Here we present a protocol for the benchtop immobilized metal affinity chromatography purification and subsequent reconstitution of a polyhistidine tagged, non-heme iron binding dioxygenase suitable for the undergraduate teaching laboratory.
Benchtop immobilized metal affinity chromatography (IMAC), of polyhistidine tagged proteins is easily mastered by undergraduate students and has become the most widely used protein purification method in the modern literature. But, the application of affinity chromatography to metal binding proteins, especially those with redox sensitive metals such as iron, is often limited to laboratories with access to a glove box – equipment that is not routinely available in the undergraduate laboratory. In this article, we demonstrate our benchtop methods for isolation, IMAC purification and metal-ion reconstitution of a poly-histidine tagged, redox-active, non-heme iron binding extradiol dioxygenase and the assay of the dioxygenase with varied substrate concentrations and saturating oxygen. These methods are executed by undergraduate students and implemented in the undergraduate teaching and research laboratory with instrumentation that is accessible and affordable at primarily undergraduate institutions.
The first reports of the purification of a polyhistidine tagged protein from extracts of a host organism using chelation of the histidine tag by an immobilized metal entered the literature in 19881,2. Since that time, the addition of polyhistidine tags to recombinant proteins and their purification by immobilized metal affinity chromatography (IMAC) have become virtually ubiquitous in the biochemical literature3,4,5. IMAC purification methods can be implemented on the benchtop, using automated chromatography and in spin-column formats. While affinity purification methods, especially IMAC, are widely used in the research laboratory, they are less common in the undergraduate teaching laboratory. The most widely used laboratory textbooks for the biochemistry laboratory do not routinely teach these methods, instead opting for more traditional ion-exchange or dye-binding chromatography6,7,8,9. For example, the purification of lactate dehydrogenase by Anderson10 uses affinity by dye-binding, and the purification of Bovine milk α-lactalbumin7,11 by Boyer uses a nickel-nitriloacetic acid matrix, but no recombinant poly-histidine tag, relying instead on intrinsic affinity of the protein for the resin. Some modern undergraduate laboratory textbooks and publications do implement immobilized metal affinity chromatography on poly-histidine tagged protein targets such as green or red-fluorescent proteins12,13,14,15, antibodies16, and selected enzymes17,18,19,20, even some of unknown function21. Arguably, the purification of an enzyme is preferable in the teaching laboratory, because the target can be assayed for activity in subsequent sessions, enriching the experience of "real science" on the part of the student; indeed, these types of laboratory experiences have been published and beneficial outcomes on student learning reported17,18,20,21. And yet, applications of IMAC to enzyme purification in the biochemistry teaching laboratory remain sparse, and the published methods may even presume access to chromatography instrumentation that is typically unavailable for use in the classroom laboratory20. There are also limitations in the application of IMAC to metalloproteins, especially those which bind redox-sensitive divalent metals that are essential to activity22. Frequently, the metal ion is lost or oxidized during purification yielding an inactive enzyme ill-suited to the undergraduate laboratory.
A full one-third of enzymes bind a metal ion23, and despite a nearly universal requirement for iron in all forms of life23, iron is arguably among the most problematic metal ions in enzymology. Non-heme Fe2+ binding enzymes are particularly prone to loss and/or oxidation of the metal during IMAC; presumably due to the lack of a dedicated organic ligand like heme and the ease with which Fe2+ can dissociate from amino acid ligands24. Furthermore, the oxygen dependent oxidation of Fe2+ to Fe3+ is spontaneous in aqueous solution, due to the negative free energy change and the relative stability of Fe3+. Often, these challenges are overcome by use of anaerobic atmosphere and/or non-IMAC chromatographic methods22. In this article, we will demonstrate the use of benchtop IMAC to purify the Fe2+ dependent metalloenzyme L-DOPA dioxygenase using simple, inexpensive chromatography supplies, followed by the reconstitution of the active site Fe2+, and enzymatic assay. These methods are standard in our own undergraduate biochemistry laboratory of 6-12 student groups and can be used to expand the repertoire of enzyme investigations at the undergraduate level.
1. Preparation for Purification
2. Purification of Polyhistidine-tagged Target by IMAC
3. Reconstitution of Target Protein with Fe2+
These representative results were collected by undergraduate students as they executed this protocol during two course laboratory periods of BCM 341: Experimental Biochemistry at Muhlenberg College. Figure 1 demonstrates the results of the purification of a 20 kDa poly-histidine tagged metalloenzyme, L-DOPA dioxygenase, as performed by two undergraduate students during a 4-hour classroom laboratory period and analyzed by SDS-PAGE by the same students during a subsequent laboratory period. The poly-histidine tagged protein is effectively purified (Figure 1, lane 2). An immunoblot of the gel using antibody against the poly-histidine tag indicates that a small amount of the poly-histidine tagged target protein is lost in the flow-through (data not shown), likely because the greater than 100 mg quantity of target protein in the lysate exceeded the binding capacity of the resin.
Figure 2 demonstrates student-collected activity data on the enzymatic reaction of the poly-histidine tagged metalloenzyme target, L-DOPA dioxygenase, after reconstitution with iron (II) and subsequent gel-filtration as described by the protocol herein. The five second dead-time before data collected begins is typical of students executing this technique for the first time. The robust activity of the metalloenzyme was detected using a published assay, and yielded steady-state kinetic parameters consistent with published results29. Steady-state kinetic parameters were determined by fitting the progress curves30 shown in Figure 2; however, non-linear least squares fitting of initial rates collected over a series of substrate concentrations is equally possible31.
Figure 1. SDS-PAGE26 analysis of the poly-histidine tagged protein before, during and after purification. Lanes 1 – Molecular weight markers, 2-purified protein (20 kDa) post Ni-NTA, 3- Ni-NTA flow through, 4 – cell free crude extracts prior to purification, 5 – cell-debris pellet post lysis. Samples were prepared using 5x sample/Loading buffer and separated on pre-cast 4-20% polyacrylamide gels using a gel electrophoresis system. Please click here to view a larger version of this figure.
Figure 2. Steady state assay of the Iron (II) reconstituted metalloenzyme (i.e., L-DOPA dioxygenase, 10µM) with substrate (L-DOPA – 5 µM (red), 25 µM (green), 50 µM (blue)) in buffer (50 mM Phosphate, 200 mM NaCl, pH 8). Traces depict product formation at 414nm. Absorbance data were continuously acquired using 1 mL methacrylate cuvettes in a split-beam scanning UV-Visible spectrometer29. Raw data are fit to a model of the Michaelis-Menten steady-state approximation (KM 30.8 µM ± 14.4, kcatapparent 2.3 s-1 ± 0.05)30. Please click here to view a larger version of this figure.
While the addition of polyhistidine tags to recombinant proteins and their purification by IMAC has become virtually ubiquitous in the biochemical literature3,4,5, applications of IMAC to enzyme purification in the biochemistry teaching laboratory remain sparse, and published methods do not always consider the resource limitations of the teaching laboratory20. Furthermore, the use of IMAC in the teaching laboratory is most effective when coupled to experiments that assess activity and purity, making IMAC purification of an enzyme an ideal instructional activity. In order to extend the application of IMAC to the purification of enzymes, including metalloenzymes, in the teaching laboratory, reliable and inexpensive methods are needed. In this protocol, we demonstrate benchtop IMAC using readily available and inexpensive laboratory supplies, while also addressing the limitations in the application of IMAC to metalloproteins22, by reconstituting the iron(II) dependent metalloenzyme, L-DOPA dioxygenase, post purification. Using the reagents and materials described , we estimate the cost of consumables for eight student groups is between $500-600 per semester to run this protocol, including the analysis steps outlined in Figure 1 and Figure 2.
Due to the ease with which Fe2+ can dissociate from amino acid ligands24 and the facile oxidation of Fe2+ by O2 to Fe3+, reconstitution of non-heme, amino acid-chelated iron(II) into a recombinant metalloenzyme is a typical component of the enzyme purification. When classical chromatography is used, it is possible to avoid total loss of iron in some cases32, but more often, the iron (II) is added back in the presence of reducing agents33,34,35,36 often under an anaerobic atomosphere37,38,39, and in some cases the excess iron is not removed33,34,36, complicating any subsequent assay. Consecutive steps of classical chromatography and an anaerobic atmosphere are not realistic for the undergraduate laboratory, prompting the development of this protocol.
While the manual preparation of the Ni-NTA column and the processing of samples largely by gravity does take additional time and effort when compared to pre-packaged columns and automated chromatography instrumentation, the manual steps allow for hands-on learning by the student that result in increased understanding of the science behind the process. The addition of an iron (II) salt under the conditions outlined here is particularly sensitive to excess dithiothreitol. If a student mistakenly adds an excess of dithiothreitol, a precipitation event is likely. We have found it helpful to require students to perform calculations of reagent quantities before arriving in lab, so laboratory time can be used most effectively at the bench. The entire benchtop IMAC purification – from cell-lysis to protein elution – can be accomplished in one 4-hour laboratory period, followed by reconstitution and assay in a subsequent lab period.
The authors have nothing to disclose.
This publication is based upon work supported by the National Science Foundation under Grant No. CHE 1708237.
consumables | |||
BeadBeater 0.1mm glass beads | BioSpec Products | 11079101 | 1 pound each |
50mL Conical Tubes with Screw Caps, sterile | VWR | 21008-178 | |
Sodium chloride | Fisher Bioreagents | BP358-1 | |
Potassium phosphate, monobasic | Acros (Fisher) | AC42420-5000 | |
Sodium Ascorbate | Acros (Fisher) | AC35268-1000 | |
DTT (Dithiothreitol) | Lab Scientific | D-115 | |
Iron(II) sulfate heptahydrate | Sigmaaldrich | 310077 | |
HisPur NiNTA Resin | Fisher (pierce) | PI88221 | |
Econo-Column Chromatography Columns – 1.5 x 20cm | Bio-Rad | 737-1522 | 1.5 x 20 cm, 35 ml, 2ea, |
Stopcock Valve, one way, female to male luer | Kimble | 420163-0000 | pack of 50 |
BD 10mL luer-loc syringe (non-sterile, without needle) | VWR | 301029 | |
Fitting for tubing: 1.6 mm Barb to Female Luer | Biorad | 7318222 | |
Fitting for tubing: 1.6 mm Barb to Male Luer | Biorad | 7318225 | |
Silicon Tubing (1.6 mm ID/0.8 mm wall, for 0.2-5 ml/min on Peristaltic Pump) | Bio-Rad | 7318211 | Pkg of 1, 1.6 mm ID/0.8 mm wall, 10 m, low-pressure tubing for liquid handling |
Glycerol | Fisher (Pierce) | 17904 | |
Coomassie Plus (Bradford) Protein Assay | Thermo Scientitic | 23236 | |
Microcentrifuge Tubes, snap cap, 1.5mL | VWR | 89000-028 | |
Fisherbrand polystyrene disposable serological pipets | Fisher | 13-676-10F | |
Fiserbrand universal pipet pump | Fisher | 14-955-110 | |
Fisherbrand Transfer Pipets | Fisher | 13-711-9AM | |
Econo-Pac 10DG desalting columns | Bio-Rad | 732-2010 | box of 30 |
ExpressPlus PAGE 5x sample buffer | Genscript | MB01015 | 5mL (Dilute 1:5 with sample) |
ExpressPlus PAGE Gel, 4-20%, 12 wells | Genscript | M42012 | 20 gels |
Fisherbrand Disposable Cuvettes, Methyacrylate | Fisher | 14-955-128 | case of 500 |
Cuvette Caps Square Disposable | Fisher | 14-385-999 | |
L-DOPA (3,4-dihydroxyphenylalanine) | Acros | D9628-5G | |
Permanent Equipment: | |||
BeadBeater 50mL chamber | BioSpec Products | 110803-50SS | 1 chamber |
BeadBeater | BioSpec Products | 1107900-101 | 350 ml polycarbonate chamber, rotor assembly, motor base, ice-water cooling jacket and one pound of glass beads. |
Centrifuge tubes, High-Speed PPCO, 50mL | Fisher | 3119-0050PK | |
Mini-PROTEAN Tetra Vertical Electrophoresis Cell for Mini Precast Gels, 4-gel | Bio-Rad | 1658004 | 4-gel vertical electrophoresis system, includes electrode assembly, companion running module, tank, lid with power cables, mini cell buffer dam |
PowerPac HC Power Supply | Bio-Rad | 1645052 | 100–120/220–240 V, power supply for high-current applications, includes power cord |
UV-1800 with UV-Probe Software | Shimadzu | UV-1800 | |
Kintek Global Kinetic Explorer | Kintek Corp | version 6 | https://www.kintekexplorer.com/downloads/ |