Introduction to Affinity Chromatography (AC) By: Sunsanee Yoojun Bang Trading 1992 Co., Ltd.

Transcription

1 Introduction to Affinity Chromatography (AC) By: Sunsanee Yoojun Bang Trading 1992 Co., Ltd.

2 Content What is affinity chromatography (AC)? What is AC used for? Principles Experimental set up Summary

3 What is affinity chromatography (AC)?

4 What is affinity chromatography? Affinity chromatography is a liquid chromatography technique which separates biomolecules based on highly specific biological interactions.

5 Why use affinity chromatography? Simple to do one step Concentrates High purity, often > 90 % Easy to achieve otherwise difficult separations Separate native from denatured and functionally different forms Fast separations

6 What is AC used for?

7 What is it used for? Monoclonal and polyclonal antibodies Tagged proteins Enzymes For ANY protein which has a binding partner!

8 Antibody Purification Title or Job Number XX Month 201X 8

9 Description of an antibody Ligands: Protein A Protein G Specificity: Fc region of many IgGs

10 IgG binding specificities of immobilized protein G and protein A differ Species Protein A Protein G binding binding Human IgA variable - IgD - - IgE IgG IgG IgG IgG IgM* variable - Avian egg yolk IgY - - Cow Dog ++ + Goat - ++ Guinea Pig IgG IgG Hamster + ++ Horse Koala - + Llama - + Species Protein A Protein G binding binding Monkey (rhesus) Mouse IgG IgG 2a IgG 2b IgG IgM* variable - Pig Rabbit Rat IgG IgG 2a IgG 2b - ++ IgG Sheep +/ relative binding strength weak or no binding Protein G is a good first choice for general capture of antibodies at lab scale since it binds a broader range of IgG from eukaryotic species and also binds to more subclasses of IgG than protein A

11 Purification of monoclonal mouse IgG 1 M r SDS-PAGE : LMW 2: Starting material, diluted 10 X 3: Eluted IgG 1 pool, diluted 5 X Sample: Cell culture supernatant containing mouse IgG 1 Column: HiTrap Protein G HP, 1 ml Binding buffer: 20 mm sodium phosphate, ph 7.0 Elution buffer: 0.1 M glycine-hcl, ph 2.7 System: ÄKTA System, 1.0 ml/min

12 Tagged Proteins Purification Title or Job Number XX Month 201X 12

13 Tagged proteins Deliberately designed for affinity purification The affinity-tagged recombinant protein binds to the specific ligand Purity typically 80-90% in one step A protease cleavage site allows the tag to be removed after purification Affinity tag Poly-histidine (HIS) Glutathione-S-Transferase (GST) Maltose Binding Protein (MBP) Strep-tag II ZZ (domain B of protein A) Ligand Metal ions (Ni, Co, Zn, Cu...) Glutathione Dextrin Strep-Tactin IgG Matrix Ligand Affinity tag Cleavage site Recombinant protein

14 Purification of MBP-tagged proteins mau min Lane 1: Lane 2: Lane 3: Lane 4: Low Molecular Weight markers Start material Flowthrough Elute Sample: MBP-2-paramyosin- -Sal in clarified E. coli lysate Column: MBPTrap HP, 1 ml Binding buffer: 20 mm Tris-HCl, 200 mm NaCl, 1 mm EDTA, 1 mm DTT, ph 7.4 Elution buffer: 10 mm maltose in binding buffer System: ÄKTA System

15 Enrichment of glycoproteins Lane 1: Lane 2: Lane 3: Lane 4: Lane 5: Low Molecular Weight markers Start material Flow through Wash Elute Sample: 0.5 ml human plasma diluted to 5 ml with binding buffer Column: HiTrap Con A 4B, 1 ml Binding buffer: 20 mm Tris, 500 mm NaCl, 1 mm MnCl 2, 1 mm CaCl 2, ph 7.4 Elution buffer: 20 mm Tris, 500 mm NaCl, 300 mm methyl-d-glucoside, ph 7.4 System: ÄKTA System

16 Principles

17 The main stages in affinity chromatography

18 Purification strategy -recommendations How specific do we really want to be? Mono-specific ligands? Group-specific ligands? Keep it simple! Design the elution scheme

19 Choosing ligands Mono-specific ligands Has affinity for a single substance such as: Antigen Hormone Antibody Receptor Usually home-made affinity media Elution protocols based on general principles Group-specific ligands Has affinity for a group of structurally/functionally similar substances: Lectins Protein G Glycoproteins IgG antibodies Often ready-made affinity media Standard, tested elution protocols

20 Group-specific ligands Ligand Protein A Protein G Concanavalin A Cibacron Blue Lysine Benzamidine Calmodulin Heparin Metal ions (e.g. Ni 2+ ) Specificity Fc region of IgG Fc region of IgG Glucopyranosyl and mannopyranosyl groups Broad range of enzymes, serum albumin Plasminogen, ribosomal RNA Serine proteases Proteins regulated by calmodulin Coagulation factors, lipoproteins, lipases, hormones, steroid receptors, protein synthesis factors, nucleic acid-binding enzymes Proteins and peptides which contain histidine

21 Designing the elution scheme Mono-specific ligands Elution scheme must be worked out for each case Often general elution Little help from the literature Group-specific ligands Known elution scheme Often competitive elution Well described in standard protocols and literature

22 Designing the elution scheme General elution conditions Low ph, salt, Glycine HCl, Urea etc Specific eluents Competing free ligand Competing binding substances

23 General elution conditions Changing buffer conditions Usually decrease ph, increase ionic strength or decrease polarity adding up to 10 % dioxane or up to 50 % ethylene glycol +

24 General elution conditions Denaturing buffer Usually extremes of ph or chaotropic agents + Reconstituting buffer

25 Example of elution at low ph IgG antibodies on rprotein A Sepharose Column: HiTrap rprotein A FF Binding buffer: 20 mm sodium phosphate, ph 7.0 Elution buffer: 0.1 M glycine HCl, ph 3 Note: To preserve antibody activity collect the eluted IgG into a small volume of 1 M Tris-HCl, ph 7.5

26 Elution at high salt concentration DNA-binding proteins on Heparin Sepharose Column: HiTrap Heparin HP Binding buffer: 20 mm Tris-HCl, ph 8, 0.5 M NaCl Elution buffer: 20 mm Tris-HCl, ph 8, 1-2 M NaCl Note: Typical additives to maintain activity include 5% (v/v) glycerol and protease inhibitors

27 Specific elution conditions Competing free ligand in solution Elution of GST-tagged proteins from Glutathione Sepharose by glutathione +

28 Elution with competing free ligand GST-tagged proteins on Glutathione Sepharose Column: GSTrap 4B 1 ml Binding buffer: 10 mm sodium phosphate, 140 mm NaCl, ph 7.4 Elution buffer: 50 mm Tris-HCl, 20 mm glutathione, ph 8.0

29 Specific elution conditions Competing binding substance in solution Elution of histidine-tagged proteins from Ni Sepharose by imidazole +

30 Competing binding substance in solution Histidine-tagged proteins on Ni Sepharose mau pool ml Column: Binding buffer: Elution buffer: HisTrap HP 20 mm phosphate, 0.5 M NaCl, 20 mm imidazole As binding buffer except 500 mm imidazole

31 Experimental set up

32 Some practical details Column and equipment set-ups Sample preparation Sample application Elution Re-equilibration and regeneration

33 1. Columns and equipment set-ups Column volume: Choose the column volume according to amount of target and capacity of the chromatography medium Column length: Not usually critical, short and wide Equipment: No special demands

34 2. Sample preparation Use filter or centrifuge to remove particles Use a desalting column to adjust ph, buffer salts and additives to promote binding Make sure that components known to interfere with binding are absent

35 3. Sample application Follow the standard protocol Equilibrate the column before applying sample Buffer: usually neutral ph Establishment of flow rate: Strong affinity: High flow rate Weak affinity: Low flow rate

36 4. Elution if eluting at extreme ph Collect the target protein in a small amount of concentrated buffer at a neutral ph or use a desalting column to transfer the eluted protein quickly to a neutral buffer if necessary

37 4. Elution step or gradient elution During development and optimization of affinity purification, use a gradient elution to scan for the optimal binding and/or elution conditions

38 5. Re-equilibration and regeneration Re-equilibrate with binding buffer or storage buffer Regenerate according to instructions

39 Practical details a summary Affinity chromatography medium Mono-specific Media for affinity chromatography should be highly porous to guarantee high coupling yields and full access to the affinity ligand. Not often commercially available. Pre-activated chromatography media available for preparing own mono-specific medium. Group-specific Media are commercially available in many formats such as prepacked columns, kits, lab packs etc to guarantee proper function. Column Affinity chromatography is in most cases step-eluted. Columns are therefore typically short and fat. Affinity chromatography is in most cases step-eluted. Columns are therefore typically short and fat. Regeneration and storage Some affinity ligands are sensitive to proteases and/or low ph. Follow recommended regeneration and storage procedures to ensure full column/media lifetime. Re-use of pre-packed columns should be restricted to identical proteins to prevent cross-contamination. Some affinity ligands are sensitive to proteases and/or low ph. Follow recommended regeneration and storage procedures to ensure full column/media lifetime. Re-use of pre-packed columns should be restricted to identical proteins to prevent cross-contamination. Eluents Harsh elution conditions, which may influence ligand lifetime are sometimes required. Always wash column/media after use. Both mild and harsh elution conditions, which may influence ligand lifetime are sometimes required. Always wash column/media after use. Flow rates Binding/desorption kinetics may restrict applicable flow rate. Sample volume Sample volume is generally of less importance, as long as the maximum load capacity of the medium is not violated. Binding/desorption kinetics may restrict applicable flow rate. Sample volume is generally of less importance, as long as the maximum load capacity of the medium is not violated.

40 Summary

41 Summary 1. Affinity chromatography is a liquid chromatography technique which separates biomolecules based on highly specific biological interactions 2. It is used for purification of ANY protein which has a binding partner 3. For practical hints and tips consult Handbook: Affinity Chromatography: Principles and Methods

42 Protein purification handbooks from GE Healthcare GE Healthcare handbooks

43 Thank you!