TALON® Metal Affinity Resins User Manual PT1320-1 (PR6Z2142) Published 25 April 2007 TALON® Metal Affinity Resins User Manual Table of Contents I. Introduction 4 II. List of Components 13 III. Additional Materials Required 15 IV. Buffers for TALON® Purification & Buffer Kits 20 V. Buffers for TALON® Magnetic Beads 21 VI. Transformation & Protein Expression 22 A. Transformation of Host Cells with Expression Vectors 22 B. Protein Expression 22 VII. Sample Preparation 23 A. TALON® xTractor Buffer Sample Preparation 23 B. Standard Sample Preparation to Isolate Native Proteins 23 C. Standard Sample Preparation to Isolate Denatured Proteins 24 D. Standard Sample Preparation for TALON® CellThru Resin 25 E. Standard HT 96-Well Sample Preparation 26 F. Standard Sample Preparation for TALON® Magnetic Beads 26 G. Sample Preparation Directly from Overnight Cultures for TALON® Magnetic Beads 27 VIII. Protein Purification Protocols 28 A. General Information 28 B. Batch/Gravity-Flow Column Purification 30 TALON® Resin, Superflow Resin, or CellThru Resin C. Large-Scale Batch Purification 32 TALON® Resin, Superflow Resin, or CellThru Resin D. Medium-Pressure Column Purification 33 TALON® Superflow Resin E. 5 ml TALON® Single Step Column Purification 34 F. 20 ml TALON® Single Step Column Purification 36 G. TALONspin™ Column Purification 38 H. TALON® HT 96-Well Purification Protocol 40 I. TALON® Magnetic Beads Purification Protocol 43 IX. Resin Washing, Reuse, and Storage 46 X. Troubleshooting Guide 48 XI. References 53 Clontech Laboratories, Inc. www.clontech.com Protocol No. PT1320-1 2 Version No. PR6Z2142 TALON® Metal Affinity Resins User Manual Table of Contents continued Appendix A. Reagent Compatibilities and Incompatibilities 54 Appendix B. Mini-Scale Protein Purification Protocol for TALON® or TALON® Superflow Resin 56 Appendix C. Vector Information 58 List of Figures Figure 1. Schematic diagram of the TALON® IMAC System. 5 Figure 2. Elution mechanism of recombinant polyhistidine-tagged proteins from TALON® Resin. 6 Figure 3. Binding of histidines to the TALON® Resin metal ion. 6 Figure 4. Using the TALON® Metal Affinity Resins User Manual 7 Figure 5. Purification of polyhistidine-tagged proteins using TALON® Resin 19 Figure 6. pHAT10/11/12 combined vector map and MCS. 58 Figure 7. pHAT20 combined vector map and MCS. 59 List of Tables Table I. Protein purification using TALON® Resins. 10 Table II. TALON® Resin characteristics 12 Table III. Reagent compatibility 54 Notice to Purchaser Clontech products are to be used for research purposes only. They may not to be used for any other purpose, including, but not limited to, use in drugs, in vitro diagnostic purposes, therapeutics, or in humans. Clontech products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without written approval of Clontech Laboratories, Inc. Licensed under U.S. Patent No. 4, 569, 794 and its international equivalents for use in research related biopolymers. Licenses for commercial applications are available from Indiana Pro- teomics Consortium, Inc. (Inproteo). TALON® Resin products are covered under U.S. Patent No. 5,962,641. Sepharose® is a registered trademark of GE Healthcare. Triton™ is a trademark of The Dow Chemical Company. Superflow™, Uniflow™, and CellThru™ are trademarks of Sterogene Bioseparations, Inc. Clontech, Clontech logo and all other trademarks are the property of Clontech Laboratories, Inc., unless noted otherwise. Clontech is a Takara Bio Company. ©2007 Protocol No. PT1320-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR6Z2142 3 TALON® Metal Affinity Resins User Manual I. Introduction Proteins have evolved very complex structures in order to perform a diverse ar- ray of functions. As a result, their physicochemical properties vary greatly, pos- ing difficulties for developing versatile purification protocols. One way to cir- cumvent this problem is to incorporate a purification tag into the primary amino acid sequence of a target protein, thus constructing a recombinant protein with a binding site that allows purification under well-defined, generic conditions. Immobilized Metal Affinity Chromatography (IMAC) IMAC was introduced in 1975 as a group-specific affinity technique for sepa- rating proteins (Porath et al., 1975). The principle is based on the reversible interaction between various amino acid side chains and immobilized metal ions. Depending on the immobilized metal ion, different side chains can be involved in the adsorption process. Most notably, histidine, cysteine, and tryptophan side chains have been implicated in protein binding to im- mobilized transition metal ions and zinc (Figure 1, Porath, 1985; Sulkowski, 1985; Hemdan & Porath, 1985a; Hemdan & Porath, 1985b; Zhao et al., 1991). TALON® IMAC Resins TALON® Resins are durable, cobalt-based IMAC resins designed to purify recombinant polyhistidine-tagged proteins (Bush et al., 1991).T hese resins are compatible with many commonly used reagents (Appendix A), and allow pro- tein purification under native or denaturing conditions.T hey can be used with all prokaryotic and eukaryotic expression systems in a variety of formats, in- cluding small- (or mini-) scale batch screening, large-scale batch preparations, and methods using gravity-flow columns and spin columns. In addition, pro- tocols used with Ni+2-based IMAC columns usually work withT ALON® resins. TALON Magnetic Beads are agarose beads utilizing our patentedT ALON tech- nology.T he beads combine the advantage of highly selectiveT ALON chemistry with magnetic bead separation. Magnetic particles in the beads facilitate quick and easy purification of proteins at microscale level using a magnetic separator. Microscale purification with TALON Magnetic Beads can be used for screening of expression levels or for protein-protein interaction studies. Tetradentate metal chelator To overcome the problem of metal leakage encountered with other IMAC res- ins,T ALON® Resin utilizes a special tetradentate metal chelator for purifying recombinant polyhistidine-tagged proteins (U.S. Patent No. 5,962,641). This chelator tightly holds the electropositive metal in an electronegative pocket (Figure 1), which is ideal for binding metal ions such as cobalt. The binding pocket is an octahedral structure in which four of the six metal coordination sites are occupied by the TALON Resin ligand. This process enhances the protein binding capacity of TALON Resin by making the bound metal ion ac- cessible to surrounding polyhistidine-tagged proteins.T he tetradentate metal binding means that no metal loss occurs during protein purification under rec- Clontech Laboratories, Inc. www.clontech.com Protocol No. PT1320-1 4 Version No. PR6Z2142 TALON® Metal Affinity Resins User Manual I. Introduction continued H2C COO– COO– N HC Sepharose Co2+ N Bead N HC COO– 2 N N A B Figure 1. Schematic diagram of the TALON® IMAC System. Part A. TALON Metal Affinity Resin; A Sepharose bead bearing the tetradentate chelator of the Co2+ metal ion. Part B. The polyhi- stidine-tagged recombinant protein binds to the resin. ommended conditions, even in the presence of strong denaturants such as 6 M guanidinium. Such durability allowsT ALON Resin to be reused (See Section IX). Cobalt IMAC Resin permits milder elution conditions TALON Resin exhibits subtle yet important differences in binding of polyhis- tidine-tagged proteins when compared with nickel IMAC resins. For example, nickel-based IMAC resins often exhibit an undesirable tendency to bind un- wanted host proteins containing exposed histidine residues (Kasher et al., 1993). WhileT ALON Resin binds polyhistidine-tagged proteins with enhanced selectivity over nickel-based resins, it exhibits a significantly reduced affin- ity for host proteins. This behavior offers two practical advantages. First, virtually no background proteins are bound to the resin when the sample is applied; consequently, cumbersome washing procedures are not generally required before protein elution. Second, polyhistidine-tagged proteins elute from the resin under slightly less stringent conditions—a slightly higher pH or lower imidazole concentration—than with nickel IMAC resins. Elution occurs when the imidazole nitrogen (pKa of 5.97) is protonated (Figure 2), generating a positively charged ammonium ion, which is repelled by the positively charged metal atom. Alternatively, the bound polyhistidine-tagged protein can be competitively eluted by simply adding imidazole to the elu- tion buffer, because imidazole is identical to the histidine side chain. Protocol No. PT1320-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR6Z2142  TALON® Metal Affinity Resins User Manual I. Introduction continued N H + N N H N H H + N N 2+ ? Unprotonated Histidine Protonated Histidine binds to metal repelled by metal N Figure 2. Elution mechanism of recombinant polyhistidine-tagged proteins from TALON® Resin. Elution occurs when the imidazole ni- N trogen (pKa = 5.97) is protonated, generating a positively charged ammonium ion which is Figure 3. Binding of histidines to the repelled by the positively charged metal ion. TALON® Resin metal ion. Under conditions of Alternatively, the bound polyhistidine-tagged physiological pH, histidine binds by sharing protein can be competitively eluted by adding imidazole nitrogen electron density with the imidazole to the elution buffer. electron-deficient orbitals of the metal ion. Polyhistidine affinity tags Histidines exhibit highly selective coordination with certain transition met- als and have great utility in IMAC. Under conditions of physiological pH, histidine binds by sharing electron density of the imidazole nitrogen with the electron-deficient orbitals of transition metals (Figure 3). Although three histidines may bind transition metals under certain conditions, six histidines reliably bind transition metals in the presence of strong denaturants such as guanidinium (Hochuli et al., 1987). Such protein tags are commonly referred to as “6 x histidine,” “hexaHis,” or “(His) .” 6 HAT—a novel IMAC affinity tag With the advent of recombinant genetic technologies, the design and production of recombinant proteins containing novel polyhistidine tags on their N- or C-termini has become more straightforward (Hochuli et al., 1987; Hochuli et al., 1988). The HAT sequence (patent pending) is a novel IMAC affinity tag derived from a unique natural protein sequence (Chaga et al., 1999). It contains six histidines unevenly interleaved by other amino acid residues (see Appendix C). The HAT amino acid sequence is derived from the N-terminus of chicken muscle lactate dehydrogenase—a sequence that is unique among reported protein sequences. The novel tag does not have the excessive positive charge characteristic of the commonly used 6 x histidine tag, thus contributing to better solubility of HAT-fusion pro- teins and similar affinity towards immobilized transition metal ions and zinc. Clontech offers the HAT Protein Expression and Purification System (Cat. No. 631205)—a complete system containing reagents and vectors Clontech Laboratories, Inc. www.clontech.com Protocol No. PT1320-1 6 Version No. PR6Z2142 TALON® Metal Affinity Resins User Manual I. Introduction continued Protein Purification Strategy (Section I) • Protein Purification Methods - Resin Characteristics • Choosing Buffers • Elution Strategy Buffers (Sections III & IV) Native Denaturing Protein Expression (Section VI) A. Transformation B. Protein Expression Sample Preparation (Section VII) Native Purification Denaturing Purification A. xTractor Buffer C. Standard & B. Standard & Superflow Resin Superflow Resin D. CellThru Resin D. CellThru Resin F. TALON Magnetic E. High-throughput (96-well) Beads F. & G. TALON Magnetic Beads Protein Purification (Section VIII) B. Batch or Gravity Flow C. Large-Scale Batch D. Medium-Pressure & FPLC Column E. 5 ml Single Step Columns F. 20 ml Single Step Columns G. TALONspin™ Columns H. HT 96-Well Plate I. TALON Magnetic Beads Appendix B. Mini-Scale Figure 4. Using the TALON® Metal Affinity Resins User Manual. Overview of the procedures. Protocol No. PT1320-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR6Z2142  TALON® Metal Affinity Resins User Manual I. Introduction continued designed for bacterial expression and purification of HAT (histidine affinity tag) proteins. Each of the three vectors—pHAT10, pHAT11, and pHAT12—contain a multiple cloning site (MCS) in all three frames to allow cloning of target cDNA. (For vector map and MCS, see Appendix C of this User Manual.) A conveniently located enterokinase proteolytic site between the HAT sequence and the MCS provides a means for removing the affinity tag. For more information, see the HAT Expression & Purification System User Manual (PT3250-1), which can be downloaded from our web site at www. clontech.com. TALON® Express Bacterial Expression and Purification Kits TALON Express Bacterial Expression and Purification Kits are designed for the cloning, expression, and purification of polyhistidine-tagged proteins using E. coli. The kits contain two separate bacterial expression vectors encoding N- or C-terminal 6xHN fusion tags. These IPTG-inducible, pET-based vectors provide high levels of protein expression. The expressed proteins are ready for quick and easy purification using the TALON resin and buffers provided in the kits. Overview of TALON® Resins The following is a list of different resin formats to meet your purification needs. • TALON® Metal Affinity Resin is useful for batch and low-pressure chromatographic applications. This resin utilizes Sepharose CL-6B (GE Healthcare), a durable substrate that performs very well under native and denaturing conditions in centrifuge-mediated purification schemes. The large pore size resin has a high-binding capacity. This resin is also available pre-packed in 2 ml gravity columns. • TALON® Superflow Resin is useful for a range of applications, including medium pressure applications with FPLC systems at back pressures of up to 150 psi (1 MPa) and high flow rates up to 5 ml per cm2 per min. This resin is recommended if short purification times are essential, or if purification protocols developed at bench scale will be scaled up for larger volumes. This resin utilizes Superflow-6 (Sterogene Bioseparations, Inc.), an aga- rose-based medium featuring a unique polysaccharide composition that resists biological degradation. Superflow-6 beads are also stabilized by a chemical cross-linking reaction that allows flow rates up to 10 times higher than are possible with regular cross-linked beads. The Talon® Superflow Resin is also present in the high throughput (HT) 96-well plate. Clontech Laboratories, Inc. www.clontech.com Protocol No. PT1320-1  Version No. PR6Z2142 TALON® Metal Affinity Resins User Manual I. Introduction continued • TALON® CellThru Resin is a novel IMAC resin for purifying polyhistidine- tagged proteins from crude cell lysates, sonicates, and fermentation liquids. The larger bead size ofT ALON CellThru Resin (300–500 µm) permits cellular debris to flow through the column, eliminating the need for high-speed centrifugation. Destabilizing factors are removed more quickly with this resin than with other IMAC resins, because the number of steps are reduced. CellThru 2 ml & 10 ml Disposable Columns have a large filter pore size (90–130 µm) that allows cellular debris to flow through the column during the purification process. The 2 ml columns are suitable for 1–2 ml bed volumes, while the 10 ml columns are suitable for 5–10 ml bed volumes. • TALONspin™ Columns are ideal for rapidly and simultaneously purifying small amounts of polyhistidine-tagged proteins.T hese columns are recom- mended for single-use applications or for use as mini gravity-flow columns. Each column contains 0.5 ml of TALON-NX Resin, which is optimized for performance in a spin column. Each column will yield 2–4 mg of polyhi- stidine-tagged protein; exact yields will vary with conditions used and polyhistidine-tagged protein characteristics. In addition, yield and purity will depend upon expression level and lysate concentration. Beginning with the clarified sample, the entire procedure takes approximately 30 min. • TALON® Magnetic Beads are useful for microscale purification of polyhisti- dine-tagged proteins under native or denaturing conditions.T he beads can also be used to purify proteins directly from cleared (centrifuged) or crude cell lysates. For screening of expression levels, proteins can be purified directly from overnight cultures as small as 0.5 ml (depending on the expres- sion level). The use of TALON chemistry allows for seamless scaling-up to large-scale purification of target proteins using our standardT ALON resin. TALON Magnetic Beads are supplied as a 5% suspension in 25% ethanol, available in either a 2 x 1 ml or 6 x 1 ml format. The beads have a binding capacity of 750 µg of 6xHN-tagged AcGFP per 1 ml of suspension. When performing assays in single tubes, 100–200 µl of beads are sufficient for each assay. Smaller amounts of beads may be used, but there may be difficulties in handling the beads in small buffer volumes. Protocol No. PT1320-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR6Z2142  TALON® Metal Affinity Resins User Manual I. Introduction continued TABLE I. PROTEIN PURIFICATION USING TALON® RESINS Method Application Key Benefit TALON® Metal Affinity Resin or TALON® Superflow Resin Mini-Scale • Check for presence of tagged protein • Fast (Appendix B) • Estimate expression levels • Requires only 1 ml of cell • Test buffer conditions culture + 1 ml of resin Batch/Gravity • Purify >5 mg of tagged protein • Very high purity Flow Column using 1 ml of resin • Does not require (Sec. VIII.B) pressurized column equipment Large-Scale • Large- and production-scale • Faster than protocols that (Sec. VIII.C & D) purification; easy to scale up use gravity-flow columns • Higher purity than using batch process alone TALON® CellThru Resin Batch/Gravity • For purifying proteins from • Fast Flow Column & nonclarified cell lysates, sonicates, • Does not require high- Large-Scale or fermentation liquids speed centrifugation (Sec. VIII.B & C) TALON® Single Step Columns (5 ml, 20 ml) Miniprep • Process several different samples • Fast (~30–40 min)1 (Sec. VIII.E & F) simultaneously • Uses unlysed cell culture • Lyse bacterial cells and bind • Simplifies screening of histidine-tagged protein in one step multiple proteins • Obtain 0.2–0.6 mg (5 ml column) • Ready-to-use columns or 0.5–4 mg (20 ml column). TALONspin™ Columns Spin Column • Process several different samples • Fast (~30 min)2 (Sec. VIII.G) simultaneously • Uses only 0.6–1 ml of • Obtain 2–4 mg of purified protein cell culture lysate per spin column • Ready-to-use columns TALON® HT 96-Well Plates 96-Well Plates • High-throughput processing of samples • Fast(<30 min)2 (Sec. VIII.H) • Obtain up to 1.0 mg of purified protein • Uses up to 2 ml of per well crude lysate per load TALON® Magnetic Beads Magnetic Beads • Microscale purification • Fast (Sec. VII.F & G • Check for presence of tagged protein • Requires 0.5 ml culture and Sec. VIII.I) • Estimate expression levels • Does not require high- • Purify from crude uncleared speed centrifugation cell lysates or cultures • Amenable to high-throughput 1 Includes time for sample prep and purification. 2 Starting with clarified lysate; does not include time to prepare samples. Clontech Laboratories, Inc. www.clontech.com Protocol No. PT1320-1 10 Version No. PR6Z2142