Novel ceftazidime-resistance β-lactamases generated by a codon-based mutagenesis method and selection -: art. no. e84

被引:12
作者
Gaytán, P [1 ]
Osuna, J [1 ]
Soberón, X [1 ]
机构
[1] Univ Nacl Autonoma Mexico, Inst Biotechnol, Cuernavaca 62250, Morelos, Mexico
关键词
D O I
10.1093/nar/gnf083
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Four known and nine new ceftazidime-resistance beta-lactamases were generated by a novel, contaminating codon-based mutagenesis approach. In this method, wild-type codons are spiked with a set of mutant codons during oligonucleotide synthesis, generating random combinatorial libraries of primers that contain few codon replacements per variant. Mutant codons are assembled by tandem addition of a diluted mixture of five Fmoc-dimer amidites to the growing oligo and a mixture of four DMTr-monomer amidites to generate 20 trinucleotides that encode a set of 18 amino acids. Wild-type codons are assembled with conventional chemistry and the whole process takes place in only one synthesis column, making its automation feasible. The random and binomial behavior of this approach was tested in the polylinker region of plasmid pUC19 by the synthesis of three oligonucleotide libraries mutagenized at different rates and cloned as mutagenic cassettes. Additionally, the method was biologically assessed by mutating six contiguous codons that encode amino acids 237-243 (ABL numbering) of the TEMpUC19 beta-lactamase, which is functionally equivalent to the clinically important TEM-1 beta-lactamase. The best ceftazidime-recognizing variant was a triple mutant, R164H:E240K: R241A, displaying a 333-fold higher resistance than the wild-type enzyme.
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页数:13
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