Overproduction and purification of the M.HhaII methyltransferase from Haemophilus haemolyticus.
The HhaII methyltransferase gene from Haemophilus haemolyticus was subcloned in an expression vector under control of the hybrid trp-lac promoter. Induction with isopropyl-beta-D-thiogalactopyranoside results in overproduction of the methyltransferase to about 3% of total cellular protein. The methyltransferase was purified to near electrophoretic homogeneity by phosphocellulose, DEAE, and gel chromatography. Its monomer Mr by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 25 kDa, in good agreement with that predicted from the nucleotide sequence. Crystals of the methyltransferase were obtained in the presence of a two-fold molar excess of the duplex oligodeoxynucleotide substrate 5'd-GGACTCC.CCTGAGG.
Chandrasegaran
S
S
Department of Environmental Health Sciences, Johns Hopkins University School of Hygiene and Public Health, Baltimore, MD 21205.
Wu
L P
LP
Valda
E
E
Smith
H O
HO
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D003460:Crystallization; D006190:Haemophilus / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis*; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C053100:DNA modification methylase HinfI; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)