TRIMETHYLAMINE DEHYDROGENASE OF BACTERIUM W(3)A(1) MOLECULAR-CLONING, SEQUENCE DETERMINATION AND OVER-EXPRESSION OF THE GENE

The gene encoding trimethylamine dehydrogenase (EC 1.5.99.7) from bacterium W3A1 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 BamHI 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 thc enzyme by protein engineering.