O □ O □ O □ S cononono □ ono f-í^O- * /\>n O □ O □ O □ ono o^S^ _jyi/2 ° D ° D ° □ on .^^H ^ft □ o □ o □ □ on v y o d o^ A d o o d c^< ■ d o c^»- o d o j. □ o □ odo d vy-. dodolGJK o d o d o ľj o d o d o d ^0 dodo ^Xi1 o d o d hh ) d o d o rZ. j o d o d >^0 d o d o □ o □ o □ 4 j o d o d o "3-lODODOD JODODODO o d o □ o d □ o d o □ o o d o □ o d □ o d o □ o o d o □ o d □ o d o □ o d a c Ol^> □ O d 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 ...]. (i) 5' Promoter Tag Gene of interest 3' Terminator Transcribe and translate NC □ C Tag fused to the N-terminus of the protein of interest (ii) - 5' Promoter Gene of 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 I 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 5-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 + 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 Tag" Advantages Disadvantages GST Efficient translation High metabolic burden initiation Inexpensive affinity resin Hornodimeric 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 Lag Thioredoxin Efficient translation Not an affinity tag1" initiation Enhances solubility L! 'i. L.I II 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 £. coli of immobilizing proteins in biotin carboxyl carrier a directed orientation protein on affinity resin Does not enhance solubility Hise Low metabolic burden Specificity of IMAC is not as high as other affinity methods Inexpensive affinity resin Mild elution conditions Tag works under both Does not enhance native and denaturing solubility conditions STHEP 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; His6. hexahistidine tag; STREP, stroptavidin 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 thioredoxin) or avidin/streptavidin [3B1. , 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 of a soluble fusion partner often improves the solubility (proper folding) 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 coti GST GI Lrtathione -S -transferase Schistosoma japonicum Trx Thioredoxin Escherichia coti NusA N-Utilization substance Escherichia coti SUMO Small ubiquitin-modifier Homo sapiens SET Solubility-enhancing tag Synthetic DsbC Disulfide bond C Escherichia coti Skp Seventeen kilodalton protein Escherichia coti T7PK Phage T7 protein kinase Bacteriophage T7 GB1 Protein G B1 domain Streptococcus sp. 27 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 Target protein (a) J/ Express in jP/ E. coli N ^ Target protein His6 GST Target protein Target protein His6 MBP Target protein Target protein Protease cleavage site His6( Trx Target protein (e) Target protein SUCCESS His6 (mbp) imac ft V_y Targe! protein Good cleavage m Purify by [MAC Cleave with protease Poor \ cleavage (c) His6 (NusA Target protein His6 (NusA) 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 (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 slsisisi s Is I S I 3 I s I s I s i s I 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 ah, 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 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. Higly acidic tags (peptide) inhibit aggregation by increasing electrostatic repulsion between nascent polypepdides (Zhang et. 2004). Solubility-enhancing tag - mechanism of action Thioredoxin > Serves as a general protein disulfide oxidoreductase. > 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. > Increasing the solubility of the target proteins by overproduction of thioredoxin strongly suggests that the redox state affects the solubility of target proteins. 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 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). J 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 Proti in Solubility Protein BPTI-22 Cnnc |mAf| (Cone. | mg/ml | ■ Solubilization Factor Re L Trypsin Inhibitory" Artiiřity (%)c 1,70 (H).PÜ) 3s.4 -nik 1.70 (10.40) 1.00(1.04) 352 1.05 -n3k 2.66 (19.97) 136(2.00) 344 1.04 n5k 537 (35.60) 3.16(336) 343 1.05 ■ c1k 1.79 (10.95) 1.05(1.10) 346 1j05 ■ c3íc 2.41 (15.23) 1.42(133) 362 1j05 C.SK 7.16 (47.47) 421 í4.75) 33.0 1.02 ■Mir 1.69(10.34) 0.99(1.03) 35\5 1.02 -n3r 2.70 (1723) 139(1.72) 35.6 099 -n5r 620 (41.11) 3.65(4.11) 353 0.99 -c1r lsi (11.07) 1.06(1.11) 35.0 1.05 ■ c3r 3.02 (1926) 1.73(1.93) 34.4 1.05 -c5r 323 (54.56) 4.34(5.46) 34.3 1.03 -CfiR 10.59(73.41) 6.22 ( 7.34) 32.7 1.1 BPTI-32" 5.63 (33.11) 3.31 (331) 1.09 BPTI^' 2.01 (11.82) 1.13(1.18) XI f 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 was determ irted as ibe maximum .i.p trrutin I conccntra tio n of a supersaturated protein solution at 4^ in IÚĽ mAf acetate buffer pľl 4.7. 1 Maximum concentrations fjj._ L.i.; I l-.! in milligrams per milliliter are indicated in parenthesis, The Mw of BPTI-22, - Ní K and -ClK, -Norland -CÍĽ., - r^ K arrf -C5K, -N IR and -C IR, -N^ 6463,6776, and 6932 Di. ■■ .-. I.i.l. ilťii -Lh llit ratio between t be molar p rotein .sin LubiLit y t > f BPT1 -11 and Lhit u f lagged KPTJ-22. Vd ues En p irerlthes is indicate the r.Li i ■ - calculated in milligrams per miltiliteFS. 1 Relative trypsin inhibitory activity calculatedas the ratio helweer.1 the activity of BPTI-22 and that ol tigged BPTI-22. BPTI-22, which Lackii R'M, aisacJanine residue involved in two hydriľgen bonding interactions with the trypsin residue backbone,34 has a reduced trypiin inhibitor activity corresponding to ~ 60% o ť the wl-BPT I a nd BPTI -; 5, 5 S ] at stoic bin [net ry and a protein concentration of 26il ^ Solubility in the .same buffer as above hu t with the addition of mML-Arg. -t-L-Glu. ' Tlie e D t hernia I rn ell ing curve could not be deter mined due to the strong absorption of arginine and glutamic acid. f Protein solubility witb SOO mM Arg-HCl added to the above buffer. 6 The trypsin activity could not be determined because the high arginine concert Er at kin inhibited trypsin activity, Kato et ah, 2006 A) BPTI 22 BPAFCLEPPYAGPAKARlIfiYTyNAAAQAAQAFVYOOAAAKBNNTABAADALAACAAA B) (a) (b) FIGURE 3 Hydrophobic residues in BPTI-22. A: One letter amino add sequence of BPTI-22 widi die hydrophobic residues {A,V,l,LTiP) shown in green letters. 13: Left, 13 IT 1-22 ribbon model with ci-helices colored red and 0~ strands colored blue. Right, surface representation of 13PT1-22 with the hydrophobic area determined as low electrostatic potential regions according to MOL-MOL>15 colored green. The molecule is oriented with the rf-sheet pointing to the back in (a) and to the front in (b). The N- andl'J-termini are labeled "N" and "C,* respectively The C-terminal end is located on the same face as a iarge hydrophobic patch shown in green, whereas the N-terminal end is on die opposite side of the molecule and is shown with a light gray letter "N' in panel fb). > 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 at, 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 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. 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, 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 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-processing fusion tags 1. Inteins Precursor lutein (b) Protein splicing (intramolecular) Final protein Inteins (mtervening 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 Tag a pH intein -n^ —ph 6'°6'5—>- Tag a pH intein -n + ^ Mmt^ ^mm^ 20-25°C, 16 h ^gUy^ Removal of fusion tags - Self - cleaving fusion tag 2. HHHHHH—[ SrtAfio-206 ~|—LFXTX} — target protein System based on the catalytic domain of 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) —C X— target protein N-terminal protease (NPro) is the first protein of the pestivirus polyprotein. It posesses autoproteolytic activity and catalyzes the cleavage under cosmotropic conditions. 4. target protein —D P— SPM fiEMs or (.'[{]) FrpC modul: 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— 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 > 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 Tabic i General features of the five self-cleavage fusion systems discussed in the text Sclf- MW Purification Cleavage Advantages Disadvantages clcaving (kDa) tag condition tag lntcin 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: 15" ELP temperature shift with native sequence misclcavagc SrtA 17 His-tag, 5 niM Ca2+ Potential of enhancing target protein In vivo cleavage; incomplete cleavage; hiotin expression and solubility introduction of an extra Gly residue to the A'-tcrminus of the target protein 19 His-tag Kosmotropic Allowing generation of target protein Limited to proteins capable of refolding; conditions with native sequence in vivo cleavage; incomplete cleavage; long cleavage time I-rpC 26 His-tag, 10 mM Ca-~ Efficient and tightly controlled Lack of solubility-enhancing capacity; CBD cleavage; insensitive to protease introduction of an extra Asp residue to inhibitors the C-tcrminus of the target protein; single C-tcrminal fusion option CPD 23 His-tag 50-100 nM Potential of enhancing target protein Introduction of up to four non-native lnsP6 expression and solubility; efficient and residues to the C-tcrminus of the target tightly controlled cleavage; insensitive protein; single C-tcrminal fusion option to protease inhibitors J Molecular weight of the self-cleaving tag b 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) Aeromonas aniinopeptidase Aniinopeptidase M Car boxy peptidase A Car boxy peptidase B Exo(di)peptidase Exopeptidase Exopeptidase Exopeptidase Exopepcidase 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. > 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 aniinopeptidase I) TAGZyme stop points_ -milt: aeid DAPase stop point ( The enzymatic cleavage site has to be placed between the fusion tag and the target protein. Enzyme C lea vage site Comments Enterokinase DDDDK* Secondary sites at other basic aa Factor Xa IDGR* Secondary sites at GR Thrombin LVPR'GS Secondary sites. Biotin labeled for removal of the protease PreScission levlfq'gp GST tag for removal of the protease TEV protease EQLYFQ'C His-tag for removal of the protease 3C protease ETLFQXP GST tag for removal of the protease Sortase A LPET'G Ca!+-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 Hisc MBP Target protein Enterokinase Asp-Asp-Asp-Asp-Lys/X Table 4 Cleavage (%) of enterokinase through densitometry (Host i eld and La 1999) based on the amino acid residue X|. The sequence...,-GSDYKDDDDK-Xi-ADQLTEEQ] A... of a GST-cal- modulin fusion protein was tested using 5 nig protein digested with 0.2 Uof enterokinase for 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 I so leucine 84 A spart ic 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,"'* Kenneth G. Mann,b and Roger L. Lundbladc*d a Haematologtc Technologies, Inc., Essex Junction, VT, USA b Department of Biochemistry, University of Vermont, Burlington, IT, USA c Department of Pathology, University of North Carolina, Chapel Hilt, NC, USA d Roger L Lumthlad, 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 horn ones* Sequence cleaved Secretin ELSLSRLRDSA Secretin ELSLSRLR (much slower than above) Vasoactive intestine polypeptide DNYTRLRK Vasoactive intestine polypeptide YTR LRKQM Ch oleocystokinin APSGRVSM Ch oleocystokinin VSMJKNLQ Dynorphin A RJRPKLKW Somatostatin-28 AM APRERK Somatostatin-28 NFFWKTFT Gastrin releasing peptide KMYPRGNH Salmon calcitonin QTYPRTNT aThe reaction mixtures contained 0.5 N1H units thrombin and l.Onmol peptide in 20 uL of 50 mM NH4CO3, pH 8.0, at 25 °C. The conditions were designed to obtain an enzyme/substrate ratio of 1:60 (w/w). Protein -Expression ^Purification Accuracy of cleavage has to be precisely verified! pRSETB::AHP2 Enterokinase cleavage site N'M^SHHHHHHGMASMTGGQQMGRDLYDDDDKp^^SRSAAGTMEFMDALIA....................GIVPQVDJJNI 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 4 9 2 3 ilk iwLi 110 3 1 I________________i___ 7 5 1 D 3 9 AHP2 enterokinase 1 3 t- y a 6 1 8 2 5 8 1 AHP2 control AHP2 standard 4 0 5 3 1 1 0 3 4 il a 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). > 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: 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. The small tags are a better choice in structural biology. Affinity chromatography > 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). 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, cairier protein > AC has a concentrating effect, the high selectivities of separations derived from the natural specificities of the interacting molecules. > AC can be used (1) to purify substances from complex biological mixtures, (2) separate native forms from denatured forms of the same substance, and (3) 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 Tahle 2 Sequence and size of affinity lags Tag Residues 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 KETAAAKFERQHMDS 1.75 HAT- 19 KDHL1HNVHKEFHAHAHNK 2.31 3x FLAG DYKDHDGDYKDHD1DYKDDDDK 2.73 C al modu li n-bi ndi ng pept ide 26 KRRWKKNF1AVSAANRFKK1SSSGAL 2.% Cellu lose-binding domains 27-189 Domains 3.00- 20.00 SBP 38 M D EKTTGW RGGH V V EGL AGEL EQLR A R LEHH PQ G Q R EP 4.03 Chi ti n-bi ndi ng domain 51 TNPGVSAWQV NTA YT AG QL VTY NG KTYKCLQPHTSLAGWEPSNVPA LW Q LQ 5.59 Glutathione S-transferase 211 Protei n 2fi.00 Maltose-binding protein 396 Protein 40.00 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 p-galactosidase) Glutathione Glutathione-S-Transferase Immunoglobulin G Protein A Cu it, Co II or Ni ii poly His or po]y Cys 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 Affinity tag Matrix Poly-Arg Pol v-His FLAG Strep-tag II c-myc S HAT (natural histidine affinity tag) Calmodulin-binding peptide Cellulo se -bi nd ing dorn ai n SBP Chi ti n-bi ridi ng do m ai n Glutathione S-transferase Maltose-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 amy lose Non - chromatographic tags Tag_Matrix ELP PHB annexin Bi 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-splicing 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. JOS it' T X Additional separation step b i + DTT T O 1 Affinity tag-Inter-target protein j| Protei ctea^ge j Mi,d heatt,g and/or I salt addition + CentnhjastiQn q I Affinity tag - mtein - target protein * 1 Centrifugerjon Phasin - lutein - target protein ELP - intein - target protein Purification tags Non - chromatographic tags + DTT Mild heating and/or ^ salt addition O 1 o Centr ifugation 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 - intsin - 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. 31 Components of a matrix for affinity chromatography > 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. 32 Typical affinity purification steps adsorption of wash 1-2 cv m- x cv-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: «_ 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 His protease cleavage site -4 charged metal chelate resin pll [imidazole] ^ [EDTA] t\ protease + /I/—\ + + + 111 |H 31 1> His-tagged protein and IMAC under native conditions One-step purification of maize p-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. M (kDa) 170 - 116 - A B C D (Zouhar et ah, 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): > 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: Zm-p60.1/ /(His)EZm-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: nitrilacetic 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 (MCAC) 2. Tag removal (TEV protease) 3. Affinity purification (MCAC) 4. Size exclusion chromatography Ub- SGSG- ■HisTaq ■SA- TEV -AME- CKI1 3. Affinity purification after TEV cleavege CKI1 RD pETM-60::CKIl 0 rnM imidaz^ 200 mM imidazole uumroamu 1,^1 uuuu 1,1.1 juuuui,u pETM-60::CKl 4. Size-exclusion chromatography RD pETM-60 1 10-20 mgforTB and M9 Pekařova B. 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 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. Step-!, IJTunobilizethelus»n-lagged "baif fmm the tysate. Gil 'AlfinilfL-^Tl Hjil ?roHir-Cjnf*n,ns Lv.jIl- Step 4. Wash away unbound protein. UssbJ Slep I. Wasii away unbound protein. Slcp 5. Elule protein prnipir interaction comnlcx. ňipljctrf ImtfitUng Cum pin Slep 3. Bind "prey" protein to immobilized "bail r rote ■■ Prwf ft otiirvC griming Slep 6. Analyze proteiniDrgteh interaction com brk on SDSFAGE. > 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. tow Mtkti PuiiPtd "qj-in K-ficd Km. Gd ndw haul 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. CaEnnůdulrri blrtdJrig peptide (CBP) TEV protease demg« site —1 Pictiin A JtjC-bindlng domain-1 Ta^:ago^tf polypeptide eťpreaed near physiological lewis PROTEIN-PROTEIN INTERACTIONS Cel tyjls In mild conditions ^ »* J, r, • 11 J- affinity chromsiograpliy SDSgel Mass Spectrometry Sinj Ic Locus PCR Genomic sequences [Regu latory and Transcribed] Location DM Micioarray (a) (b) Protein A - Protein A— TEV CBP 20.7 kDa Protein G Protein G TEV SBP 18,8 kDa (c) FLAG - (d) Biotin signal 4.6 kDa 9.6 kDa Colins and Choudhary, 2008 Current Opinion in Biotechnology Tandem affinity purification 7 1^ 1 Ifi Gene of Interest CBP TEY Expression and purification '■otn a culture. 2 ^Affinity Column 1: IgQ Beads ^ ^ Cleavage I Column 2 Y/ IgG.Bead 6 I TEVProtease y •din Beads Ca 2+ jib! 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 _ - —1-1--1— Comparison of purification strategies for recombinant pGAP Purification step Total volume (nil) Activity (U/ml) Total activity (U) Yield (%) Standard purification { untagged pGAPI Cell extract 75(1 2j0 1463 Li n; Phenyl-Sepharose HS 400 2.6 1040 7 L Phenyl-Sepharose LS 160 5.6 61 Q Sepharose HP 57 10.3 40 IMACpurification {his-tagpGAPJ Cell extract 120 19j0 2280 LOO Ni-NTA Sepharose 84 26.0 2184 96 B M 1 2 3 4 M HT-pOAP Fig. 2. Comparison of purification strategies for recombinant pGAP. A 5. amyloliqttefaciem pyroglutamyl aminopeptidase (pGAP) was produced in E. co/i with and without an N-terminal his-tag (HT-pGAP, tag sequence: MEP(H)6L). 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 al, 2006