Expression and Purification of Nuclease-Free Oxygen Scavenger Protocatechuate 3,4-Dioxygenase

Summary

Protocatechuate 3,4-dioxygenase (PCD) can enzymatically remove free diatomic oxygen from an aqueous system using its substrate protocatechuic acid (PCA). This protocol describes the expression, purification, and activity analysis of this oxygen scavenging enzyme.

Abstract

Single molecule (SM) microscopy is used in the study of dynamic molecular interactions of fluorophore labeled biomolecules in real time. However, fluorophores are prone to loss of signal via photobleaching by dissolved oxygen (O₂). To prevent photobleaching and extend the fluorophore lifetime, oxygen scavenging systems (OSS) are employed to reduce O₂. Commercially available OSS may be contaminated by nucleases that damage or degrade nucleic acids, confounding interpretation of experimental results. Here we detail a protocol for the expression and purification of highly active Pseudomonas putida protocatechuate-3,4-dioxygenase (PCD) with no detectable nuclease contamination. PCD can efficiently remove reactive O₂ species by conversion of the substrate protocatechuic acid (PCA) to 3-carboxy-cis,cis-muconic acid. This method can be used in any aqueous system where O₂ plays a detrimental role in data acquisition. This method is effective in producing highly active, nuclease free PCD in comparison with commercially available PCD.

Introduction

Single molecule (SM) biophysics is a rapidly growing field changing the way we look at biological phenomena. This field has the unique ability to link fundamental laws of physics and chemistry to biology. Fluorescence microscopy is one biophysical method that can achieve SM sensitivity. Fluorescence is used to detect biomolecules by linking them to small organic fluorophores or quantum dots¹. These molecules can emit photons when excited by lasers before photobleaching irreversibly². Photobleaching occurs when the fluorescent labels undergo chemical damage which destroys their ability to excite at the desi....

Protocol

1. Induce PCD expression in E. coli

1. Combine 1 μL pVP91A-pcaHG PCD expression plasmid (20 ng/μL, Figure 1A) and 20 μL of E.coli BL21 (20 μL commercially available cells, > 2 x 10⁶ cfu/μg plasmid) in a tube. Flick the tube to mix. Place the tube on ice 5 min.
2. Place transformation at 42 °C for 30 s. Then ice 2 min.
3. Add 80 μL SOC media (super optimal broth with catabolite repression: 2.5 mM KCl, 10 mM NaCl, 2% tryptone, 0.5% yeast extract, 10 mM MgSO₄, 10 mM MgCl₂, 20 mM glucose). Shake at 225 revolutions ....

Results

Commercially available oxygen scavenger PCD is frequently contaminated with a DNA nuclease. Contaminating nuclease activity could lead to spurious results in fluorescent studies, particularly studies that analyze DNA or DNA interacting proteins.We have found that recombinant PCD, a heterodimer of hexahistidine tagged pcaH and pcaG, may be expressed in E. coli (Figure 1). The heterodimer is first purified by nickel affinity chromatography (

Discussion

Oxygen scavenging systems are commonly included in single molecule fluorescence microscopy to reduce photobleaching^3,7,8. These microscopy techniques are often used to observe nucleic acids or protein interactions with nucleic acids^1,13,14. Contamination of OSSs with nucleases may lead to spurious results.

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Materials

Name	Company	Catalog Number	Comments
2-Mercaptoethanol	Sigma-Aldrich	M3148	βME
30% acrylamide and bis-acrylamide solution, 29:1	Bio-Rad	161-0156
Acetic acid, Glacial Certified ACS	Fisherl Chemical	A38C-212
Agar, Granulated	BD Biosciences	DF0145-17-0
AKTA FPLC System	GE Healthcare Life Sciences	AKTA Purifier: Box-900, pH/C-900, UV-900, P-900, and Frac-920
Amicon Ultra-2 Centrifugal Filter Unit	EMD Millipore	UFC201024	10 kDa MWCO
Ammonium iron(II) sulfate hexahydrate	Sigma	F-2262
Ammonium Persulfate (APS) Tablets	Amresco	K833-100TABS
Ampicillin	Amresco	0339-25G
Bacto Tryptone	BD Biosciences	DF0123173
BD Bacto Dehydrated Culture Media Additive: Bottle Yeast Extract	VWR	90004-092
BIS-TRIS propane,>=99.0% (titration)	Sigma-Aldrich	B6755-500G
Bromophenol Blue	Sigma-Aldrich	B0126-25G
Coomassie Brilliant Blue	Amresco	0472-50G
Costar 96–Well Flat–Bottom EIA Plate	Bio-Rad	2240096EDU
DTT	P212121	SV-DTT
Dulbecco's Phosphate Buffered Saline 500ML	Sigma-Aldrich	D8537-500ML	PBS
Ethidium bromide	Thermo Fisher Scientific	BP1302
Glycerol	Fisher Scientific	G37-20
Granulated LB Broth Miller	EMD Biosciences	1.10285.0500
Hi-Res Standard Agarose	AGTC Bioproducts	AG500D1
Imidazole	Sigma-Aldrich	I0250-250G
IPTG	Goldbio	I2481C25
Leupeptin	Roche	11017128001
Lysozyme from Chicken Egg White	Sigma-Aldrich	L6876-1G
Magnesium Chloride Hexahydrate	Amresco	0288-1KG
Microvolume Spectrophotometer, with cuvet capability	Thermo Fisher	ND-2000C
NaCl	P212121	RP-S23020
Ni-NTA Superflow (100 ml)	Qiagen	30430
Novagen BL21 Competent Cells	EMD Millipore	69-449-3	SOC media included
Orange G	Fisher Scientific	0-267
Pepstatin	Gold Biotechnology	P-020-25
PMSF	Amresco	0754-25G
Protocatechuic acid	Fisher Scientific	ICN15642110	PCA
Sodium dodecyl sulfate	P212121	CI-00270-1KG
SpectraMax M2 Microplate Reader	Molecular Devises
Sterile Disposable Filter Units with PES Membrane > 250mL	Thermo Fisher Scientific	09-741-04
Sterile Disposable Filter Units with PES Membrane > 500mL	Thermo Fisher Scientific	09-741-02
Superose 12 10/300 GL	GE Healthcare Life Sciences	17517301
TEMED	Amresco	0761-25ML
Tris Ultra Pure	Gojira Fine Chemicals	UTS1003
Typhoon 9410 variable mode fluorescent imager	GE Healthcare Life Sciences
UltraPure EDTA	Invitrogen/Gibco	15575
ZnCl2	Sigma-Aldrich	208086