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Poly (His) fusion proteins His MicroSpin Purification Module, HisTrap Kit, HiTrap Chelating HP, Chelating Sepharose Fast Flow The (His) 6 tag is one of the most common tags used to facilitate the purification and detection of recombinant proteins and a range of products for simple, one step purification of (His) 6 fusion proteins are available (see Purification options). Polyhistidine tags such as (His) 4 or (His) 10 are also used. They may provide useful alternatives to (His) 6 for improving purification results. For example, since (His) 10 binds more strongly to the affinity medium, a higher concentration of eluent (imidazole) can be used during the washing step before elution. This can facilitate the removal of contaminants which may otherwise be co-purified with a (His) 6 fusion protein. Chelating Sepharose, when charged with Ni 2+ ions, selectively binds proteins if complexforming amino acid residues, in particular histidine, are exposed on the protein surface. (His) 6 fusion proteins can be easily bound and then eluted with buffers containing imidazole. Purification and detection of His-tagged proteins, together with information on how to handle fusion proteins when they are expressed as inclusion bodies, are dealt with in depth in The Recombinant Protein Handbook: Protein Amplication and Simple Purification, available from Amersham Pharmacia Biotech. Purification options Binding capacity Maximum Comments operating flow His MicroSpin Purification Module 100 µg/column n.a. Ready to use, prepacked columns, buffers and chemicals. High throughput when used with MicroPlex 24 Vacuum (up to 48 samples simultaneously). HisTrap Kit 12 mg*/column 4 ml/min As above, but includes buffers for up to 12 purifications using a syringe. HiTrap Chelating HP 1 ml 12 mg*/column 4 ml/min Prepacked column, ready to use. HiTrap Chelating HP 5 ml 60 mg*/column 20 ml/min Prepacked column, ready to use. Chelating Sepharose Fast Flow 12 mg*/ml medium 400 cm/h** Supplied as suspension for packing columns and scale up. *Estimate for a (His) 6 fusion protein of M r 27 600, binding capacity varies according to specific protein. **See Appendix 4 to convert linear flow (cm/h) to volumetric flow rate. Maximum operating flow is calculated from measurement in a packed column with a bed height of 10 cm and i.d. of 5 cm. 46
Purification examples Figures 24 and 25 show the purification of recombinant proteins expressed in soluble form or as inclusion bodies and Figure 26 gives an example of simultaneous on-column purification and refolding of a recombinant protein expressed as an inclusion body. Soluble recombinant proteins Sample: 9 ml E. coli periplasm containing Protein A-(HisGly) 4 His. Diluted with 9 ml binding buffer. Column: HiTrap Chelating HP, 5 ml Metal ion: Zn 2+ Flow: 1.0 ml/min Binding buffer: 50 mM sodium phosphate, 0.1 M NaCl, pH 8.0 Elution buffer: 50 mM sodium phosphate, 0.1 M NaCl, pH 4.0 Gradient: 20 ml elution buffer, step gradient Electrophoresis: SDS-PAGE, PhastSystem, PhastGel Gradient 8–25, 1 µl sample, Coomassie stained 0.10 0.05 A 280 nm M r 97 000 66 000 45 000 30 000 20 100 14 400 1 2 3 Lane 1. Low Molecular Weight Calibration Kit (LMW), reduced Lane 2. Crude periplasmic fraction, reduced Lane 3. Pool I, purified Protein A-(HisGly) 4 His, reduced 0 pool I 45 65 ml Fig. 24. Purification of recombinant proteins on HiTrap Chelating HP, 5 ml, charged with Zn 2+ . Recombinant protein expressed in inclusion bodies Sample: 8 ml cell extract containing (His) 10 -tagged protein. The clone was a kind gift from Dr. C. Fuller and S. Brasher, Department of Biochemistry, University of Cambridge, UK. Column: HiTrap Chelating HP, 1 ml Metal ion: Ni 2+ Binding buffers: 20 mM sodium phosphate, 0.5 M NaCl, 100 mM imidazole, 8 M urea or 6 M guanidine hydrochloride, pH 7.4 Elution buffers: 20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole, 8 M urea or 6 M guanidine hydrochloride, pH 7.4 Flow: Approx. 4 ml/min Equipment: Syringe Electrophoresis: SDS-PAGE, PhastSystem, PhastGel 10–15, 1 µl sample, silver staining Purification in 8 M urea M r 97 000 66 000 45 000 30 000 20 100 14 400 Purification in 6 M guanidine hydrochloride M r 97 000 66 000 45 000 30 000 20 100 14 400 1 2 3 4 5 6 7 1 2 3 4 5 6 7 Lane 1. Low Molecular Weight Calibration Kit (LMW) Lane 2. Starting material, cell extract, diluted 20-fold Lane 3. Flow-through, diluted 10-fold Lane 4. Wash Lane 5. Elution (first two ml) Lane 6. Elution (last two ml) Lane 7. LMW Lane 1. Low Molecular Weight Calibration Kit (LMW) Lane 2. Starting material, cell extract, diluted 10-fold Lane 3. Flow-through Lane 4. Wash Lane 5. Elution (first two ml) Lane 6. Elution (last two ml) Lane 7. LMW Fig. 25. Purification of (His) 10 -tagged protein from inclusion bodies on HiTrap Chelating HP, 1 ml, charged with Ni 2+ . 47
Page 1 and 2: Affinity Chromatography Principles
Page 3 and 4: Affinity Chromatography Principles
Page 5 and 6: Serine proteases and zymogens with
Page 7: Appendix 4 ........................
Page 10 and 11: This handbook describes the role of
Page 12 and 13: Affinity chromatography is also use
Page 14 and 15: BioProcess Media for large-scale pr
Page 17 and 18: Chapter 2 Affinity chromatography i
Page 19 and 20: Avoid using magnetic stirrers as th
Page 21 and 22: pH elution A change in pH alters th
Page 23 and 24: 0.6 0.4 0.2 0 A280 pH selected for
Page 25 and 26: Situation Cause Remedy Protein does
Page 27 and 28: Chapter 3 Purification of specific
Page 29 and 30: Table 2. Relative binding strengths
Page 31 and 32: Purification examples Figure 11 sho
Page 33 and 34: MAbTrap Kit Fig. 13. MAbTrap Kit, r
Page 35 and 36: Media characteristics Ligand Compos
Page 37 and 38: A 280 nm 2.0 1.5 1.0 0.5 Column: rP
Page 39 and 40: Desalt and/or transfer purified IgG
Page 41 and 42: Performing a separation Column: Rec
Page 43 and 44: M r 97 000 66 000 45 000 30 000 20
Page 45 and 46: in The Recombinant Protein Handbook
Page 47: Cleaning These procedures are appli
Page 51 and 52: Nickel solution: 0.1 M NiSO 4 Bindi
Page 53 and 54: Protein A fusion proteins IgG Sepha
Page 55 and 56: Purification or removal of serine p
Page 57 and 58: Sample: 2 ml clarified E. coli homo
Page 59 and 60: Serine proteases and zymogens with
Page 61 and 62: DNA binding proteins HiTrap Heparin
Page 63 and 64: Sample: Column: Binding buffer: Elu
Page 65 and 66: Media characteristics Ligand densit
Page 67 and 68: Sample: Pooled and frozen human pla
Page 69 and 70: The harsh conditions required to br
Page 71 and 72: Purification or removal of albumin
Page 73 and 74: IFN-act. A units 280 nm 250 0.12 20
Page 75 and 76: Purification options Binding capaci
Page 77 and 78: Sepharose NH (CH 2) n NH N N O N N
Page 79 and 80: If detergent or denaturing agents h
Page 81 and 82: Glycoproteins or polysaccharides Co
Page 83 and 84: For complex samples containing glyc
Page 85 and 86: For complex samples containing glyc
Page 87 and 88: Chemical stability Avoid exposure t
Page 89 and 90: Proteins and peptides with exposed
Page 91 and 92: Several methods can be used to dete
Page 93 and 94: Media characteristics Composition M
Page 95 and 96: Performing a separation Binding buf
Page 97 and 98: Do not store the suspension for lon
Page 99 and 100:
Sepharose has been modified and dev
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Ligand coupling Several methods are
Page 103 and 104:
Couple a ligand through the least c
Page 105 and 106:
Table 9 summarizes recommended liga
Page 107 and 108:
Coupling through the primary amine
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Ligand and column preparation 1. Di
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Options Product Spacer Coupling con
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100 80 Coupled substance, % 60 40 2
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Coupling small ligands through amin
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Ligand coupling 1. Add the ligand s
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Alternative coupling solutions: Dis
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Media characteristics Product Compo
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Ligands containing amino groups can
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Chapter 6 Affinity chromatography a
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Resolution is achieved by the selec
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For any capture step, select the te
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Appendix 1 Sample preparation Sampl
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Remove salts from proteins with mol
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1. Filter (0.45 µm) or centrifuge
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For laboratory scale operations, Ta
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Removal of lipoproteins Lipoprotein
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Appendix 2 Selection of purificatio
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4. Estimate the amount of slurry (r
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Appendix 5 Conversion data: protein
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Middle unit residue (-H 2 0) Charge
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Expected results when changing cond
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Expected results with competitive e
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Appendix 8 Analytical assays during
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Appendix 9 Storage of biological sa
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Ordering information Product Quanti
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STREAMLINE, Sepharose, HiTrap, ÄKT
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