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Application note 29-0147-56 AA Affinity chromatography

Purification of MBP-(His)6 using HiTrap™ TALON® crude and ÄKTApurifier™ plus We purified histidine-tagged maltose binding protein MBP-(His)6 using ÄKTApurifier 10 plus with UNICORN™ control software and HiTrap TALON crude column prepacked with TALON Superflow™, which is a cobalt-based IMAC medium. The method is efficient because unclarified E. coli lysate can be directly loaded onto the column without centrifugation and filtration. This results in a faster purification process, which can be crucial to retaining the activity of the target protein. The column screening kit on ÄKTApurifier plus enables automatic switching between different column positions so that a new column can easily be selected for a new sample. Finally, we showed that the protein activity of the purified MBP-(His)6 was unchanged using a binding assay on Biacore™ T200 system.

IntroductionThe use of tagged recombinant proteins has increased greatly in recent years, as has the wealth of techniques and products used for their amplification and purification. The purification of tagged proteins is relatively simple and saves time due to the high specificity between the tag on the expressed protein and the ligand on the affinity chromatography medium. You may get high purity (> 80%) in a single step. However, certain downstream applications (e.g., structural studies, lead design etc.), often require higher purity of the target protein. Further purification steps may then be necessary. The three-stage purification strategy of Capture, Intermediate Purification, and Polishing (CIPP) gives guidelines for how to develop and implement an appropriate purification protocol (Strategies for Protein Purification, GE Healthcare, 28-9833-31 Edition AA, pp. 39–50 (2010).

The histidine tag is widely used to facilitate both the purification of soluble proteins and also, the detection and on-column refolding of insoluble proteins. Precharged cobalt IMAC medium is commonly used for the purification of histidine-tagged proteins. Since many proteins in the cell have intrinsic histidine and/or cysteine amino acid residues, they may also bind to the IMAC medium together with the target protein.

In such cases, it is often necessary to optimize binding, wash, and elution conditions by varying the concentration of imidazole in these buffers. Increasing the concentration of imidazole in the binding and wash buffers generally decreases nonspecific binding, whereas lower concentrations give stronger affinity interaction. The key is finding the right balance.

Introduction of automation into the IMAC purification process has led to significant efficiency gains such as a reduction in both the time and amount of sample required for the development of different chromatographic steps. For example, HiTrap TALON crude (Fig 1), which is designed for purifying histidine-tagged recombinant proteins can be used in conjunction with ÄKTApurifier systems (Fig 2). These are versatile, modular liquid chromatography systems for fast and reliable purifications.

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Fig 1. HiTrap TALON crude and a schematic workflow of the purification of histidine-tagged proteins. There is no need for you to centrifuge or filter your sample before loading onto the column thus increasing the efficiency of your purification process.

Fig 2. We used ÄKTApurifier 10 plus in this study. The system is preassembled with automation kits for media screening and method optimization.

Enzymatic and mechanical cell lysis

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In this application note, we describe the development and application of a method for using HiTrap TALON crude, ÄKTApurifier 10 plus, and different imidazole concentrations to purify MBP-(His)6 with high yields and purity in a fast and efficient manner.

Materials and MethodsHiTrap TALON crude, ÄKTApurifier 10 plus, Amersham™ ECL Gel, Ettan™ DIGE Imager, Deep Purple™ Total Protein Stain, and Biacore T200 were used according to the manufacturer’s (GE Healthcare) instructions. The binding buffer contained either 5, 10 or 20 mM imidazole with 300 mM NaCl, 50 mM sodium phosphate, pH 7.4. The elution buffer was 150 mM imidazole, 300 mM NaCl, 50 mM sodium phosphate, pH 7.4. The samples were prepared by spiking pure MBP-(His)6 into E. coli lysate containing 5, 10 or 20 mM imidazole. The final concentration of MBP-(His)6 was 0.9 mg/ml. SDS gel electrophoresis was performed using Amersham ECL Gel with a gradient of 8 to 16%. The samples were mixed at a ratio of 1:1 with a 2-fold sample buffer (reduced) and heated for 5 min at 95°C. The sample mixture (20 µl) was applied to the wells on the gel, ran, and stained with Deep Purple Total Protein Stain.

We used a binding assay involving surface plasmon resonance (SPR) to determine the activity of purified MBP-(His)6 using Biacore T200. The samples [pure MBP-(His)6 reference sample and one eluted fraction from purification, see Figure 4A] were diluted to 20 µg/ml in the immobilization buffer (10 mM sodium acetate, pH 4.5), and immobilized onto Sensor Chip CM5 using standard amine coupling with an injection time of 400 s. The immobilization levels were about 4000 resonance units (RU) for the reference sample and about 7000 RU for the eluted fraction. A maltose concentration series of 40 nM to 10 µM was injected over the immobilized MBP-(His)6 in order to determine the affinity of the interaction. The running buffer was 20 mM PBS, 137 mM NaCl, 2.7 mM KCl, pH 7.4.

Results and discussionDetermination of the binding efficiency of MBP-(His)6To determine whether the target protein could bind to the column in the presence of 5 mM imidazole, a sample containing 4.5 mg pure MBP-(His)6 (no E. coli) in binding buffer was loaded onto HiTrap TALON crude and the results (Fig 3) show that binding was efficient with a yield of about 100%.

Effect of different imidazole concentrationsIt is common to include low concentrations of imidazole in the sample, binding, and wash buffers during an IMAC purification in order to reduce the binding of host cell proteins with metal binding capabilities. At relatively higher concentrations, imidazole may decrease the binding of histidine-tagged proteins. The imidazole concentration in each step must therefore be optimized to ensure the best balance of high purity (low binding of specific cell proteins) and high yield (strong binding of histidine-tagged target protein).

Fig 3. Determination of the binding capacity of pure MBP-(His)6 at 5 mM imidazole. The blue curve shows the absorbance at 280 nm.

Fig 4. Purification of MBP-(His)6 on HiTrap TALON crude at three different imidazole concentrations in the sample and binding buffer. (A) 5 mM imidazole; (B) 10 mM imidazole; and (C) 20 mM imidazole.

Column: HiTrap TALON crude, 1 mlSample: 4.5 mg pure MBP-(His)6 in 5 ml binding bufferBinding buffer: 5 mM imidazole, 300 mM NaCl, 50 mM sodium phosphate,

pH 7.4Elution buffer: 150 mM imidazole, 300 mM NaCl, 50 mM sodium

phosphate, pH 7.4Flow: 1 ml/minSystem: ÄKTApurifier 10 plus

Column: HiTrap TALON crude, 1 mlSample: 4.5 mg MBP-(His)6 in 5 ml E. coli lysateBinding buffer: 5, 10 or 20 mM imidazole, 300 mM NaCl, 50 mM sodium

phosphate, pH 7.4.Elution buffer: 150 mM imidazole, 300 mM NaCl, 50 mM sodium

phosphate, pH 7.4.Flow: 1 ml/minSystem: ÄKTApurifier 10 plus

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Fig 5. SDS gel electrophoresis (reduced conditions) of pooled fractions from the purification of MBP-(His)6, see chromatograms in Fig 4. The gel was stained with Deep Purple Total Protein Stain.

Lane1. Low molecular weight (LMW) markers 2. Start material, MBP-(His)6 in E. coli lysate3. Flowthrough, 5 mM imidazole4. Flowthrough, 10 mM imidazole5. Flowthrough, 20 mM imidazole6. Eluted sample, 5 mM imidazole during binding7. Eluted sample, 10 mM imidazole during binding8. Eluted sample, 20 mM imidazole during binding

Determination of the activity of purified MBP-(His)6The relative activity of MBP-(His)6 before and after purification was assessed using a binding assay on Biacore T200. The MBP portion of MBP-(His)6 has an affinity for maltose and by immobilizing MBP-(His)6 (ligand) on a chip and measuring the binding activity towards maltose (analyte) the relative activity could be established. The results (Fig 6) show that both samples had similar affinities for maltose [KD = 7.6 × 10-7 for pure MBP-(His)6 reference sample and KD = 5.3 × 10-7 for MBP-(His)6 after purification]. showing that the protein activity was unaffected by the purification process.

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Fig 6. Comparative binding activity of MBP-(His)6. The sensograms and steady- state data were fitted to a 1:1 model for (A) pure MBP-(His)6 reference sample, KD = 7.6 × 10-7 and (B) (MBP-His)6 after purification, KD = 5.3 × 10-7.

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The chromatography medium in HiTrap TALON crude charged with Co2+ ions normally requires lower concentrations of imidazole in the binding buffer compared to Ni Sepharose™ charged with Ni2+ ions. We examined the effects of 5, 10, and 20 mM imidazole in the sample and binding buffer on the yield and purity of MBP-(His)6 using HiTrap TALON crude. TALON crude column was used for each run utilizing the automatic switching of the Column Screening Kit on ÄKTApurifier plus. The results (Fig 4) show that relatively small variations in imidazole concentrations had significant effects on the yield of MBP-(His)6: 98% at 5 mM, 52% at 10 mM, and 6% at 20 mM imidazole.

The eluted protein was analyzed by SDS gel electrophoresis (Fig 5). The final purity was over 95% for all the three different imidazole concentrations we investigated. The decreasing strength of the major protein band in lanes 6 to 8 represent a decrease in yield at higher imidazole concentrations. This in turn correlates with an increase in the amount of the target protein in the flowthrough at higher imidazole concentrations as seen in lanes 3 to 5. The results show that the specificity of HiTrap TALON crude for MBP-(His)6 is optimal at 5 mM imidazole, resulting in a high yield of ≥ 98% and purity of ≥ 95%.

GE, imagination at work and GE monogram are trademarks of General Electric Company.

ÄKTApurifier, Amersham, Biacore, Deep Purple, Ettan, HiTrap, Sepharose, and UNICORN are trademarks of GE Healthcare companies.

TALON is a registered trademark of Clontech Inc. Superflow is a trademark of Sterogene Bioseparations Inc.

Deep Purple Total Protein Stain is exclusively licensed to GE Healthcare from Fluorotechnics Pty Ltd. Deep Purple Total Protein Stain may only be used for applications in life science research. Deep Purple is covered under a granted patent in New Zealand entitled “Fluorescent Compounds”, patent number 522291 and equivalent patents and patent applications in other countries.

Purification and preparation of fusion proteins and affinity peptides comprising at least two adjacent histidine residues may require a license under US patent numbers 5,284,933 and 5,310,663, and equivalent patents and patent applications in other countries (assignee: Hoffman La Roche, Inc).

© 2012 General Electric Company—All rights reserved. First published Mar. 2012.

All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information.

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29-0147-56 AA 03/2012

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Ordering informationSystems Quantity Code no.

ÄKTApurifier 10 plus* 1 28-9914-36

ÄKTApurifier 100 plus* 1 28-9914-35

Biacore T200 1 28-9750-01

Columns

HiTrap TALON crude 5 × 1 ml 28-9537-66

HiTrap TALON crude 5 × 5 ml 28-9537-67

HiTrap TALON crude 100 × 1 ml** 28-9538-05

HiTrap TALON crude 100 × 5 ml** 28-9538-09

* For a complete ÄKTApurifier plus offer, please contact your local GE Healthcare representative.

** Pack size available by special order

Related products

Strategies for Protein Purification, Handbook 28-9833-31

ÄKTApurifier, Data file 18-1119-48

TALON Superflow and prepacked formats, Data file 28-9664-10

Column Screening Kit 11-0035-95

Series S Sensor Chip CM5 BR-1005-30

Amine Coupling Kit BR-1000-50

Buffer PBS 10X BR-1006-72

Immobilization buffer Acetate 4.5 BR-1003-50

Amersham ECL Gel 8-16% 28-9901-58

Amersham ECL Gel Box 28-9906-08

Deep Purple Total Protein Stain, 1 × 5 ml RPN6305

Conclusions• The data shows that it is feasible to purify MBP-(His)6 using

HiTrap TALON crude and ÄKTApurifier 10 plus.

• The purification method is fast and simple because it allows unclarified E. coli lysate to be loaded directly onto HiTrap TALON crude column without centrifugation and filtration. This cuts down the purification time and this can be critical for maintaining the activity of the target protein.

• The method is efficient because of the inherent automation of buffer selection and column switching using ÄKTApurifier plus together with UNICORN control software.

• Different imidazole concentrations in the binding buffer had significant effects on the yield of MBP-(His)6.

• The purified MBP-(His)6 retained activity as determined via binding assay using a Biacore T200 system.