PRODUCTION OF MUTANT DIHYDROFOLATE REDUCTASES OF LACTOBACILLUS-CASEI FOR NUCLEAR-MAGNETIC-RESONANCE SPECTROSCOPY

被引:4
作者
ANDREWS, J [1 ]
MINTER, SJ [1 ]
DAVIES, RW [1 ]
机构
[1] UNIV MANCHESTER,INST SCI & TECHNOL,DEPT BIOCHEM & APPL MOLEC BIOL,MANCHESTER M60 1QD,LANCS,ENGLAND
关键词
RECOMBINANT DNA; ANTIBIOTIC; ANTINEOPLASTIC; SITE-DIRECTED MUTAGENESIS; HIGH-LEVEL EXPRESSION; NMR PEAK ASSIGNMENT; ISOTOPIC LABELING;
D O I
10.1016/0378-1119(91)90370-Q
中图分类号
Q3 [遗传学];
学科分类号
071007 ; 090102 ;
摘要
Seven mutations (L4P, W21L, D26E, D26N, R57H, R57K and T63Q) affecting residues of dihydrofolate reductase of Lactobacillus casei, suspected of being important in substrate, inhibitor, or cofactor binding, were made by gapped-duplex site-directed mutagenesis. Expression of the L. casei dhfr gene required the removal of nucleotide sequences flanking the coding region. Temperature-inducible expression from the lambda-p(L) promoter of plasmid pPLc28 allowed synthesis and subsequent affinity purification of five mutant proteins in amounts and purity sufficient for nuclear magnetic resonance (NMR) spectroscopic analysis (100 mg or more) from 10-liter cultures. W21L required the growth of 40-liter batches, and L4P was not found. Using a two-plasmid system with pc1857 providing lambda-repressor and pMAC5-14 expressing the mutant gene, any auxotrophic strain of Escherichia coli can be used as a host, allowing isotopic labelling of each amino acid of any protein for rapid NMR peak assignment.
引用
收藏
页码:219 / 224
页数:6
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