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Anaerobic Protein Purification and Kinetic Analysis via Oxygen Electrode for Studying DesB Dioxygenase Activity and Inhibition

Published: October 3rd, 2018



1Department of Chemistry, Wesleyan University

Here we present a protocol for anaerobic protein purification, anaerobic protein concentration, and subsequent kinetic characterization using an oxygen electrode system. The method is illustrated using the enzyme DesB, a dioxygenase enzyme which is more stable and active when purified and stored in an anaerobic environment.

Oxygen-sensitive proteins, including those enzymes which utilize oxygen as a substrate, can have reduced stability when purified using traditional aerobic purification methods. This manuscript illustrates the technical details involved in the anaerobic purification process, including the preparation of buffers and reagents, the methods for column chromatography in a glove box, and the desalting of the protein prior to kinetics. Also described are the methods for preparing and using an oxygen electrode to perform kinetic characterization of an oxygen-utilizing enzyme. These methods are illustrated using the dioxygenase enzyme DesB, a gallate dioxygenase from the bacterium Sphingobium sp. strain SYK-6.

Enzymes that utilize iron or other metals to activate oxygen are often susceptible to inactivation during the purification process because of their removal from the reducing environment of a cell. Therefore, these proteins must be used as cell lysates, be subjected to external reducing agents, or be purified anaerobically to ensure that they have optimal enzymatic activity1,2,3,4. For those enzymes that are oxygen-sensitive (specifically iron-containing enzymes), performing all the purification and characterization steps while maintaining an....

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1. General Materials and Methods

  1. Prepare all the required media as described in Table 1. Autoclave at 120 ˚C for 15 min. Sterile filter the SOC solution, after the addition of MgCl2 and glucose, by passing it through a 0.2 µm filter. Adjust the pH of the Miller's Lysogeny Broth (LB media) solution prior to autoclaving. Supplement the LB-Amp media solution after autoclaving with sterile solutions of 0.2 mM L-cysteine, then 0.1 mM ferrous ammonium sulfate to enhance pro.......

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Shown is the SDS-PAGE gel analysis of individual fractions from purification of the DesB-maltose binding protein (MBP) fusion construct (Figure 3). The gel reveals that the protein is pure (MW = 91.22 kDa), except for the presence of DesB (MW = 49.22 kDa) and MBP protein domain (42 kDa) cleaved from each other. Fractions E2 and E3 were selected for concentration (step 4.2).

Reproducible results from.......

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The critical steps in obtaining active, purified DesB protein involve the forming and maintaining of the reduced Fe(II) active site in the enzyme. As such, correct performance of the induction, purification, concentration, and desalting steps are essential to successfully obtaining active enzyme. Inducing protein expression in the presence of 1 mM ferrous ammonium sulfate ensures that Fe(II) is correctly incorporated into the active site of DesB. This method is inspired by studies like those with amidohydrolase metalloen.......

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We would like to thank Dr. Camille Keller of Wesleyan University for technical support. Special thanks to Professor Lindsay D. Eltis and Jenna K. Capyk from the University of British Columbia, as well as Christian Whitman from the University of Texas at Austin, for their advice regarding anaerobic protein purification methods and the use of an O2-sensitive electrode.


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Name Company Catalog Number Comments
Isopropyl β-D-1-thiogalactopyranodise Gold Bio Technologies I2481C50
Coomassie Brilliant Blue R-250 Bio-Rad 161-0400
Ammonium persulfate Bio-Rad 161-0700
30% Acrylamide Bio-Rad 161-0158
N,N'tetramethyl-ethylenediamine Bio-Rad 161-0801
Amylose Resin High Flow New England Biolabs E8022S
BL21 (DE3) competent Escherichia coli cells New England Biolabs C2527I
L-cysteine Sigma Aldrich C7352
gallic acid Sigma Aldrich G7384
4-nitrocatechol Sigma Aldrich N15553
Ferrous ammonium sulfate Mallinckrodt 5064
Sodium dithionite Alfa Aesar 33381-22
wheaton serum bottles Fisher Scientific 06-406G
25 mm Acrodisc PF Syringe Filter with Supor Membrane Pall Corportation 4187
400 mL Amicon Stirred Cell Concentrator EMD Millipore UFSC40001
76 mm Millipore Ultracel 10 kDa cutoff reconsituted cellulose membrane filter EMD Millipore PLGC07610
DL-dithiothreitol Gold Bio Technologies DTT50
Sephadex G-25 coarse desalting gal column GE Healthcare 17-0033-01
2 mL Crimp-Top Vials Fisher Scientific 03-391-38
Oxygraph Plus Electrode Control Unit Hansatech Instruments OXYG1 Plus
Oxygen Eletrode Chamber Hansatech Instruments DW1
Electrode Disc Hansatech Instruments S1
PTFE (0.0125 mmX25mm) 30m reel Hansatech Instruments S4
Electrode cleaning Kit Hansatech Instruments S16
Spacer paper Zig Zag available at any gas station
He-series Dri-Lab glove box Vacuum/Atmospheres Company
HE-493 Dri-Train Vacuum/Atmospheres Company
Double-Ended Micro-Tapered Stainless Steel Spatula Fisher Scientific 21-401-10
DWK Life Sciences Kimble Kontes Flex Column Economy Column Fisher Scientific k420400-1530
10 μL, Model 701 N SYR, Cemented NDL 26s ga, 2 in, point stlye 2 syringe Hamilton 80300
DWK Life Sciences Kimble Kontes Flex Column Economy Column Fisher Scientific K420401-1505
Emulsiflex-C5 high-pressure homogenizer Avestin
B-PER Complete Bacterial Protein Extraction Reagent Thermo Fisher Scientific 89821
Lysozyme from chicken egg white Sigma Aldrich 12650-88-3
Sodium dodecyl sulfate Thermo Fisher Scientific 151-21-3
ampicillin Sigma Aldrich 7177-48-2
Tryptone Fisher Scientific BP-1421-500
Yeast extract Fisher Scientific BP1422-2
Sodium Chloride Fisher Scientific S271-10
Potassium Chloride Fisher Scientific P217-3
Magnesium Chloride Fisher Scientific M33-500
Dextrose Fisher Scientific D16-3
Sodium Hydroxide Fisher Scientific S318-1
Tris hydrochloride Fisher Scientific BP153-500
Maltose Fisher Scientific BP684-500
Glycine Fisher Scientific G46-500

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