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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 Redβ and Erf from the bacteriophages λ and P22, RecT from the prophage rac and the Sak protein from the lactococcal phage ul36. The SSAPs are structurally characterized by a typical β-β-β-α 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).

Protokół

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 selection. The resulting plasmid pMAL-c2E-MGM101 is then introduced into the E. coli strain BL21-CodonPlus(DE3)-RIL by selecting ampicillin and chloramphenicol resistant colonies.

1. Expression, Induction, Cell Lysis and DNase I Treatment

1. Inoculate fresh transformants in 10 ml of LB medium (1% Bacto tryptone, 0.5% yeast extract, 1% NaCl) supplemented with glucose (0.2%), ampicillin (100 μg/ml) and chloramphenicol (50 μg/ml). Incubate at 37 °C O/N with shaking at 200 rpm.
2. Inoculate the 10 ml preculture into 2 liters of the supplemented LB medium as above. Grow the cells at 37 °C with shaking until OD₆₀₀ reaches 0.5.
3. Induce expression of the MBP-Mgm101 fusion protein by adding Isopropyl β-D-1-thiogalactopyranoside (IPTG) at a final concentration of 0.3 mM. Grow the cells with shaking at 30 °C for 5 hr.
4. Collect the cells by centrifugation using a Beckman JA-10 rotor (5,500 x g, 4 °C, 10 min). After discarding the supernatant, resuspend the cell pellet in 30 ml of lysis buffer (20 mM Tris-HCl, pH 7.4 and 1 mM EDTA, pH 7.4) containing protease inhibitors (25 μM leupeptin, 1 μM pepstatin A and 1 mM PMSF).
5. Sonicate cell suspension on ice for 20 sec using an ultrasonic processor (Heat Systems; Model W-385) at 50% duty cycle, allow to cool on ice for 30 sec, and repeat 2x.
6. Add 1 ml of DNAse 1 stock at 2 mg/ml. Rock the cell lysate at 4 °C for 2 hr.
7. Adjust NaCl to a final concentration of 500 mM.
8. Remove the cell debris by centrifugation at 10,000 x g at 4 °C for 30 min.

2. Purification with Amylose Affinity Chromatography

1. Equilibrate 1.5 ml of amylose resin (50% slurry) in the lysis buffer. Add the equilibrated amylose resin to the cell lysate. Rock gently at 4 °C O/N.
2. Load the lysate-resin mix onto an Econo-Column chromatography column installed in a cold room. Allow the unbound proteins to pass the column by gravity.
3. Wash the amylose resin with 300 ml of the wash buffer I (20 mM Tris-HCl, pH 7.4; 400 mM NaCl; 1 mM EDTA, pH 7.4; and 0.2 mM PMSF).
4. Repeat the wash with 150 ml of wash buffer II (20 mM Tris-HCl, pH 7.4; 200 mM NaCl, 1 mM EDTA, pH 7.4; and 0.2 mM PMSF).
5. Add 5 ml of elution buffer (20 mM Tris-HCl, pH 7.4; 200 mM NaCl; 1 mM EDTA, pH 7.4; and 0.2 mM PMSF, 10 mM maltose) to the column, incubate at 4 °C for 10 min, collect the eluate and repeat for more times.
6. Combine the eluates, determine the yield and purity of MBP-Mgm101 by loading an aliquot of the eluate on a SDS-PAGE gel followed by coomassie staining (Figure 4).

3. PreScission Protease Cleavage and Cation Exchange Chromatography

1. Add 50 units of PreScission protease to the MBP-Mgm101 eluate. Perform the cleavage at 4 °C O/N and allow it to continue during dialysis against the dialysis buffer (20 mM Tris-HCl, pH 7.4; 100 mM NaCl; 2 mM DTT; 1 mM EDTA, pH 7.4; and 0.2 mM PMSF)
2. Check the efficiency of cleavage on a SDS-PAGE gel (Figure 5A).
3. Load the cleaved MBP-Mgm101 by multiple injections on a Bio-Scale Mini Macro-Prep High S cartridge connected to a Bio-Rad Biologic DuoFlow FPLC system.
4. Start the cation exchange chromatography by applying a step salt gradient of 5 mM, 300 mM, 500 mM, 750 mM and 1,000 mM of NaCl that is created by mixing Buffer A (5 mM NaCl; 10 mM Na-phosphate, pH 7.2; 1 mM PMSF) and Buffer B (1 M NaCl; 10 mM Na-phosphate, pH 7.2; 1 mM PMSF). Set the flow rate at 0.5 ml/min. Collect the Mgm101 fraction and check the purity of the protein on a SDS-PAGE gel (Figure 5B).

4. Size Exclusion Chromatography

1. Combine the protein fractions from cation exchange chromatography. Dialyze the protein in GF equilibration buffer (20 mM MOPS, pH 7.0; 150 mM NaCl; 1mM EDTA, pH 7.4; 5 mM 2-Mercaptoethanol; 0.2 mM PMSF).
2. Concentrate the protein with VIVASPIN 15R Ultrafiltration spin column to reduce the volume to ~1 ml by spinning at 3,000 x g at 4 °C.
3. Load Mgm101 on a calibrated Superose 6 prep grade column equilibrated with the chromatography buffer (20 mM MOPS, pH 7.0; 150 mM NaCl; 5 mM β-mercaptoethanol; 1 mM EDTA, pH 7.4; 0.2 mM PMSF).
4. Start the size exclusion with the chromatography buffer run at a flow rate of 0.5 ml/min.
5. Collect the fractions of the purified Mgm101 peaks. Load aliquots of the fractions on a 12% SDS-PAGE for checking the final quality of the protein.
6. Concentrate the protein with VIVASPIN 6, then quickly freeze the samples in liquid N2 and store at -80 °C.
7. Use the Mgm101 samples within 6-12 months for ssDNA-binding assay (Figure 8) and for structural visualization by transmission electron microscopy (Figure 9).

Wyniki

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...

Dyskusje

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...

Materiały

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