Name: preparation of the mgm101 recombination protein by mbp-based tagging strategy
Uploaded: 2013-06-25T14:00:00
Description: Watch this Scientific Journal Video about Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy at JoVE.com

We thank Stephan Wilkens for help in transmission electron microscopy. This work was supported by the National Institutes of Health Grant R01AG023731.

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 protea...

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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...

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 also take place in a RecA/Rad51-independent manner (Figure 1, right panel). For instance, the yeast Rad52 and Rad59 proteins can directly catalyze the annealing of complementary ssDNA strands which are exposed by resectioning of dsDNA breaks. This recombination process, known as single strand annealing, generally does not involve homologous pairing with dsDNA templates. After annealing, heterologous tails are removed by exonucleases and nicks are ligated to restore genome continuity 8-10. Repair by the single strand annealing mechanism is often accompanied by deletions of genomic sequences between directly repeated regions.
Rad52 belongs to a diverse group of recombination proteins that are widespread among bacteriophages 11. These proteins are also known as Single Strand Annealing Proteins (SSAPs), based on their activity in promoting the annealing of homologous single stranded DNA molecules. The best characterized bacteriophage SSAPs are Red&beta; and Erf from the bacteriophages &lambda; and P22, RecT from the prophage rac and the Sak protein from the lactococcal phage ul36. The SSAPs are structurally characterized by a typical &beta;-&beta;-&beta;-&alpha; fold, although similarity is virtually undetectable in their primary sequences. They all form large homo-oligomeric rings of 10 - 14 fold symmetry in vitro 12-14. The functional implications of this characteristic higher order structural organization is not well understood.
We are interested in understanding the mechanism of homologous recombination in the mitochondrial genome. We have previously identified the MGM101 gene that is essential for the maintenance of mtDNA in Saccharomyces cerevisiae 15. MGM101 was subsequently found to be associated with mitochondrial nucleoids and is required for the tolerance of mtDNA to DNA-damaging agents 16. However, the study of Mgm101 has been held back in the last decade by the difficulty to produce recombinant Mgm101. We have recently succeeded in producing soluble Mgm101 at large quantities from E. coli using the MBP-fusion strategy. This has enabled us to demonstrate that Mgm101 shares biochemical and structural similarities with the Rad52-family of proteins 17,18. In this report, a three-step purification procedure is described, which produces homogeneous Mgm101for biochemical and structural analyses (Figure 2).

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

preparation of the mgm101 recombination protein by mbp-based tagging strategy

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.

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...

Watch this Scientific Journal Video about Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy at JoVE.com

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

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.

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 ...

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