Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy

Summary

The yeast mitochondrial nucleoid protein, Mgm101, is a Rad52-type recombination protein that forms large oligomeric rings. A protocol is described to prepare soluble recombinant Mgm101 using the Maltose Binding Protein (MBP)-tagging strategy coupled with cation exchange and size exclusion chromatography.

Abstract

The MGM101 gene was identified 20 years ago for its role in the maintenance of mitochondrial DNA. Studies from several groups have suggested that the Mgm101 protein is involved in the recombinational repair of mitochondrial DNA. Recent investigations have indicated that Mgm101 is related to the Rad52-type recombination protein family. These proteins form large oligomeric rings and promote the annealing of homologous single stranded DNA molecules. However, the characterization of Mgm101 has been hindered by the difficulty in producing the recombinant protein. Here, a reliable procedure for the preparation of recombinant Mgm101 is described. Maltose Binding Protein (MBP)-tagged Mgm101 is first expressed in Escherichia coli. The fusion protein is initially purified by amylose affinity chromatography. After being released by proteolytic cleavage, Mgm101 is separated from MBP by cationic exchange chromatography. Monodispersed Mgm101 is then obtained by size exclusion chromatography. A yield of ~0.87 mg of Mgm101 per liter of bacterial culture can be routinely obtained. The recombinant Mgm101 has minimal contamination of DNA. The prepared samples are successfully used for biochemical, structural and single particle image analyses of Mgm101. This protocol may also be used for the preparation of other large oligomeric DNA-binding proteins that may be misfolded and toxic to bacterial cells.

Introduction

Homologous recombination is important for the repair of double-strand breaks (DSBs) and interstrand crosslinks, and for the reinitiation of DNA replication from collapsed replication forks ¹. In conventional homologous recombination, the central reaction is catalyzed by the ATP-dependent recombinases including RecA in prokaryotes, and Rad51 and Dmc1 in eukaryotes ^1-3. These recombinases form nucleoprotein filaments on ssDNA, which are essential for initiating homology search and strand invasion within duplex DNA templates (Figure 1, left panel) ^4-7. In addition to the conventional scheme, homologous recombination can a....

Protocol

Previous studies have shown that the first amino-terminal 22 residues of Mgm101 are cleaved after import into mitochondria ¹⁹. For expression in Escherichia coli, the MGM101 open reading frame lacking the first 22 codons is amplified by PCR and placed downstream of the malE sequence encoding the maltose binding protein (MBP) in a modified version of the expression vector pMAL-c2E. This generates the MBP-Mgm101 fusion with a linker containing a cleavage site for the PreScission protease (Figure 3). The plasmid is first constructed by selecting E. coli transformants without the IPTG and Xgal blue/white sele....

Results

Mgm101 is a Rad52-related recombination protein in mitochondria. Rad52 has been extensively studied for its role in mitochondrial DNA recombination (Figure 1). Recombinant Mgm101 can be prepared by a three-step procedure (Figure 2). This is facilitated by the use of the MBP-tagging strategy that allows Mgm101 to be expressed in a soluble form and subsequently released from the tag by proteolytic cleavage (Figure 3).

In a typical preparation, a.......

Discussion

It has been a challenge to produce a stable, native recombinant Mgm101 protein from E. coli possibly due to its insolubility in bacterial cells. In this report, we show that the MBP-fusion strategy allows the protein to be expressed at a reasonably high level. By using negative staining transmission electron microscopy and size exclusion chromatography, we have previously shown that the MBP-fusion protein forms uniform oligomers in vitro ¹⁸. It is possible that the folding and oligomerization.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Expression vector pMAL-c2E	New England Biolabs	#N8066S
PreScission Protease	GE Healthcare Life Sciences	#27-0843-01
BL21-CodonPlus(DE3)-RIL cells	Strategene	#230245
Leupeptin	Roche Applied Science	#11034626001
Pepstatin	Roche Applied Science	#11359053001
Phenylmethylsulfonyl fluoride (PMSF)	Roche Applied Science	#10837091001
DNAse I	Sigma	#DN25-1G
Isopropyl β-D-1-thiogalactopyranoside (IPTG)	Roche Applied Science	#11411446001
Amylose resin	New England Biolabs	#E8021L
Econo-Column chromatography column	BIO-RAD	#7372512
Bio-Scale Mini Macro-Prep High S cartridge (1 ml)	BIO-RAD	#732-4130
VIVASPIN 15R Ultrafiltration spin column (10,000 MWCO)	Sartorius Stedium	#VS15RH02
Superose 6 prep grade column	Amersham Bioscirnces	#17-0489-01
VIVASPIN 6 Ultrafiltration spin column (5,000 MWCO)	Sartorius Stedium	#VS0611