Gel Filtration (GF) or Size Exclusion Chromatography (SEC)

Transcription

1 Gel Filtration (GF) or Size Exclusion Chromatography (SEC) 1

2 Gel Filtration chromatography (GF) Principles of GF Fractionation range Parameters for resolution optimization Use of GF Column performance Troubleshooting Examples 2

3 What is gel filtration? Gel filtration is a technique of liquid chromatography which separates molecules according to their ratio (sizes, oligomeric state) Beads with pores of well-defined sizes: fractionation range Mobile phase: almost all kind of buffers 3

4 Agarose Dextran Gel structure Void volume V o Volume of the gel matrix V s A hypothetical structure for Superdex Pore volume V i Beads of defined porosity: fractionation range The degree of cross-linking determines the size of the pores and therefore the fractionation range of the resin Unlike many other chromatographic procedures, size exclusion is not an adsorption technique. 4

5 Terms and explanations Void volume V o V o = Void volume: volume of the solution outside the beads, or elution from very large molecules V e = the volume from the time the protein is placed until it appears in the effluent V i = volume of the solution inside the beads = V c - V s - V o V c = Total (geometric) volume of the column Vt = Elution volume for very small molecules V e Volume of the gel matrix V s V o V t Pore volume V i V c

6 Steric exclusion Gel bead Molecules are excluded from the gel bead to different extents according to their sizes. Largest molecules - excluded from pores, travel with the mobile phase, elute rapidly from column The volume at which large molecules elute is called the void volume, Vo (same as the volume of solution that surrounds the beads) Intermediate size molecules spend different amounts of time both inside and outside the beads (partition between the mobile and stationary phase) The volume at which intermed.molecules elute is called the elution volume (Ve) and depends on the partition of the molecule between the Vo and Vs which is proportional to the distribution coefficient (K) Ve = Vo + KVs Smallest molecules enter the pores of the beads, are included in the matrix and retarded in their movement, spend most of the time in the stationary phase, elute last The volume at which small molecules elute corresponds to Vt (total volume of solution surrounding (Vo) and inside the beads, Vs) Vt = Vo + Vs 6

7 Constructing a selectivity curve K av 1 Kav Ve V Vt V o o Run standards and determine the elution volume for each Calculate K av values log (M r ) Plot log (M r ) for each standard against the calculated K av Selectivity curve is usually moderately straight over the range Kav=.1 to Kav=.7 Extrapolate MW of your protein according to his Ve 7

8 Shape effects Different fractionation ranges for: K av 1 Native, globular proteins.7.1 Native proteins Denatured proteins log (M r ) Partially folded molecules Proteins inside detergent micelle: MW of protein + MW of micelle 8

9 How to choose GF type K av 1 Linear polysaccharides Globular proteins Molecules with different shapes have different selectivity curves log (M r ) RESIN FR Glob Prot FR Dextrans Sephacryl S1 1-1 kda ND Sephacryl S kda 1-8 kda Sephacryl S kda 2-4 kda Sephacryl S4 2-8 kda 1-2 kda Protein 1: 3kDa Protein 2: 8kDa Vo Vt 9

10 Gel Filtration chromatography (GF) Principles of GF Fractionation range Parameters for resolution optimization Use of GF Column performance Troubleshooting Examples 1

11 Increasing exclusion limit RESIN FR Glob Prot FR Dextrans Results depend on selectivity (fractionation range) Sephacryl S1 1-1 kda ND Sephacryl S kda 1-8 kda AU 28 Sephacryl S kda 2-4 kda Sephacryl S-3 Sephacryl S4 2-8 kda 1-2 kda Better for larger proteins Sephacryl S-2 BSA IgG b-l Cyt C Cytidine Best for these proteins Sephacryl S-1 Better for smaller proteins ml 11

12 Resolution depends on efficiency and selectivity Low selectivity high efficiency High efficiency can compensate for low selectivity. low efficiency High selectivity high efficiency low efficiency If selectivity is high, low efficiency can be tolerated (if large peak volume is acceptable). 12

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14 Efficiency Efficiency depends on: Particle size of matrix Particle size distribution of matrix Packing quality of the column Sample (volume, purity and viscosity) Flow rate (more important for bigger beads) 14

15 Peak width depends on particle size Superdex Peptide µm Superdex 3 prep grade µm AU AU Retention time (min) Retention time (min) 15

16 Resolution depends on column length Increasing column length increases resolution Retention time (min) AU Retention time (min) 1 x Superdex Peptide HR 1/3 2 x Superdex Peptide HR 1/3 mau mau 15 SUMO-Atox1 SUMO 15 SUMO-Atox1 SUMO Atox1 Atox ml Superdex Peptide 6 x 1.6cm ~ 12ml ml Superdex Peptide 1 x 1.6cm ~ 2ml 16 Michal Shoshan from Edith Tshuva lab.

17 Resolution depends on sample volume Column: Superdex Peptide HR 1/3 A µl AU µl µl Retention volume (ml) Retention volume (ml) 2 µl AU µl Retention volume (ml) 4 µl 17

18 Why use gel filtration? Group separations: Desalting, Buffer exchange, Removing reagents (replace dialysis) Purification of proteins and peptides: complex samples, monomer/dimer QC: Estimation size & size homogeneity Protein-Protein Interaction 18

19 Desalting proteins Desalting in a simple column HSA NaCl Column: Sample: Buf fer: PD-1 HSA, 25 mg NaCl.5M volume Volume for desalting: up to 25% column volume 19

20 Adjusting ph, buffer type, salt concentration during sample Use in group separations preparation, e.g. before an assay. Removing interfering small molecules: EDTA, Gu.HCl, etc Gravity Desalting Columns Multi Spin Desalting Columns Removing small reagent molecules, e.g. fluorescent labels, radioactive markers. Spin Desalting Columns FPLC Desalting Columns 2

21 Gali Prag Use buffer that avoid protein precipitation mau HiTrapDesalt1ml1:1_UV1_28nm HiTrapDesalt1ml1:1_Cond HiTrapDesalt1ml1:1_Fractions HiTrapDesalt1ml1:1_Inject HiTrapDesalt1ml1:1_Logbook OD 28nm Conductivity Desalting in the presence of buffer + 1mM NaCl ml HiTrapDesalt1ml2:1_UV1_28nm HiTrapDesalt1ml2:1_Cond HiTrapDesalt1ml2:1_Fractions HiTrapDesalt1ml2:1_Inject HiTrapDesalt1ml2:1_Logbook mau 1 Desalting in the presence of buffer + 25mM NaCl Waste Waste ml 21

22 Fractionation of multiple components Separate multiple components in a sample on the basis of differences on their size Best results with samples that contains few components or partially purified samples (polishing step) : Not recommended for proteins close by MW Limited sample volume (.5-4% of total column volume). Not so suitable if the sample volume is large Flow-rate limitation : Time consuming Removes higher oligomeric states and other aggregates Protein elutes with the column equilibration buffer 22

23 Separating dimer and oligomers from monomer.5 A 28 nm Monomer.25 Column: Superdex 75 HR 1/3 Sample: A special preparation of rhgh in distilled water Oligomer V O Dimer 1 2 V C T ime (min) 23

24 Case study: HLT-p53CT- Affinity start with pellet of 1.5L culture Ni-Sepharose FF 14ml HLTp53CTNiNTA16ml4:1_UV1_28nm HLTp53CTNiNTA16ml4:1_UV2_26nm HLTp53CTNiNTA16ml4:1_Conc HLTp53CTNiNTA16ml4:1_Fractions HLTp53CTNiNTA16ml4:1_Inject HLTp53CTNiNTA16ml4:1_Logbook mau 25 Load + 1cv %B + 3cv 8%B + 4cv 15%B + 4cv 1%B 2 15 POOL 17-22: 3.5OD x 35ml ~ 276mg /26/215 F4 Waste ml

25 mau HLT-p53CT- Cation Exchange after TEV protease cleavage ON 4ºC HLTp53CTHiTrapSP5mlml5:1_UV1_28nm HLTp53CTHiTrapSP5mlml5:1_UV2_26nm HLTp53CTHiTrapSP5mlml5:1_Cond SP-Sepharose HLTp53CTHiTrapSP5mlml5:1_Conc FF 5ml HLTp53CTHiTrapSP5mlml5:1_Fractions HLTp53CTHiTrapSP5mlml5:1_Inject HLTp53CTHiTrapSP5mlml5:1_Logbook /26/215 F ml

26 p53c-terminal - Polishing column Column: Sephacryl S1 prep. 96 x 26mm (~5ml) 6ml/fract. Ni column TEV protease cleavage ON dilution CEIX concentration & GF Ronen Gabizon from Assaf Friedler lab. mau HLTp53CTSephacrylS1of5ml4:1_UV1_28nm HLTp53CTSephacrylS1of5ml4:1_UV2_26nm HLTp53CTSephacrylS1of5ml4:1_Fractions HLTp53CTSephacrylS1of5ml4:1_Inject HLTp53CTSephacrylS1of5ml4:1_Logbook 15 POOL Fractions around 23 and 32 are higher MW impurities 5 26 F ml

27 Quality control: Use for size estimation / oligomeric state Gives an estimate of molecular size in native or denative solution (Guanidine HCl, urea, detergents) MW of globular protein using native buffers (Precision is not so good) Oligomeric state of the protein / homogeneity / complex Complementary information to PAGE-SDS 27

28 SEC-MALS: Size Exclusion Chromatography - Multi Angle Light Scattering Size exclusion chromatograph in line with multi angle light scattering, added value to characterize proteins mass and shape in native solution conditions Calculating Mw and radius from the light scattering equations much more accurate. Calculate the Mw during the elution peaks- detect homogeneity sample. Detect low amount of aggregation large molecules amplify the intensity of LS. Useful for protein/protein or protein/ligand interaction

29 Refolding by Dilution with Non Detergent Sulfo Betaine mau Multimer Monomer mau Refolding with 1MNDSB at.1mg/ml refolding with 1M NDSB at.2mg/ml ml ml mau mau refolding with 1M NDSB at.5mg/ml Refolding with 1M NDSB at 1mg/ml ml ml

30 Complex formation: Leptin and Leptin Receptor mau Superdex75prep2:1_UV3_22nm Superdex75prep2:1_Fractions Superdex75prep2:1_Inject Superdex75prep2:1_UV3_22nm1 Superdex75prep2:1_UV3_22nm2 Superdex75prep2:1_Logbook 9. Superdex 75 16x1.6cm column - Buffer: 2mMTrisHCl ph8. 5mMNaCl.2%NaN Complex: Leptin + Receptor Leptin alone Receptor alone Waste ml 3

31 Gel Filtration chromatography (GF) Principles of GF Fractionation range Parameters for resolution optimization Use of GF Column performance Troubleshooting Examples 31

32 Increasing resolution Choose appropiate fractionation range Increase column volume (Connect two columns) Reduce the flow rate Change to a gel with smaller beads (higher efficiency) Reduce the sample volume / protein quantity Check the column efficiency Clean and/or re-pack 32

33 Optimization of ATOX1 purification Michal Shoshan from Edit Tshuva lab SUMO alone and ATOX1 ATOX1 alone ATOX1: SUMO protease treatment after IMAC purification. Ammonium Sulphate precipitation. Load on 12ml Superdex 3 column (2mM MES ph mM NaCl) SUMO-Atox1 IMAC purification mau 15 1 SUMO alone 5 ATOX1 alone ATOX1: SUMO protease treatment after IMAC purification. Concentration by UF instead of AS precipitation. Load on 2ml Superdex 3 column (2mM MES ph mM NaCl) ml ATOX1: SUMO protease treatment after IMAC purification. Concentration by UF in the presence of 4M NaCl. Load on 2ml Superdex 3 column (2mM MES ph mM NaCl)

34 Optimization of Complex Formation Strategy: Co-purification of two proteins (separately expressed) Complex stabilization:.1% Tween-2 Nadav Komornik / Oded Livnah lab %B chelating2mlair Imp7Beta :1_UV1_28nm chelating2mlair Imp7Beta :1_UV2_26nm chelating2mlair Imp7Beta :1_Conc chelating2mlair Imp7Beta :1_Fractions chelating2mlair Imp7Beta :1_Logbook 1%B Chelating2mlAir, Imp7598 ImpBeta1-442 from 1L 17C ON Fractions Peak-833 mau 2%B 15%B F4 1 F ml Capture: IMAC column Imp7BetaSuperose12prepar2ml :1_UV1_28nm Imp7BetaSuperose12prepar2ml :1_UV2_26nm Imp7BetaSuperose12prepar2ml :1_Fractions Imp7BetaSuperose12prepar2ml :1_Inject Imp7BetaSuperose12prepar2ml :1_Logbook mau Imp7BetaSuperdex75prep32ml :1_UV1_28nm Imp7BetaSuperdex75prep32ml :1_UV2_26nm Imp7BetaSuperdex75prep32ml :1_Fractions Imp7BetaSuperdex75prep32ml :1_Logbook mau 14 Superose12prepar2ml Imp7 598 ImpBeta Tween2.1% after Ni 2/12/12 Extra ImpBeta Peak - 16ml Imp7 (598-C) + Imp Beta (1-442) after Ni and conc - Superdex75prep32ml Peak agregate: 115 ml Peak1: 137 ml 12 1 Complex Complex Peak - 87ml Free protein 3. Peak2: 182 ml Aggregate 1. 4 Aggregate Peak - 65ml ml ml Superose 12 2ml column Superose 12 32ml column.1% Tween-2 through purification

35 Increasing resolution - Example: Pegylated protein 12617M165Superose12Anal1:1_UV1_28nm 12617M165Superose12Anal1:1_UV2_26nm 12617M165Superose12Anal1:1_Fractions 12617M165Superose12Anal1:1_Inject 12617M165Superose12Anal1:1_Logbook 12618Superose12Prep3columns5ml1:1_UV1_28nm 12618Superose12Prep3columns5ml1:1_UV2_26nm 12618Superose12Prep3columns5ml1:1_Fractions 12618Superose12Prep3columns5ml1:1_Inject 12618Superose12Prep3columns5ml1:1_Logbook mau 15 mau 15 Vo F Waste ml Superose 12 analytical 3 x 1cm = 23ml column Load : ~1mg protein ml Superose 12 preparative 3 tandem columns 25 x 1.6cm = 52ml column Load : ~25mg protein / 5ml How can we get better separation between 2 and 3?? Can we scale-up protein loading to separate 1 from 2 and 3??

36 Column size Desalting and other group separations Volume four times the expected sample volume Length is not so important Fractionation Volume ~.5-4% times the expected sample volume Sample volume can be increase if resolution is OK Length 3-1 cm or more (depends of the resolution) 36

37 Why choose gel filtration? Advantage Separates by size. Complementary to IEX and HIC Very gentle, high yields Works in any buffer solution Removes aggregates Fast for buffer exchange Mostly use in a final polishing step Mandatory for QC Complementary results than PAGE-SDS Disadvantage Limited sample volume Poor resolution in a complex mixture Flow-rate limitation time consuming Elution of diluted sample Poor selectivity compared with SDS- PAGE Not efficient in capture or intermediate steps 37

38 Troubleshooting Lower yield than expected Protease degradation of the protein Adsorption to filter, valves or top of the column Non-specific adsorption Sample precipitate MW of protein is not as expected Oligomerization state of the protein is different Protein bounds to another protein or complex Unfolded or naturally unfolded protein Protein has changed during storage Ionic or Hydrophobic interactions between protein and matrix Protein precipitate Very broad peak elution Different oligomeric states or protein aggregation Sticky protein Non specific adsorption to matrix Protein is part of complex with different sizes Overloading Peak of interest is poorly resolved Sample volume is too high Short column Poor selectivity or efficiency of the column Flow rate too high Column is dirty or not well packed Viscous sample 38

39 Field flow fractionation (FFF) Some molecules are highly hydrophobic, making them incompatible with fractionation via sizeexclusion chromatography (SEC). Field flow fractionation (FFF) separates macromolecules and nanoparticles by size without a stationary phase, eliminating most of the non-ideal surface interactions prevalent in SEC. In an Asymmetric-Flow FFF separation channel, macromolecules and nanoparticles are gently pushed against a semipermeable membrane by crossflow. Smaller particles diffuse back up towards the center of the channel. Laminar channel flow induces a parabolic flow velocity profile, causing smaller particles to elute earlier. 39

40 Inhibition of HIV-1 Replication by Modulating the Oligomerization Equilibrium of the Viral Integrase Zvi Hayouka. et al. PNAS 14 (2): (27) Friedler lab The effect of ligand binding on the oligomeric state of Integrase Molecular weight (gr/mol) Analytical gel filtration (superose 12) IN IN + LEDGF/p IN + LEDGF/p (2h incubation) IN + DNA LTR IN + DNA LTR + LEDGF/p

41 NATIVELY UNFOLDED PROTEIN Case study: HTL - A natively unfolded proline-rich domain in ASPP2 that regulates its protein interactions by intramolecular binding to the Ank- SH3 domains. Shahar Rotem et al. JBC Friedler lab mau kda HTL435aaSuperdex2prep5mlB5:11_UV3_22nm HTL435aaSuperdex2prep5mlB5:11_Logbook FoldIndex : a simple tool to predict whether a given protein sequence is intrinsically unfolded. Jaime Prilusky, Clifford E. Felder, Tzviya Zeev-Ben-Mordehai, Edwin Rydberg, Orna Man, Jacques S. Beckmann, Israel Silman, and Joel L. Sussman, 25, Bioinformatics. 435aa MW: 49.7kD Superdex 2 prep. 1x2.6cm : trimer?? Analytical ultracentrifugation: monomer 5 HTL 435aa ml