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Purification and characterization of the MspI DNA methyltransferase cloned and overexpressed in E. coli
A K Dubey1, B Mollet, R J Roberts
1Cold Spring Harbor Laboratory, NY 11724.
Abstract:
The MspI restriction-modification system, which recognizes the sequence 5'-CCGG-3', has been previously cloned and sequenced (1). We subcloned the methyltransferase gene (M.MspI) downstream of the ptac promoter in the multicopy vector pUC119 and overexpressed it in E. coli. Upon induction with IPTG, M.MspI constitutes more than 10% of cellular protein. A scheme has been devised to purify large amounts of biologically active M.MspI to apparent homogeneity from these overexpressing E. coli cells. Approximately 0.8 mg of pure M.MspI per gram of cells (wet weight) can be obtained. The apparent molecular weight of M.MspI is 49 kD, by SDS gel electrophoresis and 48-54 kD by gel filtration. At low concentrations (less than 0.4 mg/ml), the methyltransferase is a monomer in solution but at higher concentrations (greater than 3.0 mg/ml) it exists predominantly as a dimer. Polyclonal antibodies raised against M.MspI cross-react with the DNA-methyltransferases of several other restriction-modification systems.
Insights
Researchers successfully overexpressed and purified the MspI methyltransferase (M.MspI) from E. coli. This provides a scalable method for obtaining active M.MspI for further study of DNA modification systems.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- The MspI restriction-modification system targets the 5'-CCGG-3' DNA sequence.
- Previous cloning and sequencing of the MspI system laid the groundwork for further investigation.
Purpose of the Study:
- To develop a method for overexpressing and purifying biologically active MspI methyltransferase (M.MspI).
- To characterize the purified M.MspI protein.
Main Methods:
- Subcloning the M.MspI gene into a pUC119 vector under the control of the ptac promoter.
- Overexpression in E. coli following IPTG induction.
- Purification of M.MspI to apparent homogeneity.
- SDS-PAGE and gel filtration for molecular weight determination.
- Analysis of M.MspI oligomerization state in solution.
Main Results:
- M.MspI was overexpressed to constitute over 10% of cellular protein.
- A purification scheme yielded approximately 0.8 mg of pure M.MspI per gram of cells.
- The apparent molecular weight was determined to be 49 kD (SDS-PAGE) and 48-54 kD (gel filtration).
- M.MspI exists as a monomer at low concentrations and a dimer at high concentrations.
- Antibodies against M.MspI showed cross-reactivity with other DNA methyltransferases.
Conclusions:
- A robust protocol for large-scale purification of active M.MspI was established.
- The study characterized the molecular properties of M.MspI, including its oligomeric state.
- Cross-reactivity of antibodies suggests conserved epitopes among MspI and related DNA methyltransferases.