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

Summary

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.

Abstract

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.

Introduction

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 activity^1,2,3,4. For those enzymes that are oxygen-sensitive (specifically iron-containing enzymes), performing all the purification and characterization steps while maintaining an....

Protocol

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 protein expression and solubility.
2. Prepare the Laemmli buffer and running buffer for polyacrylamide gel electrophoresis (PAGE) as described in ....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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