Overproduction, purification and characterization of M.HinfI methyltransferase and its deletion mutant.
We have used the polymerase chain reaction to alter transcriptional and translational signals surrounding the hinfIM gene [encoding M.HinfI methyltransferase (MTase)] so as to achieve overexpression in Escherichia coli. The PCR-generated hinfIM gene was subcloned in a high-expression vector under control of the hybrid trp-lac promoter. In addition, the positive retroregulator stem-loop sequence derived from the crystal protein-encoding gene of Bacillus thuringiensis was inserted downstream from hinfIM. Using a similar approach, we have also constructed overproducer clones of a deletion mutant of M.HinfI MTase that has 97 amino acids from the C terminus deleted. The plasmid from the mutant clones is fully protected from HinfI restriction endonuclease digestion. It appears that the functional properties (the recognition and catalytic functions) are encoded within this mutant gene. The overproducer clones yield the wild type (wt) and the mutant enzymes to about 10% of total cellular protein upon induction with 1 mM IPTG. The wt M.HinfI and the mutant MTase were purified to near electrophoretic homogeneity by phosphocellulose, DEAE and gel chromatography. Their monomer sizes by SDS/polyacrylamide-gel electrophoresis are 43 kDa and 31 kDa, respectively, in good agreement with that predicted from the nucleotide sequence. DNA methylation experiments with purified enzymes using single-strand and double-strand M13mp18 DNA substrates indicate that while wt enzyme methylates both forms of DNA substrates, the mutant enzyme appears to preferentially methylate ss DNA substrate.
Bassing
C H
CH
Department of Environmental Health Sciences, Johns Hopkins University, School of Hygiene and Public Health, Baltimore, MD 21205.
Kim
Y G
YG
Li
L
L
Chandrasegaran
S
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D006190:Haemophilus / Q000201:enzymology / Q000235:genetics*; D007700:Kinetics; D008745:Methylation; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D014158:Transcription, Genetic
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; C053100:DNA modification methylase HinfI; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)