Cloning and expression of aminoglycoside phosphotransferase gene (APH) from a butirosin producing strain of Bacillus circulans
Cloning and expression of aminoglycoside phosphotransferase gene (APH) from a butirosin producing strain of Bacillus circulans
A 2.7 kb SalI fragment containing the APH gene from B. circulans had previously been cloned into pBR322. A tentative sequence of the coding region had also been described [Herbert, C.J., Giles, I.G. and Akhtar, M. (1983) FEBS Lett. 160, 67-71]. This work has been extended in this thesis. The sequences of the coding and regulatory regions were corrected and extended using specially designed oligodeoxynucleotides. The sequence information was exploited to isolate a 1229 bp DdEI fragment of the gene for cloning into a high expression vector, pKK223-3, which contains a strong tac promoter. The successful cloning protocol involved filling the recessed termini of 1229 bp APH fragment and EcoRI cut pKK223-3 and ligating the two fragments together. The high expression construct (pMS5) containing the APH gene was characterised and several features of the expression of the phosphotranferase studied. The phosphotransferase activity was best expressed in a growth medium lacking glucose. The maximum production of the enzyme was achieved by growing the bacteria to stationary phase. The induction of phosphotransferase expression with IPTG (inducer) was found to be undesirable as overproduction of the enzyme led to the killing of bacteria. The subcellular location of the phosphotransferase and also the in vivo site of the phosphorylation of neomycin was determined. The phosphotranferase was purified and its N-terminal amino acid sequence determined. The method of purification was also extended to the phosphotransferase of Streptomyces fradiae cloned into S. lividans. Kinetic parameters of the phosphotransferases from B. circulans and S. fradiae were studied using several antibiotics as substrates. Brenner [Nature (1987) 329, 21] identified a common motif [HxDhxxxNhhh(x)9-13D; where x is any amino acid and h is a hydrophobic amino acid] in aminoglycoside phosphotransferases as well as in a number of protein kinases and suggested that the sequence may play an important role in the phosphoryl transfer process. In order to evaluate the validity of this hypothesis, site-directed mutagenesis of the phosphotranferase of B. circulans was undertaken and its aspartate 187 was mutated to asparagine 187. The mutant enzyme has only 0.2% of the activity of the wild type enzyme. Whether this impairment of catalytic activity is due to the obligatory involvement of the carboxylate group of aspartate 187 in catalysis or the importance of this residue in protein folding has not yet been determined.
University of Southampton
1989
Sarwar, Muhammad
(1989)
Cloning and expression of aminoglycoside phosphotransferase gene (APH) from a butirosin producing strain of Bacillus circulans.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
A 2.7 kb SalI fragment containing the APH gene from B. circulans had previously been cloned into pBR322. A tentative sequence of the coding region had also been described [Herbert, C.J., Giles, I.G. and Akhtar, M. (1983) FEBS Lett. 160, 67-71]. This work has been extended in this thesis. The sequences of the coding and regulatory regions were corrected and extended using specially designed oligodeoxynucleotides. The sequence information was exploited to isolate a 1229 bp DdEI fragment of the gene for cloning into a high expression vector, pKK223-3, which contains a strong tac promoter. The successful cloning protocol involved filling the recessed termini of 1229 bp APH fragment and EcoRI cut pKK223-3 and ligating the two fragments together. The high expression construct (pMS5) containing the APH gene was characterised and several features of the expression of the phosphotranferase studied. The phosphotransferase activity was best expressed in a growth medium lacking glucose. The maximum production of the enzyme was achieved by growing the bacteria to stationary phase. The induction of phosphotransferase expression with IPTG (inducer) was found to be undesirable as overproduction of the enzyme led to the killing of bacteria. The subcellular location of the phosphotransferase and also the in vivo site of the phosphorylation of neomycin was determined. The phosphotranferase was purified and its N-terminal amino acid sequence determined. The method of purification was also extended to the phosphotransferase of Streptomyces fradiae cloned into S. lividans. Kinetic parameters of the phosphotransferases from B. circulans and S. fradiae were studied using several antibiotics as substrates. Brenner [Nature (1987) 329, 21] identified a common motif [HxDhxxxNhhh(x)9-13D; where x is any amino acid and h is a hydrophobic amino acid] in aminoglycoside phosphotransferases as well as in a number of protein kinases and suggested that the sequence may play an important role in the phosphoryl transfer process. In order to evaluate the validity of this hypothesis, site-directed mutagenesis of the phosphotranferase of B. circulans was undertaken and its aspartate 187 was mutated to asparagine 187. The mutant enzyme has only 0.2% of the activity of the wild type enzyme. Whether this impairment of catalytic activity is due to the obligatory involvement of the carboxylate group of aspartate 187 in catalysis or the importance of this residue in protein folding has not yet been determined.
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Published date: 1989
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Local EPrints ID: 462431
URI: http://eprints.soton.ac.uk/id/eprint/462431
PURE UUID: a15199b4-9626-4c85-ac38-c77862f02dfa
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Date deposited: 04 Jul 2022 19:08
Last modified: 04 Jul 2022 19:08
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Author:
Muhammad Sarwar
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