Overproduction and purification of the M·HhaII methyltransferase from Haemophilus haemolyticus

Srinivasan Chandrasegaran; Louisa P. Wu; Eduardo Valda; Hamilton O. Smith

doi:10.1016/0378-1119(88)90240-5

Overproduction and purification of the M·HhaII methyltransferase from Haemophilus haemolyticus

Srinivasan Chandrasegaran, Louisa P. Wu, Eduardo Valda, Hamilton O. Smith

School of Medicine

Research output: Contribution to journal › Article › peer-review

13 Scopus citations

Abstract

The HhaII methyltransferase gene from Haemophilus haemolyticus was subcloned in an expression vector under control of the hybrid trp-lac promoter. Induction with isopropyl-β-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 M_r 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.

Original language	English (US)
Pages (from-to)	15-21
Number of pages	7
Journal	Gene
Volume	74
Issue number	1
DOIs	https://doi.org/10.1016/0378-1119(88)90240-5
State	Published - Dec 25 1988

Keywords

DNA methylation
Recombinant DNA
nucleotide sequencing
protein crystals
tac promoter

ASJC Scopus subject areas

Genetics

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10.1016/0378-1119(88)90240-5

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title = "Overproduction and purification of the M·HhaII methyltransferase from Haemophilus haemolyticus",

abstract = "The HhaII methyltransferase gene from Haemophilus haemolyticus was subcloned in an expression vector under control of the hybrid trp-lac promoter. Induction with isopropyl-β-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.",

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T1 - Overproduction and purification of the M·HhaII methyltransferase from Haemophilus haemolyticus

AU - Chandrasegaran, Srinivasan

AU - Wu, Louisa P.

AU - Valda, Eduardo

AU - Smith, Hamilton O.

PY - 1988/12/25

Y1 - 1988/12/25

N2 - The HhaII methyltransferase gene from Haemophilus haemolyticus was subcloned in an expression vector under control of the hybrid trp-lac promoter. Induction with isopropyl-β-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.

AB - The HhaII methyltransferase gene from Haemophilus haemolyticus was subcloned in an expression vector under control of the hybrid trp-lac promoter. Induction with isopropyl-β-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.

KW - DNA methylation

KW - Recombinant DNA

KW - nucleotide sequencing

KW - protein crystals

KW - tac promoter

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Overproduction and purification of the M·HhaII methyltransferase from Haemophilus haemolyticus

Abstract

Keywords

ASJC Scopus subject areas

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