Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase

Summary

Recombinant prototype foamy virus integrase protein is often contaminated with a bacterial nuclease during purification. This method identifies nuclease contamination and removes it from the final preparation of the enzyme.

Abstract

The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared to IN from other retroviruses, PFV IN is more soluble and more amenable to experimental manipulation. Additionally, it is sensitive to clinically relevant human immunodeficiency virus (HIV-1) IN inhibitors, suggesting that the catalytic mechanism of PFV IN is similar to that of HIV-1 IN. IN catalyzes the covalent joining of viral complementary DNA (cDNA) to target DNA in a process called strand transfer. This strand transfer reaction introduces nicks to the target DNA. Analysis of integration reaction products can be confounded by the presence of nucleases that similarly nick DNA. A bacterial nuclease has been shown to co-purify with recombinant PFV IN expressed in Escherichia coli (E. coli). Here we describe a method to isolate PFV IN from the contaminating nuclease by heparin affinity chromatography. Fractions are easily screened for nuclease contamination with a supercoiled plasmid and agarose gel electrophoresis. PFV IN and the contaminating nuclease display alternative affinities for heparin sepharose allowing a nuclease-free preparation of recombinant PFV IN suitable for bulk biochemical or single molecule analysis of integration.

Introduction

Biochemical and single molecule studies of protein interactions with DNA require exceptionally pure recombinant proteins. Contaminating nucleases from bacteria can obscure the results of these assays. A contaminating nuclease has been found in preparations of recombinant proteins oxygen scavenger protocatechuate-3,4-dioxygenase (PCD) and prototype foamy virus (PFV) integrase (IN) isolated from Escherichia coli (E. coli)^1,2,3.

Retroviral integration assays rely on the conversion of supercoiled DNA to nicked or linear products as a ....

Protocol

1. Induce PFV IN Expression in E. coli

1. Add 1 μL of PFV IN expression plasmid (10 ng/μL) to 20 μL of E. coli BL21(DE3) pLysS (20 μL aliquot of commercially available cells has >2 x 10⁶ CFU/μg plasmid) in a 1.5 mL tube. Mix gently by finger tapping or flicking the tube. Incubate on ice 5 min.
   1. Heat shock for exactly 30 s in a 42 ˚C water bath and then immediately return to ice for 2 min.
   2. Add 80 μL of room temperature super optimal broth with catabolite repression (SOC) media. Incubate for 1 h at 37 ˚C with 225 revolutions per minut....

Results

Recombinant PFV IN is often contaminated with a bacterial nuclease¹. Biochemical integration assays depend on the quantitation of the conversion of supercoiled plasmid DNA to relaxed circles and linear products. The presence of a contaminating nuclease could lead to spurious quantitation of these assays. Expression of PFV IN with a hexahistidine tag is induced in E. coli (Figure 1) and first purified by nickel affinity chromatography (Fig.......

Discussion

Recombinant proteins that interact with DNA, such as DNA repair proteins, oxygen scavengers for single molecule microscopy applications, or retroviral integrases, should be free of contaminating bacterial nucleases^2,3. These contaminants may confuse the interpretation of results during bulk biochemical or single molecule assays.

We have found that a bacterial nuclease frequently co-purifies with PFV IN. However, PFV IN displays an affi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BL21/DE3 Rosetta E. coli	EMD Millipore	70956-4
LB broth	EMD biosciences	1.10285.0500
Ampicillin	Amresco	0339
Chloramphenicol	Amresco	0230
PBS	Sigma-Aldrich	D8537
IPTG	Denville Scientific	CI8280
ZnCl2	Sigma-Aldrich	208086
Tris Ultra Pure	Gojira Fine Chemicals	UTS1003
NaCl	P212121	RP-S23020
PMSF	Amresco	0754
Imidazole	Sigma-Aldrich	I0250
DTT	P212121	SV-DTT
UltraPure EDTA	Invitrogen/Gibco	15575
MgSO4	Amresco	0662
Agarose	Denville Scientific	CA3510
Ethidium bromide	Thermo Fisher Scientific	BP1302
Glycerol	Fisher Scientific	G37-20
Ni-NTA Superflow	Qiagen	30430
Heparin Sepharose 6 Fast Flow	GE Healthcare Life Sciences	17-0998-01
HRV14 3C protease	EMD Chemicals	71493-3