HAMSTER MONOMORPHIC ARYLAMINE N-ACETYLTRANSFERASE - EXPRESSION IN ESCHERICHIA-COLI AND PURIFICATION

N-Acetylation is a major pathway in the metabolism of hydrazine and ar ylamine drugs, and has been associated with carcinogen bioactivation. Monomorphic hamster liver N-acetyltransferase (NAT1) cDNA was cloned f rom hamster liver cells by reverse transcriptase-coupled polymerase ch ain reaction. The determined nucleotide sequence was identical to that reported for NAT1. The NAT1 coding region was subcloned into the pG1 yeast expression vector, but cell extracts provided only transient ace tyltransferase activity. In addition, cDNA was subcloned into the expr ession vectors pFLAG-ATS and pFLAG-MAC, The latter vectors encoded a t ac promoter and appended a low-molecular weight (1 kDa) hydrophilic FL AG; marker peptide to the amino terminus of NAT1. Unexpectedly, peripl asmic export of FLAG(ATS)-NAT1 by the ompA signal peptide of pFLAG-ATS proved to be detrimental to enzyme activity. High acetyltransferase a ctivity, however, was obtained when the fusion protein was expressed i n the cytosol. Enzyme purified to homogeneity by immunoaffinity chroma tography exhibited substrate specificity comparable to that of the ham ster-derived protein. (C) 1995 Academic Press, Inc.