O'Gara, M., Adams, G. M., Gong, W., Kobayashi, R., Blumenthal, R. M., Cheng, X. (August 1997) Expression, purification, mass spectrometry, crystallization and multiwavelength anomalous diffraction of selenomethionyl PvuII DNA methyltransferase (cytosine-N4-specific). European Journal of Biochemistry , 247 (3). pp. 1009-18. ISSN 0014-2956 (Print)
Abstract
The type II DNA-methyltransferase (cytosine N4-specific) M.PvuII was overexpressed in Escherichia coli, starting from the internal translation initiator at Met14. Selenomethionine was efficiently incorporated into this short form of M.PvuII by a strain prototrophic for methionine. Both native and selenomethionyl M.PvuII were purified to apparent homogeneity by a two-column chromatography procedure. The yield of purified protein was approximately 1.8 mg/g bacterial paste. Mass spectrometry analysis of selenomethionyl M.PvuII revealed three major forms that probably differ in the degree of selenomethionine incorporation and the extent of selenomethionine oxidation. Amino acid sequencing and mass spectrometry analysis of selenomethionine-containing peptides suggests that Met30, Met51, and Met261 were only partially replaced by selenomethionine. Furthermore, amino acid 261 may be preferentially oxidized in both native and selenomethionyl form. Selenomethionyl and native M.PvuII were crystallized separately as binary complexes of the methyl donor S-adenosyl-L-methionine in the monoclinic space group P2(1). Two complexes were present per asymmetric unit. Six out of nine selenium positions (per molecule), including the three that were found to be partially substituted, were identified crystallographically.
| Item Type: | Paper |
|---|---|
| Uncontrolled Keywords: | Amino Acid Sequence Cloning, Molecular Crystallization Escherichia coli/genetics Molecular Sequence Data Protein Conformation Recombinant Proteins/chemistry/genetics/isolation & purification Research Support, U.S. Gov't, Non-P.H.S. Research Support, U.S. Gov't, P.H.S. Site-Specific DNA Methyltransferase (Cytosine-Specific)/chemistry/ genetics/isolation & purification Spectrum Analysis, Mass |
| Subjects: | Investigative techniques and equipment > cloning Investigative techniques and equipment > assays > cloning organism description > bacteria > escherichia coli bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > genes, structure and function > gene expression Investigative techniques and equipment > spectroscopy > mass spectrometry biotechnology > chromatography > protein purification |
| CSHL Authors: | |
| Communities: | CSHL labs > Kobayashi lab |
| Depositing User: | Kathleen Darby |
| Date: | 1 August 1997 |
| Date Deposited: | 07 May 2014 18:37 |
| Last Modified: | 07 May 2014 18:37 |
| Related URLs: | |
| URI: | https://repository.cshl.edu/id/eprint/30008 |
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