 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 8 aa N- or C-terminus Purification, secretion Glutathione ^-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 Tag* Advantages Disadvantages GST Efficient translation High metabolic burden initiation Inexpensive affinity resin Homodfrneric protein Mild elution conditions □oes not enhance solubility MBP Efficient translation High metabolic burden initiation Inexpensive affinity resin Enhances solubility Mild elution conditions MusA Efficient translation High metabolic burden initiation Enhances solubility Not an affinity tag Thioredoxin Efficient translation Not an affinity tagh initiation Enhances solubility Ubiq uitin Efficient translation Mot 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. cofi of immobilizing proteins in biotin carboxyl carrier a directed orientation protein on affinity resin □oes not enhance solubility Low metabolic burden Specificity of IMAC is not as high as other affinity methods Inexpensive affinity resin Mild elution conditions Tag works under both □oes not enhance native and denaturing solubility conditions STREP Low metabolic burden Expensive affinity resin High specificity □oes not enhance solubility Mild elution conditions SET Enhances solubility Mot an affinity tag CBP Low metabolic burden Expensive affinity resin High specificity □oes not enhance solubility Mild elution conditions S-tag Low metabolic burden Expensive affinity resin High specificity Harsh elution conditions (oron-column cleavage) □oes not enhance solubility "GET, glutathione S-transferase; MBP, maltose binding protein; NusA, N utilization substance Ar FLAG. FLAG-tag peptide: BAP. biotin acceptor peptide: Hise. hexahistidine tag: STREP, streptavidin binding peptide; SET, solubility enhancing tag; UBP, calmodulin binding peptide. derivatives of thioredoxin have been engineered to have affinity for immobilized metal ions (His-patch thioredoxin'i or avidin/streptavidin [38], XI7 , Waugh, 2005 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. Otázka T. 1: Jaké jsou důvody pro vyuiívaní taguikotev? 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, NusAu ) - 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 coil GST GI utathio ne-S -transfer as e Schistosoma japonicum Trx ThioredoAin Escherichia coil NusA N-Utilization substance Escherichia coil SUMO Small ubiquitin-modifier Homo sapiens SET Solubility-enhancing tag Synthetic DsbC Disulfide bond C Escherichia coil Skp Seventeen kilodalton protein Escherichia coil T7PK Phage TT 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 Dead end: insolubility His6 GST Target protein His6 NusA His6 MBP His6 Trx (a) Express in Target protein £■coli Target protein His6 GST Target protein His6 NusA Target protein lb Target protein Protease cleavage site His6 [MBP His6 ( Trx Target protein Target protein 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. 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). Nhfe ^2 NH Cht 1 Cht Cht 1 1 Cht Cht 1 J Cht Cht HjN+—C —CQs 1 H3N+—C —COfc-1 1 H 1 H Arginine (R) Lysine (K) Z- E Í : - I = •r 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 al, 2006 Biochemical properties of poly-Arg and poly- Lys tagged BPTI-22 protein Protein Solubility Conn. 1 m jVÍ | Soliih ilization Re I. Trypsin Inhibitory Protein (Cone. 1 mg/ml 1 )* Factorb Activity (%)c bpti 11 1.70 (10.00) XIK 1.70 (10.40) 1.00 (1.04) 35.2 1.05 -N3K l.bb ■ 1 W>7' 136(2.00) 34.4 1.04 K5K 5.37 (35.60) ji.]f>(3.56) 34.3 1.05 -C1K 1.79 (10.95) 1.05(1.10) 34.6 1.05 The addition of 0.5 M Arg -C3K: 2.41 (15.28) 7.16 ■47.471 1.42(1.53) ^^U^5) 36.2 35.0 1.05 1.02 barely increased its solubility, and trypsin activity was inhibited by the high arginine concentration. On the other -N3R ^R^??!?4) 2.70 (17.23) 6.20 (41.11) ^f?9(u!?) 1.59 (1.72) 3.65(4.11) 353 TÍ2™ 0.99 0.99 -OR 1.81 (11.07) 1.06(1.11) 35.0 1.05 hand, addition of 50 mM -OR 3.02 (19.26) 1.78(1.93) 34,4 1.05 Arg+Glu was more effective -C5R 8.23 (54.56) 10.59(73.41) 4^4 (5.46) 6.22 (7.34) 34.8 32.7 1.08 1.1 and increased protein solubility more than threefold. BpT! 22J bptj 111 5.63 (33.1 lj) 2.01 (11.32) 3Ji(JJij l.lft(l.lS) Utf 1.09 1 Prolan solubility was del tr mined as the maximum supernatant concentration of a supersaturabed protein solution at 4"C in I0U mM acetatebufter pi! 4.7. A Maximum concentrations calculated in milligrams per rnflliliLu" art' in lIIlatin parentltesLv The Mw of BPTI-22, - NIK and -OK, -N3Kand -C3K, -N5Kand-C5K,-NIR and-ClR,-N3R and-C3R,-N5R and -CSR, and -C6Rare,respectively: 5880,6123,6379,6636,6151,6463,6776, and 6932 Da. b Calculated as the. ratio between [tie miliar p rotein solubilit y o f BPTI -22 and that of tagged BPTI-22. Values in parenthesis indicate tlte ratio calculated in milligrams per mi Hi liter h. " Relative trypsin inhibitory act ivity ca k dated as the ratio between tlte activity ox BPTI-22 and that of tagged BPTI-2 2. BPTI-2 2, which lacks R39, an arginine residue involved in two hydrogen bonding interactions with tlte trypsin residue backbone, has a reduced trypsin inhibitor activity tor responding to -60% of the wt-BPTI and BPTI-. 5,55| at stoichiometry and a protein concentration of 280 riAl" Solubility in the same buffer as above but with the addition of 50 mM t-Arg +■ t-Glu. " The CD thermal melting curve could not be deter mi ned due to the strong absorption of arginine and glutamic acid. f Protein solubility with 50U mMArg-HCI added to the above buffer. * The trypsin activity could not he determined because the hig h ar gin ine conttn [ration inhibited trypsin activity. Kato et ah, 2006 A) BPTI-22: HPAFCLEPPYAOPAKABIIRYFyNAAAOAAQArVYOOAAAKBNNFABAADALAACAAA B) (a) . ['■: [.c IL, IWYl 22 ribbon model with a-helices colored red and ^-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 ii-sheet pointing to the back in fa) and to the front in (b). The N- and C-termini are Libeled "N" and 11C," respectively. The C-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. ^ 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 vyuiily pro zvýpení 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 I MRGSHHHHHH M12 M15 / / GMASMEKNNQ M28V / GNGQGHNVPN 40 I 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 al, 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 l-A pH intein N C Thiol intein A4 pH 6.0-6.5 -i 20-25°C, 16 h 15-30 mM thiol -i 4QC, 16 h A- pH intein N + ^ ^ + C Thiol intein A Tag Target Protein r intein Cysl N-S acvl shift r 0 H2N S* Thiocstcr 1 intein thiol reagent SR + intein 0 C-terminal a-thioester Perler, (2005) Removal of fusion tags - self cleaving fusion tag 2. HHHHHH—I SrtAf,n.2i»>~|—LPXIX?— 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(proj —C X— 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. 4. target protein I—D P— SPM A E Ms Of (.'[ill 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 —VDALADGK— CPD —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 Table 3 General features of the five self-cleavage fusion systems discussed in the text Self- MW* Purification Cleavage Advantages Disadvantages cleaving (kDa) tag condition tag Jntcin 51; CBD, CBM, Thiols; pH flexible fusion and cleavage options; Lack of solubility-enhancing capacity; in phasin, and/or allowing generation of target protein vivo cleavage; incomplete cleavage; 17: LLP temperature with native sequence misclcavagc 15" shift SrtA 17 His-tag, hi o tin 5 mM Ca_+ Potential of enhancing target protein expression and solubility In vivo cleavage; incomplete cleavage; introduction of an extra Gly residue to the A'-tcrminus of the target protein 19 His-tag Kos inotropic conditions Allowing generation of target protein with native sequence Limited to proteins capable of refolding; in vivo cleavage; incomplete cleavage; long cleavage time LrpC 26 His-tag, CBD 10 mM Ca-~ Lfficicnt and tightly controlled cleavage; insensitive to protease inhibitors Lack of solubility-enhancing capacity; introduction of an extra Asp residue to the t'-tcrminus of the target protein; single C-tcrminal fusion option CPD 23 His-tag 50-100 tiM 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 C-tcrminus of the target protein; single C-tcrminal fusion option J Molecular weight of the self-cleaving tag b Inteins with different sizes arc available Li, 2011 Removal of fusion tags enzymatic cleavage Cleavage site Protease Target 4-37°C, time varies Site-specific proteolytic cleavage: y Exopeptidases > Endopeptidases Exopeptidases (aminopeptidases and carboxypeptidases): DA Päse (TAGZyme) Aeromortas aminopeptidase Aminopeptidase M Carboxy peptidase A Carboxy peptidase B Exo(di)peptidase Exopeptidase Exopeptidase Exopeptidase Exopeptidase Cleaves N-terminal. His-tag (C-terminal) for purification and removal Cleaves N-terminal, effective on M,L. Requires Zn Cleaves N-terminal, does not cleave X-P Cleaves C-terminal. No cleavage at X-R, P Cleaves C-terminal R, K. y 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 Jtop point (1) sequence' Lysine (Lys. Kj Arginine [Ang. R) Proline (Pro. P) Proline (Pro. P) GLtamine (Gin. QJ1 Xaa-Xaa. Xaa-Xaa. Xaa-Xaa. Xaa-Xaa. Xaa-Xaa. Xaa-Xaa 1 Lys-Xaa ... Xaa-Xaa 1 Arg-Xaa Xaa-Xaa I Xaa-Xaa ProXaa.. Xaa-Xaa i- Xaa-Pro Xaa-Xaa.. Xaa-Xaa I Gln-Xaa... DAPase cleavage [1 T stop M K HQ i HQ HQ HQ H H P 1 HT-Tix HHP-Tix M 1 2 3 4 5 6 7 ■ — 4-HT-Tix *-HHP-Ti x Arnau et at., 2006 Removal of fusion tags - enzymatic cleavage Endopeptidases Enzyme Cleavage site Comments Enterokinase DDDDK* Secondary sites at other basic aa Factor Xa IDGR* Secondary sites at GR Thrombin LVPR'CS Secondary sites, Biotin labeled for removal of the protease Pre Scission LEVLFQ'GP GST tag for removal of the protease TEV protease EQLYFQ*G His-tag for removal of the protease 3C protease ETLFQ*GP GST tag for removal of the protease Sortase 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, r^X. M*N.S*X Serine protease. Risk for unspecific cleavage , Protease site HiSc MBP Target protein Enterokinase Asp-Asp-Asp-Asp-Lys/X Table 4 Cleavage (%) of enterokinase through densitometry (Hosfield and Lu 1999) based on the amino acid residue Xi. The sequence.. „-GSD YKDDDDK-X, - ADQLTEEQ1A-... of a GST-cal- modulin fusion protein was tested using 5 mg protein digested with 0,2 Uof enterokinase for I ň h at 37 "C Amino acid in position X| Cleavage of enterokinase (%) Alanine 88 Methionine 86 Lysine 85 Leucine 85 Asparagine 85 Phenylalanine 85 1 so leucine 84 Aspartic acid 84 Glutamic acid 80 Glutamine 79 Valine 79 Arginine 78 Threoni ne 78 Tyrosine 78 Histidine 76 Serine 76 Cysteine 74 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. Lundbladc,d fl Haematofoglc Ted mo fogies. Inc., Essex Junction, VT, USA Department of Biochemistry, University of Vermont, Burlington, VT, USA e Department of Pathology, University of North Carolina, Chapel Hill, JVC. USA d Roger L Lun&Iad LLC, Chapel Hill, NC, USA Received 27 February 2003, and in revised farm 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 cleLived by thrombin in polypeptide hormones Polypeptide horn ones* Sl'l-jl':::i HLSLSRLRDS A Secretin ELSLSRLR (much slower than above) Vasoactive intestine polypeptide DNYTRLRK Va so active intestine polypeptide YTR LRKQM Ch oleocystokinin APSGRVSM Ch oleocystokinin VSMJKNLQ Dynorphin A RIRPKLKW Somatostatin-28 AM APR HR K Somatostatin-28 NFFWKTFT Gastrin releasing peptide JCMYPRGNH Salmon calcitonin QTYPRTNT aThe reaction mixtures contained 0.5N1H units thrombin and l.Onmol peptide in 20 uL of 50 mM NH4CO3, pH SO, at25°C The conditions were designed to obtain an enzyme/ subs t r ate ratio of 1:60 (w/w). Protein ^Expression ^Purification Accuracy of cleavage has to be precisely verified! pRSETB::AHP2 Enterokinase cleavage site NT^GSHHHHHHGMASMTGGQ .u u u u u .GIVPQVDIN C Theoretically: 3,4 kDa 18,9 kDa Intact mass spectrometry analysis a.i. 1000 800 600 400 200 - r 4041 3 22 1 NLkJii 3887 1988 y V 3894 ' 4053 AHP2 enterokinase 4823 1 *1 [88 11031 I I 18256 I 1 1 1 11D39 75 1 1 1 3A 12 AHP2_control J AHP2 standard 22^37 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-vyptapujícím tágem odvozeným od inteinu? Affinity chromatography (AC) 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 p-galactosidase fi-galactosidase) G1 uta th ione G! utathion e -S- T ra n sferase Immunoglobulin G Protein A Cu II, Co II or Ni II poly His or poly Cys ^ 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 I m mob I i zed binding partner of Affinity tag fused to N- orC-terminus affinity lag of protein TPEG (substrate analogue of [}-galactasidase JJ-galactosidase) Glutathione Glutathione-S-Transferase Immunoglobulin G Protein A Cu II» Co II or Ni II poly His or poly Cys Tabid Sequence and size of affinity tags Tag Re si dues Sequenee Size (kDa) Poly-Arg 5-6 RRRRR 0.80 (usual Iv 5) Poly-His 2-10 HHHHHH 0.84 (usuallv 6) FLAG 8 DYKDDDDK 1.01 Strop-tag II 8 WSHPQFEK 1.06 c-myc 1 1 EQKL1SEEDL 1.20 S- 15 KETAAAKFERQHMDS 1.75 HAT- 19 KDHL1HNVHKEFHAHAHNK 2.31 3x FLAG 22 DYKDHDGDYKDHDIDYKDDDDK 2.73 Calmoduliri-biriding peptide 26 KRRWKKNF1AVSAANRFKK1SSSGAL 2.96 Cellu lose-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 Chi tin-binding domain 51 TN PG V S AW Q V NT A YT AGQL VT Y NGKT YKC LQ P HT S L AGWEPSNVPALWQLQ 5.59 Glutathione ^transferase 211 Protein 26.00 Ma It ose-bin ding 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 Affin it v tag Matrix Poly-Arg Poly-His FLAG Strep-tag II c-myc S HAT (natural histidine affinity tag) Calmodulin-binding peptide Cellulose -binding domain SBP Chi ti n-bi ndi ng domain Glutathione S-transferase Maltose-binding protein Cation-exchange resin Ni'+-NTA, Coy+-CMA (Talon) Anti-FLAG monoclonal antibody S trep -Tact in (m odi fie d st re pta v i di n) Monoclonal antibody S-fragment of RNaseA Co2+-CMA (Talon) Calmodulin Cellulose S trep tavi din Chi tin Glutathione Cross-linked amvlose Non - chromatographic tags Tag_Matrix ELP PHB annexin Gl 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 y Traditional purification tags y The tag binds strongly and selectively to an immobilized ligand on a solid support. ^ After optimization one could achieve > 90% purity. T X Additional separation + DTT T O 1 o v AfFir»ity tag - Imker - target protein vf protease cleavage Affinity tag - mtein - target protein 4 Pliasiri - intein - target protein Centrifugation E Mild heating and/or ^ salt addition ELP - mtein - target protem Purification tags The PHB system (c): Non - chromatographic tags > 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. > 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 / \ Inefficient binding target el utes djring binding and eliition ~i-T LO 15 20 25 Eiution volume, ml Efficient binding target elutes in a single peak ~^~\--r--1-1— 0 5 10 15 20 25 ELution volume, ml Fig. 56* UsiDg spacer arms, a) Ligand attached directly to the matrix, b} Ligand attached to the matrix VLa 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 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 32+, and Zn2+. 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. x I l_ \ W i J r CH—CH. II' 0 M (kDa) 170 • 116 -86 ■ 58 Binding strength of His tag to metal ions: Cu2+ > Ni2+ > Zn2+ ~ Co2+ 27 20 -(HisißZm-peO. Zn2+ Ni2+ Co2+ Cu2+ ^ 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 (Zouhar et ah, 1999) 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 Hisn protease cleavage site — PROTETN + charged metal chelate resin I PH [imidazole] [EDTA] [\ 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. A B C D (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): y 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 (3-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 kcatwas hampered by the fact that the refolding process yielded a number of improperly folded polypeptides. Zm-p60.1/ /(His)EZm-p60.r (Zouhar et al, 1999) 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)6AHPi ^ 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 3. Affinity purification after TEV cleavege Ub- SGSG- •HisTaq- ■SA- TEV -AME- CKI1 200 mM imidazole CKIl RD 10 cv pETM-60 pETM-60::CKIlRD 10 rhMJmidaz. \ iui,i:jju^uui,uiuuuuujir pETM-60::CK RD ■ pETM-60 4. Size-exclusion chromatography 1L" -10-20 mg for TB and M9 Pekařova B. Otázka T.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 .Identification of novel interaction is carried out by mass spectrometry analysis. Pull-down assay y 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. y 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). 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. y Centrifugation is used to collect the GST fusion probe protein and adhering proteins. y The complexes are washed to remove nonspecifically adhering proteins. Stspl, Immobilize the luswn-tagged '■i):=!l ! h: 11 == {H\■AT1G20930CDKB2;2 7)AT3G54180CDKB1;1 S)AT3G48750CDKA;1 9) ArachO5g16630 (Eugene) unknown 10) AT4G16143 knportin alpha-2 II) ATSÖ40460 unknown 12) AT1G10690 unknown 13) AT5O27*i0Cyi!H;1 14) AT4Q3082DMAT1 15) AT1G03190UVH6 16) AT1G55750 TFIIH-falated 17) AT1G32380PRS2 1S)AT2G35390PRS1 19) AT2G44530 PRS, putative 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.