High-level expression and isotopic labeling of Lactobacillus casei dihydrofolate reductase for nuclear magnetic resonance spectroscopy.
Two efficient systems have been used for high-level expression of Lactobacillus casei dihydrofolate reductase in Escherichia coli, including the production of protein generally and specifically labeled with 13C and 15N. A system based on T7 RNA polymerase led to the production of dihydrofolate reductase at a level of 37% of the total soluble protein of the host strain: 50 mg of pure enzyme was obtained from a 1 liter of culture (or 14 mg/g wet weight of cells). In this system, a small amount of the enzyme (less than 5%) was identified as a catalytically active 21-kDa fusion protein. Introduction of a second in-frame (ochre) stop codon did not eliminate the production of this fusion protein. The same expression system was also used to prepare dihydrofolate reductase generally labeled with 15N and to prepare single and double mutants of the enzyme. In order to have an expression system which can be used with a range of auxotrophic strains of E. coli, a system based on the tac promoter was used. This led to the production of dihydrofolate reductase at a level of 29% of total soluble protein; a yield of 40 mg enzyme per liter of culture (or 11 mg/g wet weight of cells). This system was successfully used to produce mutants of the enzyme as well as the enzyme selectively labeled with [gamma-13C]aspartic acid.
Badii
R
R
Biological NMR Centre, University of Leicester, United Kingdom.
Basran
J
J
Casarotto
M G
MG
Roberts
G C
GC
D001483:Base Sequence; D002247:Carbon Isotopes; D004926:Escherichia coli / Q000235:genetics; D007780:Lacticaseibacillus casei / Q000201:enzymology* / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins; D013762:Tetrahydrofolate Dehydrogenase