stability window for Taq polymerase pH ionic strength size isoelectric point (pI) hydrophobicity solubility * temperature stability - need to work rapidly at lowered temperature * pH stability - selection of buffers for extraction and purification; selection of conditions for ion exchange, affinity or reversed phase chromatography * organic solvents stability - selection of conditions for reverse phase chromatography * detergent requirement - consider effects on chromatographic steps and the need for detergent removal. consider choice of detergent. * salt (ionic strength) - selection of conditions for precipitation techniques and hydrophobic interaction chromatography * cofactors for stability and activity - selelection of additives, pH, salts, buffers * protease sensitivity - need for fast removal of proteases or addition of inhibitors * sensitivity to metal ions - need to add EDTA or EGTA in buffers * redox sensitivity - need to add reducing agents * molecular weight - selection of gel filtration media * charge properties - selection of ion exchange conditions * biospecific affinity - selection of ligand for affinity medium * post translational modifications - selection of group specific affinity medium * hydrophobicity - selection of medium for hydrophobic interaction chromatography protein properties used during purification * charge - ion exchange (IEX) * size - gel filtration (GF) * hydrophobicity - hydrophobic interaction (HIC), reversed phase (RPC) * biorecognition (ligand specifity) - affinity (AC) * charge, ligand specificity or hydrophobicity - expanded bed adsorption (EBA) follows the principles of AC, IEX or HIC suitability of purification techniques for the Three Phase Purification Strategy: IEX: main features: - high resolution - high capacity - high speed capture: 3stars intermediate: 3stars polish: 3stars sample start condition: - low ionic strength - sample volume - not limiting sample end condition: - high ionic strength or pH change - concentrated sample HIC: main features: - good resolution - good capacity - high speed capture: 2stars intermediate: 3stars polish: 1stars sample start condition: - high ionic strength - sample volume - not limiting sample end condition: - low ionic strength - concentrated sample AC (affinity chromatography): main features: - high resolution - high capacity - high speed capture: 3stars intermediate: 3stars polish: 2stars sample start condition: - specific binding conditions - sample volume - not limiting sample end condition: - specific elution conditions - concentrated sample GF (gel filtration): main features: - high resolution - using Superdex(TM) media intermediate: 1stars polish: 3stars sample start condition: - limited sample volume (<5% total column volume) - limited flow rate range sample end condition: - buffer exchanged (if required) - diluted sample RPC: main features: - high resolution intermediate: 1stars polish: 3stars sample start condition: - requires organic solvents sample end condition: - in organic solvent - risk loss of biological activity "Ammonium sulphate precipitation is a common sample clarification and concentration step at laboratory scale and in this situation HlC (which requires high salt to enhance binding to the media) is ideal as the capture step. The salt concentration and the total sample volume will be significantly reduced after elution from the HlC column. Dilution of the fractionated sample or rapid buffer exchange using a Sephadex G-25 desalting column will prepare it for the next IEX or AC step." "GF is well suited for use after any of the concentrating techniques (IEX, HlC, AC, EBA) since the target protein will be eluted in a reduced volume and the components from the elution buffer will not affect the gel filtration separation (gel filtration is a non-binding technique with limited volume capacity and unaffected by buffer conditions)." Logical combinations of chromatographic steps crude sample -> GF (desalt mode) -> AC -> ___ -> GF crude sample -> GF (desalt mode) -> IEX -> ___ -> GF crude sample -> Hlc (dilution may be needed) -> IEX -> GF crude sample -> GF (desalt mode) -> IEX -> HlC -> GF clear or very dilute samples -> AC -> GF or RPC clear or very dilute samples -> IEX -> GF or RPC clear or very dilute samples -> IEX -> HlC -> GF clear or very dilute samples -> precipitation (e.g., in high ionic strength) -> resolubilise -> treat as for sample in high salt concentration "A sample is purified using a combination of techniques and alternative selectivities. For example, in an IEX-HlC-GF Three Phase Strategy the capture step selects according to differences in charge (IEX), the intermediate purification step according to differences in hydrophobicity (HlC) and the final polishing step according to differences in size (GF)." IEX-HlC-GF is regarded as the standard if you know nothing about the proteins. RPC = reversed phase chromatography Purification step Media Quantity Code No. Capture STREAMLINE SP 300 ml 17-0993-01 Capture STREAMLINE DEAE 300 ml 17-0994-01 Capture HiPrepTM 16/10 SP XL 1 column 17-5093-01 Capture HiPrep 16/10 Q XL 1 column 17-5092-01 Intermediate purification HiPrep Phenyl (high sub) 1 column 17-5095-01 Polishing HiLoadTM 16/60 Superdex 75 prep grade 1 column 17-1068-01 Polishing HiLoad 16/60 Superdex 200 prep grade 1 column 17-1069-01 Sample clarification/conditioning Pre-packed PD-10 Column 30 columns 17-0851-01 Sample clarification/conditioning HiTrap Desalting 5 columns 17-1408-01 Sample clarification/conditioning HiPrep 26/10 Desalting 1 column 17-5087-01 Pre-packed column Sample volume Sample volume Code No. loading per run recovery per run HiPrep Desalting 26/10 2.5 -15 ml 7.5 - 20 ml 17-5087-01 HiTrap Desalting 0.25 - 1.5 ml 1.0 - 2.0 ml 17-1408-01 Fast Desalting PC 3.2/10 0.05 - 0.2 ml 0.2 - 0.3 ml 17-0774-01 PD-10 Desalting 1.5 - 2.5 ml 2.5 - 3.5 ml 17-0851-01 Table 6. Recommended HiTrap affinity columns for laboratory scale separation. Code No. Quantity/ Approximate Application HiTrap column components binding capaci- ty per ml gel 17-5079-01 5 x 1 ml human Isolation of HiTrap rProtein A 17-5080-01 1 x 5 ml IgG 50 mg/ml immunoglobulins 17-5029-02 2 x 1 ml IgG classes, fragments and subclasses 17-0402-01 5 x 1 ml human IgG classes, fragments HiTrap Protein A 17-0402-03 2 x 1 ml IgG 20 mg/ml and subclasses 17-0403-01 1 x 5 ml 17-0404-01 5 x 1 ml human IgG classes, fragments HiTrap Protein G 17-0404-03 2 x 1 ml IgG 25 mg/ml and subclasses 17-0405-01 1 x 5 ml including human IgG3 strong affinity to monoclonal mouse IgG1 and rat IgG Monoclonal and poly- MAbTrapTM GII 17-1128-01 HiTrap Protein G as above clonal IgG from ascites column (1 ml), fluid, serum and cell accessories, pre-made culture supernatant buffers for 10 purifica- tions Mouse recombinant RPAS Purification 17-1362-01 HiTrap Anti-E column, 0.17 mg Single chain antibody Module accessories, pre-made ScFv/5 ml Fragment variable buffers for 20 purifica- (ScFv) produced in tions E.Coli IgY antibodies from HiTrap IgY 17-5111-01 1 x 5 ml IgY 20 mg/ml egg yolk Purification IgM HiTrap IgM 17-5110-01 5 x 1 ml IgM 5 mg/ml Purification Group Specific Media: HiTrap Con A 17-5105-01 5 x 1 ml transferrin Glycoproteins or 4 mg/ml polysaccharides Specificity: branched mannoses, carbohydrates withterminal mannose or glucose(a Man> a Glc> GlcNAc) Specificity: HiTrap Lentil 17-5106-01 5 x 1 ml thyroglobulin branched mannoses Lectin 4 mg/ml with fucose linked a(1,6) to the N- acetyl- glucosamine, (a Man> a Glc> GlcNAc) N- acetyl- glucosamine binding lectins HiTrap column Code No. Quantity/ Approximate Application components binding capa- city per ml gel HiTrap Peanut 17-5108-01 5 x 1 ml asialofetuin Specificity: Terminal ß Lectin 3 mg/ml -galactose, (Gal ß 1,3 GalNAc > a and ß Gal) HiTrap Wheat 17-5107-01 5 x 1 ml ovomuroid Specificity: chitobiose Germ Lectin 4 mg /ml core of N-linked oligo- saccharides, [GlcNAc (ß 1,4GlcNAc) 1- 2 > ß GlcNAc] HiTrap Lectin Test 17-5109-01 4 x 1 ml columns of as listed above Specificity: as listed for Kit HiTrap Con A each column HiTrap Lentil Lectin HiTrap Peanut Lectin HiTrap Wheat Germ Lectin Group Specific Media HiTrap Blue 17-0412-01 5 x 1 ml HSA 20 mg/ml Various Nucleotide- 17-0413-01 1 x 5 ml requiring enzymes, coagulation factors, DNA binding proteins, a2-macro-globulin Proteins and peptides HiTrap Chelating 17-0408-01 5 x 1 ml (His)6-tagged with exposed amino 17-0409-01 1 x 5 ml protein (27.6 acids: His (Cys, Trp) kD) 12 mg /ml e.g. a-2-macro-globu- lin and interferon Histidine-tagged fusion HisTrapTM 17-1880-01 HiTrap Chelating as above proteins column (1 ml), acces- sories, pre-made buffers Biotin and biotinylated HiTrap 17-5112-01 5 x 1 ml biotinylated substances Streptavidin BSA 6 mg/ml Coagulation factors, HiTrap Heparin 17-0406-01 5 x 1 ml ATIII (bovine) lipoprotein lipases, 17-0407-01 1 x 5 ml 3 mg/ml steroid receptors, hormones, DNA binding proteins, interferon, protein syntheses factors Matrix for prepara- HiTrap 17-0716-01 5 x 1 ml ligand specific tion of affinity media NHS-activated 17-0717-01 1 x 5 ml Coupling of primary amines Recommended separation conditions All HiTrap columns are supplied with a detailed protocol to ensure optimum results Maximum flow rates: HiTrap 1 ml column: up to 4 ml/min HiTrap 5 ml column: up to 20 ml/min Table 7. Volatile buffer systems. Volatile buffer systems used in ion exchange chromatography pH range Buffer system Counter-ion pK-values for buffering ions 2.0 Formic acid H+ 3.75 2.3-3.5 Pyridine/formic acid HCOO- 3.75; 5.25 3.0-5.0 Trimethylamine/formic acid HCOO- 3.75; 9.25 3.0-6.0 Pyridine/acetic acid CH3COO- 4.76; 5.25 4.0-6.0 Trimethylamine/acetic acid CH3COO- 4.76; 9.25 6.8-8.8 Trimethylamine/HCl Cl- 9.25 7.0-8.5 Ammonia/formic acid HCOO- 3.75; 9.25 8.5-10.0 Ammonia/acetic acid CH3COO- 4.76; 9.25 7.0-12.0 Trimethylamine/carbonate CO32- 6.50; 9.25 7.9 Ammonium bicarbonate HCO3- 6.50; 9.25 8.0-9.5 Ammonium carbonate/ammonia CO32- 6.50; 9.25 8.5-10.5 Ethanolamine/HCl Cl- 10.0 8.5 Ammonium carbonate CO32- 6.50; 9.25 Extraction process Typical conditions Protein source Comment Gentle erythrocytes E.coli 2 volumes water to lower product yield but 1 volume packed periplasm: intracellular reduced protease Cell lysis (osmotic shock) pre-washed cells proteins release Enzymatic digestion lysozyme 0.2 mg/ml, bacteria: intracellular lab scale only, often 37 °C, 15 mins. proteins combined with mechanical disruption Hand homogenisation follow equipment liver tissue instructions Mincing (grinding) " muscle Moderate follow equipment muscle tissue, most instructions animal tissues, plant Blade homogeniser tissues Grinding with abrasive " bacteria, plant tissues e.g. sand Vigorous follow equipment cell suspensions: small scale, release of Ultrasonication instructions intracellular proteins in nucleic acids may cause or cytoplasm, periplasm, viscosity problems bead milling inclusion bodies inclusion bodies must be resolubilised Manton-Gaulin follow equipment cell suspensions large scale only homogeniser instructions French press follow equipment bacteria, plant cells instructions Fractional precipitation see section on fractional extracellular: secreted precipitates must be precipitation recombinants, resolubilised monoclonal antibodies, cell lysates Details from Protein Purification, Principles and Practice, R.K. Scopes and other sources. Table 9. Common substances used in sample preparation. Typical conditions for use Purpose Buffer components 20 mM, pH7.4 maintain pH minimise Tris acidification caused by lysosomal disruption NaCl 100 mM maintain ionic strength of medium EDTA 10 mM reduce oxidation damage, chelate metal ions Sucrose or glucose 25 mM stabilise lysosomal membra- nes, reduce protease release Detergents See Table 10 extraction and purification of lonic or non-ionic detergents integral membrane proteins solubilisation of poorly soluble proteins DNAse and RNAse 1 μg/ml degradation of nucleic acids, reduce viscosity of sample solution Protease inhibitors* Inhibits 0.5 - 1 mM PMSF serine proteases 0.4 - 4 mM APMSF serine proteases 0.2 mM Benzamidine-HCl serine proteases 1 μM Pepstatin aspartic proteases 10 - 100 μM Leupeptin cysteine and serine proteases 10 - 100 μM Chymostatin chymotrypsin, papain, cysteine proteases 1 - 100 μM Antipain-HCl papain, cysteine and serine proteases 2 - 10 mM EDTA metal dependent proteases, zinc and iron 2 - 10 mM EGTA metal dependent proteases e.g. calcium Reducing agents 1 - 10 mM keep cysteine residues 1, 4 dithiothreitol, DTT reduced 1 - 10 mM 1, 4 dithioerythritol, DTE " 0.05% Mercaptoethanol " Others 5 - 10% for stabilisation, up to 50% can Glycerol be used if required PMSF - Phenylmethylsulfonyl fluoride APMSF - 4-Aminophenyl-methylsulfonyl fluoride PMSF is a hazardous chemical. Half-life time in aqueous solution is 35 min. PMSF is usually stored as 10 mM or 100 mM stock solution (1.74 or 17.4 mg/ml in isopropanol) at - 20° C. * Protease inhibitors are available in pre-made mixes from several suppliers. Details taken from Protein Purification, Principles and Practice, R.K. Scopes. 1994, Springer., Protein Purification, Principles, High Resolution Methods and Applications, J-C. Janson and L. Rydén, 1998, 2nd ed. Wiley Inc. and other sources. Table 10. Examples of ionic and non-ionic detergents. Sodium dodecyl sulphate 0.1 - 0.5% denatures proteins, used for SDS-PAGE use non-ionic detergents to avoid denaturation Triton-X-100 0.1 % non-ionic detergent for membrane solubilisation. Note: may absorb strongly at 280 nm! Nonidet-P-40 0.05 - 2% " Dodecyl b D maltoside 1% " Octyl b D glucoside 1 - 1.5% " For further information on detergents: Protein Purification, Principles, High Resolution Methods and Applications, J-C. Janson and L. Rydén, 1998, 2nd ed. Wiley Inc. Pre-packed column Sample volume Sample volume Code No. loading per run recovery per run HiPrep Desalting 26/10 2.5 -15 ml 7.5 - 20 ml 17-5087-01 HiTrap Desalting 0.25 - 1.5 ml 1.0 - 2.0 ml 17-1408-01 Fast Desalting PC 3.2/10 0.05 - 0.2 ml 0.2 - 0.3 ml 17-0774-01 PD-10 Desalting 1.5 - 2.5 ml 2.5 - 3.5 ml 17-0851-01 Precipitation agent Typical conditions for use Sample type Comment Ammonium sulphate as described >1mg/ml proteins stabilizes proteins, no especially immuno- denaturation, super- globulins natant can go directly to HIC Dextran sulphate as described samples with high precipitates lipoprotein levels of lipoprotein e.g ascites Polyvinylpyrrolidine Add 3% (w/v), stir 4 hours, alternative to dextran " centrifuge, discard pellet sulphate Polyethylene glycol up to 20% wt/vol plasma proteins no denaturation super- (PEG, M.W. >4000) natant goes direct to IEX or AC. Complete removal may be difficult Acetone up to 80% vol/vol at 0 °C useful for peptide may denature protein precipitation or con- irreversibly centration of sample for electrophoresis Polyethyleneimine 0.1% w/v precipitates aggregated nucleoproteins Protamine sulphate 1% " Streptomycin sulphate 1% precipitation of nucleic acids Details taken from Protein Purification, Principles and Practice, R.K. Scopes. 1994, Springer., Protein Purification, Principles, High Resolution Methods and Applications, J-C. Janson and L. Rydén, 1998, 2nd ed. Wiley Inc.and other sources Table 13. Examples of denaturing agents. Denaturing agent Typical conditions for use Removal/comment Urea 2 - 8M remove using Sephadex G25 Guanidine hydrochloride 3 - 8M remove using Sephadex G25 or during IEX Triton X-100 with 2% ” Sarcosyl 1.5% ” N-octyl glucoside 2% ” Sodium dodecyl sulphate 0.1 - 0.5% exchange for non-ionic detergent during first chromatographic step, avoid anion exchange chromatography alkaline pH > pH 9, NaOH may need to adjust pH during chromatography to maintain solubility Details taken from Protein Purification, Principles and Practice, R.K. Scopes. 1994, Springer., Protein Purification, Principles, High Resolution Methods and Applications, J-C. Janson and L. Rydén, 1998, 2nd ed. Wiley Inc.and other sources