Purification of recombinant proteins Protein purification from a complex mixture of macromolecules present in a biological sample 2D electrophoresis of the cell extract • Several thousand proteins with different properties (~ 5000-8000) and in different amounts (actin ~ 10%, unique transcription factor <0.001% of total proteins) • DNA, RNA, polysacharides, lipids Biomas desintegration Physical methods: sonication by ultrasound, pressure in a French press, osmotic shock, shear forces in various types of grinders and homogenizers - must be cooled during sonication or mechanical methods! Chemical: detergents, chelators in lysis buffers, organic solvents - substances may interfere with the subsequent purification method Enzymatic: must be chosen according to the expression system Lysozyme for bacteria, lyticase or zymolase (glucanase) for yeasts. In all lysis procedures, the cells are destroyed and their contents are released, including proteolytic enzymes. Therefore, it is advisable to add protease inhibitors to the lysis buffer, which will prevent degradation of the product during disintegration and other steps. Before starting 1. Why??? For what purpose? 2. How??? How to analyze target protein? 3. What??? What features has target protein? 1. Why??? For what purpose? Aplication Amount Purity Identification 0,002-0,2 jig High >95% Antibody production (ig-mg middle-high Enzymology 1-5 mg High > 95 % Biofysical studies mg-g High (>95%) 3D structure (crystalization, NMR) 10-20 mg High (>95%) Farmaceutical purposes mg-kg high (99,9%) 2. How??? How to analyze the protein? 1. Polyacrylamide gel electrophoresis with specific detection: Detection of target protein during purification Using antibodies SDS PAGE followed by western bloting with antibody detection Biological activity monitoring during purification • For enzymes e.g. gel staining using chromogenic substrates (or specific constant determination in complex samples) Native PAGE followed by substrate staining, zymogram gel 2. How??? How to analyze the protein? 2. Polyacrylamide gel electrophoresis with nonspecific detection Protein purity and homogeneity monitoring • e.g. coommasie blue, silver staining SDS PAGE Native PAGE — Dimer - — Monomer 3. Determination of protein concentration • e.g. Bradford, Lowry methods What??? What features has target protein? Information about target protein and related proteins from databases, literature or from pilot experiments • Protein size (SDS PAGE, gel filtration nebo analytical centrifugation) • Izoelectric point (izoelectric focusing) • Stability (pH, temperature, presence of salts, proteases, additives ensuring protein solubility) • Purification strategy (methods, buffers, protein stability,.....) 2D and native PAGE • Sample complexity, features of target protein and other contaminating proteins Properties/purification methods Solubility Stability Size pi (surface charge) Hydrophobicity Specific binding site precipitation e.g. ammonium sulphate, low/high pH thermal precipitation gel filtration (gel permeation chromatography) ion exchange chromatography hydrophobic or reverse phase chromatography afinity chromatography Affinity Chromatography - A type of adsorption chromatography, in which the molecule to be purified is specifically and reversibly adsorbed to a complementary binding substance immobilized on an insoluble support. - Mostly it is a specific interaction of affinity fusion tags (eg. polyhistidine, glutathione-S-transferase, etc.) with ligands (eg. metal, glutathione, etc.) in chromatographic matrix. Ion Exchange Chromatography Ion exchange chromatography involves the separation of ionizable molecules based on their total charge. Generally, media, which have cationic or anionic groups, are used as stationary phases and the counter-ion added buffers are used as mobile phases Time In the sample application step, molecules with opposite charge to the media bind to them by ionic interaction. Next, in the elution step, by increasing the concentration of the counter-ions in the mobile phase, molecules with the lowest net charge are eluted first and those with higher charge are eluted later. Ion Exchange Chromatography r*fft[«lfl bindi pi Jf ü-DTjj'UTa^n V Hpf'V^ COO tlind! Protein stability and ion exchange media binding vary with total protein charge, which depends on pH. pH Reslri Type Cation Exchanger Anion Exchanger Not charge ot molecule of interest + — Charge oi resin — + Running conditions 0,5-1.5 pH units below the pi of the mdecule of interest 0.5-1.5 pH units above the pi of the molecule of interest Functional groups used in ion Exchange chromatography media: Anion exchanger Quaternary ammonium (Q) strong Diethylaminoethyl (DEAE)* weak Diethylaminopropyl (ANX)* weak Cation exchanger Sulfopropyl (SP) strong Methyl sulfonate (S) strong Carboxymethyl (CM) weak -CH2-N+-(CH3)3 -CH2-CH2-N+-(CH2-CH3)2 -CH2-CHOH-CH2-N+-(CH2-CH3)2 -CH2-CH2-CH2-S03--CH2-S03-- CH2-COO- A "weak" exchanger is ionized over only a limited pH range, while a "strong" exchanger shows no variation in ion exchange capacity with changes in pH.... Strong exchangers do not vary and remain fully charged over a broad pH range, which can make optimization of separation simpler than with weak exchangers.. Hydrophobic Chromatography Time Ligand used in purification matrix Phenyl -0- Butyl-S -S-!CH2]3-CH3 Butyl -O-lCH^-ChL Octyl -O-ICH^CH, Ether -O-CHj-CHOH-CHa-OH Isopropyl -o-CH-ICHjf Gel permeation Chromatography (gel filtration) • Size-exclusion chromatography separates proteins on the basis of size. • Molecules move through a bed of porous beads. Smaller molecules diffuse further into the pores of the beads and therefore move through the beads more slowly, while larger molecules enter less or not at all and thus move through the beads more quickly. • Both molecular weight and three-dimensional shape contribute to the degree of retention. How many steps are needed? The number of steps used will always depend on the purity requirements and purposed use for the target protein. For most laboratory-scale work a two- or three-step purification protocol will be sufficient. Difficult purifications may require several additional steps. ___ C[]^inal purification*^ High purity of target protein-removal of almost all impurities, mainly product related impurities. a a O Next purification Gain of target protein Removal of major part of impurities other proteins, nucleic acids......, concentration of target proteins. of sample by target protein, icentration and stabilization of target protein. for chromatography Removal of insoluble part of crude extract and lipids, concentration of the sample (salt precipitation or ultrafiltration) Purification steps Logic combination of purification steps Every separation technique should be evaluated with regard to different parameters such as: Speed is important with respect to the protein degradation by proteases in the complex sample Resolution Speed Resolution how efficiently proteins are separated from each other Capacity Capacity (max.) the sample amount that can be handled. Yield-minimalization of protein loss Gain of target protein Goal: fast isolation, stabilization and concentration. Purification techniques: affinity chromatography ion exchange chromatography hydrophobic chromatography Resolution Speed Capacity Yield Column: rProtein A Sepharose Fast Flow, XK16/20, bed height 4.8 cm (9.6 ml) Sample: 600 ml clarified cell culture containing 87.6 mg of IgG^ Starting buffer: 20 mM sodium phosphate, pH 7.0 Elution buffer: 20 mM sodium citrate, pH 40 Flow rate: 5ml/min(150cm/h) 2 0 15 10 0.5 0.0 0 200 400 600 Volume Iml) Fig 4.5. Example of capture step: Purification of lgG?0from clarified cell culture. Next purification of protein Goal: Purification and concentrating. Purification techniques: ion exchange chromatography hydrophobic chromatography Column; XK 16/20 Butyl Sephorose 4 Fast Flow Sample: 5 ml of partially purified Annexin V expressed in E. coli Buffer A: 20 mM Sodium phosphate, pH 7.0,1 M INHJzSOs Buffer B: 20 mM Sodium phosphate, pH 7.0 Flow rate: 100 cm/h Gradient: 0 to 50% B, 20 column volumes A™ 0 60 Time (mini Fig 4.7. Example of an intermediate purification step: Purification of recombinant Annexin V by HIC. Final purification and adjustment of conditions for target protein storage (pH, salts, additives) Goal: Product in high purity. Purification techniques: gel permeation chromatography ion exchange chromatography hydrophobic chromatography Resolution Speed Capacity Yield Column: XK16/60 packed with Superdex 75 prep grade Sample: 1.0 ml of portiolly purified ZZ-bfain IGF Suffer: 300 mM ammonium acetate, pH 6.0 Rewrote. 0.5 ml/min 115 cm/hi 0 12 3 4 Time(h) Fig 4.9. Example of polishing step: removal of dimers and multimers by GF. Column: Mono ST" 5/50 GL Sample: 14.5 ml of partially punfied and desalted transposase TniA Binding buffer: 20 mM MES pH 6.5.1 mM EDTA, 2 mM MgCI,, 1 mM DTT Elurion buffer: 20 mM MES pH 6.5,1 mM EDTA, 2 mM MgCI;. 1 mM DTT. 1 M NaCI Flow rare 1 ml/min Gradient: 0%-100% elution buffer, 20 CV Fig 4.10. Example of polishing: removal of trace contaminants by high-resolution CIEX. Purification of the transposase TniA. His-tagged protein purification 1 step 2 steps 3 steps © Buffer exchange to prepare far I EX. IHiTrap Desalting, ftiPiep 26/10 Desalting coPumnsí Steps in circles a re optional and are applied if necessary. Buff E r exch a nge to remove imidazole oi salts. 7; 500 mM NaCI; no chelators Other proteins: low concentration of imidazole High concentration of imidazole, pH > 7/500 mM NaCI GF ++ + + +++ Most conditions acceptable, limited sample volume Buffer exchange possible diluted sample IEX +++ +++ +++ +++ +++ Low ionic strength. pH depends on protein and lEXtype High ionic strength or pH changed HIC +++ ++ ++ +++ +++ High ionic strength addition of salt required Low ionic strength Basic rules for order of steps in recombinant protein purification • In the early stages, the methods characterized by high capacity and low yield and resolution are needed —» high amount of input material. • Later, methods characterized by high resolution and yield are important, capacity is less relevant —» amount of protein is smaller. • The method should be rank rationally, without intermediate steps like changes of buffers between two separation techniques e.g. after precipitation by ammonium sulfate or after ion exchange chromatography (protein is eluted in high salt concentration) to order hydrophobic chromatography (sample is injected on the column in high salt concentration). • Individual separation methods not to repeat. • The fewer steps, the higher yield of protein. Fusion proteins Translation fusion of sequences coding a recombinant protein and tag. Tags: a) short peptides [ex. (His)n, (Asp)n, (Arg)n ... ]. b) protein domains, entire proteins [ex. MBP, GST, thioredoxin ...]. • Facilitating the purification of recombinant proteins (purification uniformity) • Increasing the yield of recombinant proteins • Enhancing the solubility of recombinant proteins • Improving protein detection • Enabling secretion • Tag can be selectively removed. 5 Promoter Tag Gene of interest ^Transcribe and translate Tag fused to the N-termiiuis of the protein of interest Gene of interest I Transcribe and translate X [ Tag fused to the C-terminus of the protein of" interest Fusion partner (tag) Size Tag placement Uses His-tag 6, 8, or 10 aa N- or C-terminus Purification, detection Thioredoxin 109 aa (11.7 kDa) N- or C-terminus Purification, solubility enhancement Calmodulin-binding domain (CBD) 26 aa N- or C-terminus Purification Avidin/streptavidin Strep-tag 8 aa N- or C-terminus Purification, secretion Glutathione S'-transferase (GST) 26 kDa N-terminus Purification, solubility enhancement Maltose binding protein (MBP) 396 aa (40 kDa) N- or C-terminus Purification, solubility enhancement Green fluorescent protein (GFP) 220 aa (27 kDa) N- or C-terminus Localization, detection, purification Poly-Arg 5-16 aa N- or C-terminus Purification, solubility enhancement N-utilization substance A (NusA) 495 aa (54.8 kDa) N-terminus Solubility enhancement 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 - Advantage of N-terminal tags - Rather proteins (highly soluble proteins) than peptides -Fusion with a soluble fusion partner often helps to properly fold their fusion partners leading to improved solubility (in vivo and in vitro) of the target protein. -The choice of a fusion partner is still a trial-and-error experience. - Fusion partners do not perform equally with all target proteins, and each target protein can be differentially affected by several fusion tags (Esposito and Chatterjee, 2006) >PROTEINS Some commonly used solubility-enhancing fusion partners Tag Protein Source organism MBP Maltase-bincjing protein £ sehetk his coli GST Glutathione-S-transferase Schistosoma japoncum Trx Thioredoxin Escherichia coli NusA N-Utilization substance Escherichia coli SUMO Small ubigutir-modifier Homo sapiens SET Solubilrty-enriancrig tag Synthetic DsbC Disulfide bond C Escherichia coli Skp Seventeen kikxJalton protein Escherthia coli T7PK Phage TV protein kinase Bacteriophage T7 GB1 Protein G B1 domain Streptococcus sp. zz Protein A IgG ZZ repeat domaan Staphylococcus aureus Adopted from Esposito and Chatterjee, 2006 > PEPTIDES Poly-Arg Poly-Lys Generate parallel expression clones Dead end: insolubility His6 I GST]— Target protein His6 (NusA)— Target protein (a) Express in E. coli His6 I MBP)—Target protein I rargi Protease cleavage site Target protein Target protein Target protein Target protein His6 MBP Target protein IMAC FT SUCCESS Target protein Good cleavage Hisü 0 M Purity by IMAC Cleave with protease Poor cleavage Target protein (d) Target protein (c) His6 (NusA Target protein His6 NusA Target protein Dead end: protein insoluble after cleavage ot tag Dead end: difficult to separate cleaved protein from fusion Current Opinion in Bidechnology Schematic representation of the pathway from protein expression to purification using solubility tags (Esposito and Chatterjee, 2006). Solubility-enhancing tags - the mechanism of action -The mechanism by which partners exert their solubilising function is not fully understood and possibly differs between fusion proteins. Examples of possible mechanisms Maltose binding protein (MBP) has an intrinsic chaperone-like activity. MBP might bind reversibly to exposed hydrophobic regions of nascent target polypeptide, steering the polypeptides towards their native conformation by a chaperone like -mechanism. N-utilization substance (NusA) decreased translation rates by mediating transtriptional pausing, that might enable critical folding events to occur. MBP and N-utilization substance (NusA) attract chaperones. The fusion tag drives its partner protein into a chaperone-mediated folding pathway. MBP and N-utilization substance (NusA) interact with GroEL in E. coli (Huang and Chuang, 1999). Small ubiquitin related modifier (SUMO) promotes the proper folding and solubility of its target proteins possibly by exerting chaperoning effects in a similar mechanism to the described for its structural homolog Ubiquitin (Ub; Khorasanizadeh et al, 1996). Negative charged tags (highly acidic peptide) inhibit aggregation by increasing electrostatic repulsion between nascent polypepdides (Zhang et. 2004). Purification tags Tag Chromatographic technique Principle of separation technique poly [His] afinity Bind to metal IgG binding domain afinity Bind to metal Poly [Asp] ion exchange Bind to anion binding matrix Poly [Phe] hydrophobic Bind to hydrophobic matrix Strep-tag afinity Bind to streptavidin Poly [Arg] ion exchange Bind to cation binding matrix These separation techniques are characteristic by equilibrium of all parameters. Speed Resolution Capacity Yield Immobilized metal 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. > 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. M (kDa) 170 - L Ligand 116 — (Ni2+) 86 - -4—(HisioZm-peO.r 56 - 27- Functional/reactive group 5 Support matrix 20 — (agarose) Zn2+ Ni2+ Co2+ Cu 2+ Bond strength: Cu2+ > Ni2+ > Zn2+ ~ Co2+ Metal Chelate Affinity Chromatography Matrix Protein with polyhistidin tag „spacer arm Ligand V (Ni2+)fi / li _ \ Function/reactive group Support matrix (porous polysacharide beads) „spacer arm >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. inefficient Cxnding target «Hulas during binding and eiut^n / EUcibtI binding ľ target ftlutH >n s single peak Elution (íi jt*. mi In;. 56. spater jrm.v dl I _.n..: ittJthcJ dirctll y to [he matrix, hi 1 attached to the matrix vid a *rwvcr arm. Immobilized metal affinity chromatography (IMAC) Purification 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 cleat age site PROTEiN + -1 -i charged metal chelate resin pH [imidazole] [EDTA] protease + -4 f—J$M foe") Ü + -i - + —^0 + + + + + > 1> 1> 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). i60.1/ Tn-p60.r 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 (Zouhar et ctí, 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 v^ and kcat was hampered by the fact that the refolding process yielded a number of improperly folded polypeptides. 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 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 N I x:i*i'i Irrtsin □ NA: FINA: NI I Pfotein; JV-exfein Protein: C I .1 ^ tra rise riptic i translation Intel" pre, *-- precursor protein COOH C-exlein ^ protein splicing Inteins (mfervening protems^) 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. mature protern excised injein hA- pH intern N pH 6.O-6.S 20-25'C, 1 6 h Tag >A pH intern -N -\- C- Thiol intein A 1 5-30 mM thiol 4°C, 1 6 h -+- C- Thiol intein -A{ Perler, (2005) 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): DAPuse (TAGZyme) Exoldi (peptidase Cleaves N-terminal. His-tag (C-terminal) tor purification and removal Aaromonas aminopeplidase Exopeptidase Cleaves N-terminal. effective on M. L. Requires Zn Aminopeplidase M Exopeptidase Cleaves N-terminal, does not cleave X-P Carboxy peptidase 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_ Amiro acid DAPase Mop point (A) sequence-' Lysine (Lys, KJ Artjinire (Arg. Rj Proline [Pro. Pj Proline (Pro. P) ( ill J.nnni.-- (Gin. Q' XaaXaa . h...Xaa XaaXra ..Xaa-Xaa j Arq-Xaa ... Xaa-Xja. ..Xaa Xaa 4. XaaXaa fto-Xaa.. Xaa-Xna ..XaaXaa X Xaa-Pro Xoa-Xaa.. Xaa-Xoa...XaaXaa X Gln-Xoa... DAPase cleavage DAPase stop M K HQ HQ HQ HQ HHP K HTTrx HHP-Tiy M 12 3 4 5 6 7 HT-Tix HHP-Trx Arnau etal, 2006 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 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'g His-tag for removal of the protease 3C protease ETLFQXP GST tag for removal of the protease Sortase A LPET'g Ca2+-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 j Protease site Hisc MBP Target protein Enterokinase Asp-Asp-Asp-Asp-Lys/X Table 4 Cleavage (%) of e nterokinasc through densitometry (Hosficld and Lu 1999) based on the amino acid residue Xi. The sequence. ...GSDYKDDDDK-Xi-ADQLTEEQIA... 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 Xj Cleavage of enterokinase Alanine 88 Methionine 86 Lysine 85 Leucine 85 Asparagine 85 Phenylalanine 85 I so leucine 84 Aspartic acid 84 Glutamic acid 80 Glutamine 79 Valine 79 Argininc 78 Threonine 78 Tyrosine 78 Histidine 76 Serine 76 Cysteine 74 Glycine 74 Tryptophan 67 Proline 61 Removal of fusion tags - enzymatic cleavage > Optimization of protein cleavage conditions (mainly enzyme-to-substrate ratio, temperature, pH, salt concentration, length of exposure). > Cleavage efficiency (Optimization is needed. The efficiency varies with each fusion protein in an unpredictable manner, probably due to aggregation or steric issues; the problem can be solved by introducing short linkers between the protease site and the fusion tag). > Unspecific cleavage (SOLUTION: optimization of protein cleavage conditions or using re-engineered proteases with increased specificity such as ProTEV and AcTEV proteases). Product of cleavage is reccomended to verify using mass spectrometry. > 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). > Target protein modification (some proteases like thrombin, TEV, Precision leave one or two amino-acids on the target protein near the cleavage site). > Re-purification step is needed to separate the protease from target protein. 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 wild type protein and soluble mutant form for enzymatic measurements and crystallization. M (kDa) 170 - 116 - a B c d (Zouhar et al., 1999) Purification of AHP2 protein (Arabidopsis histidin phosphotransfer protein 2) 4L cell culture Immobilized metal affinity chromatography Buffer: 50 mM Tris pH 7.9, 300 mM NaCI, 10 % glycerol, 20 mM imidazole, 3.9 mM mercaptoethanol Gradient elution: 20 -500 mM imidazole Size exclusion chromatography Anion exchange chromatography Buffer: 20 mM Tris pH 7.9, 250 mM NaCI Isocratic elution Buffer: 20 mM Tris pH 7.9 Gradient elution: 0 -1M NaCI 45 kDa 36 kDa 29 kDa 24 kDa 15%SDSPAGE 10% NATIVE PAGE 15 % SDS PAGE ' 10% NATIVE PAGE His-tagged protein and IMAC under native conditions Four-step purification of Arabidopsis ( K11 RI) 1. Affinity purification (MCAC) 2. Tag removal (TEV protease) 3. Affinity purification (MCAC) 4. Size exclusion chromatography Ub- SGSG HisTaq- SA-TEV-AME-( IKIl 3. Affinity purification after TEV cleavege 200 mM imidazole CKI1 rd pETM-60::CKIl M imida^. PETM-ÓO-CKJIrd . rd . pETM-60 4. Size-exclusion chromatography 1 -10-20 mg for TB and M9 Pekařova B.