Cloning of aminoglycoside phosphotransferase (APH) gene from antibiotic-producing strain of Bacillus circulans into a high-expression vector, pKK223-3. Purification, properties and location of the enzyme.
The aminoglycoside phosphotransferase gene from a butirosin-producing strain of Bacillus circulans was cloned in a high-expression vector (pKK223-3) to give the recombinant plasmid pMS5. Escherichia coli harbouring the plasmid, E. coli JM103[pMS5], was characterized, and several features of the expression of the phosphotransferase were studied. The phosphotransferase activity was best expressed in a medium lacking glucose, and the highest levels of the enzyme were found between 12 and 24 h of growth. The induction of the phosphotransferase expression with isopropyl beta-D-thiogalactopyranoside (inducer) was found to be undesirable as the overproduction of the enzyme led to the killing of the bacteria. The subcellular location of the phosphotransferase, and also the site in vivo of the phosphorylation of neomycin, was found to be in the cytoplasm. The phosphotransferase was purified to homogeneity in good yield (17 mg of purified protein/3 litres of culture) and was shown to be a monomer of Mr 30,000-32,000. The N-terminal amino acid sequence was in agreement with that predicted from the gene sequence and confirmed the absence of any signal sequence. The regiospecificity of the phosphotransferase reaction was studied by m.s. and by 1H-, 13C- and 31P-n.m.r. using ribostamycin as the substrate, and it was found that the antibiotic was phosphorylated at the 3'-hydroxy group.
Sarwar
M
M
Department of Biochemistry, University of Southampton, U.K.
Akhtar
M
M
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D055598:Chemical Phenomena; D002621:Chemistry; D003001:Cloning, Molecular; D004790:Enzyme Induction / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D019868:Kanamycin Kinase; D008969:Molecular Sequence Data; D010770:Phosphotransferases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D010957:Plasmids / Q000235:genetics*
D007544:Isopropyl Thiogalactoside; D010770:Phosphotransferases; D019868:Kanamycin Kinase