Production of mutant dihydrofolate reductases of Lactobacillus casei for nuclear magnetic resonance spectroscopy.
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 pL 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 pcI857 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.
Andrews
J
J
Department of Biochemistry and Applied Molecular Biology, U.M.I.S.T., Manchester, U.K.
Minter
S J
SJ
Davies
R W
RW
D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006860:Hydrogen Bonding; D007780:Lacticaseibacillus casei / Q000201:enzymology* / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D010957:Plasmids; D011487:Protein Conformation; D015183:Restriction Mapping; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D015345:Oligonucleotide Probes; D013762:Tetrahydrofolate Dehydrogenase