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Trimethylamine dehydrogenase of bacterium W 3 A 1 Molecular cloning, sequence determination and over‐expression of the gene
Author(s) -
Boyd Geoffrey,
Mathews F.Scott,
Packman Leonard C.,
Scrutton Nigel S.
Publication year - 1992
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(92)81291-s
Subject(s) - microbiology and biotechnology , gene , nucleic acid sequence , molecular cloning , biology , expression vector , open reading frame , escherichia coli , peptide sequence , plasmid , sequence analysis , dna , biochemistry , recombinant dna
The gene encoding trimethylamine dehydrogenase (EC 1.5.99.7) from bacterium W 3 A 1 has been cloned. Using the polymerase chain reaction a 530 bp DNA fragment encoding a distal part of the gene was amplified. Using this fragment of DNA as a probe, a clone was then isolated as a 4.5 kb Bam HI fragment and shown to encode residues 34 to 729 of trimethylamine dehydrogenase. The polymerase chain reaction was used also to isolate the DNA encoding the missing N‐terminal part of the gene. The complete open reading frame contained 2,190 base pairs coding for the processed protein of 729 amino acids which lacks the N‐terminal methionine residue. The high‐level expression of the gene in Escherichia coli was achieved by the construction of an expression vector derived from the plasmid pKK223‐3. The cloning and sequence analysis described here complete the partial assignment of the amino acid sequence derived from chemical sequencing (1) and will now permit the refinement of the crystallographic structure of trimethylamine dehydrogenase and also a detailed investigation of the mechanism and properties of the enzyme by protein engineering.