Related Experiment Videos
Purification, crystallization, and preliminary X-ray diffraction analysis of an M.HhaI-AdoMet complex
S Kumar1, X Cheng, J W Pflugrath
1Cold Spring Harbor Laboratory, New York 11724.
Biochemistry
|September 15, 1992
Summary
The type-II DNA-(cytosine-5)-methyltransferase M.HhaI was purified and characterized. The enzyme
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- DNA methylation is a crucial epigenetic mechanism regulating gene expression.
- Type-II DNA-(cytosine-5)-methyltransferases play vital roles in DNA modification.
- M.HhaI is a specific enzyme involved in DNA methylation.
Purpose of the Study:
- To overexpress and purify the type-II DNA-(cytosine-5)-methyltransferase M.HhaI.
- To characterize the purified enzyme, including its cofactor dependency and kinetic properties.
- To crystallize the M.HhaI-cofactor complex for structural determination.
Main Methods:
- Overexpression of M.HhaI in Escherichia coli.
- Purification using a high salt back-extraction and FPLC chromatography.
- Kinetic studies and crystallization by hanging drop vapor diffusion.
Main Results:
- M.HhaI was purified to homogeneity, yielding 0.75-1.0 mg per gram of bacterial paste.
- Two forms of the enzyme were isolated: cofactor-bound (S-adenosylmethionine) and cofactor-free.
- Kinetic parameters (KmAdoMet, KiAdoHye, Kcat) were determined, and crystals suitable for diffraction were obtained.
Conclusions:
- A robust purification protocol for M.HhaI was established.
- The purified M.HhaI enzyme exhibits catalytic activity and can be crystallized.
- The obtained crystals are suitable for X-ray crystallography to elucidate the enzyme's structure.