Soluble expression in Escherichia coli, one-step purification, and characterization of Chinese hamster dihydrofolate reductase.
Chinese hamster dihydrofolate reductase (ch-DHFR) was overexpressed in Escherichia coli DH5 alpha under the transcriptional control of PRPL promoters regulated by temperature-sensitive repressors. The desired recombinant product is soluble and constitutes about 30% of the total soluble proteins of the bacterial cell. With repeated cycles of freezing and thawing as a first step, the purification of the recombinant ch-DHFR to homogeneity requires only one further step, gel filtration on a Sephadex G-75 column with 85-90% enzyme recovery, two to three times higher than that obtained with the commonly used affinity chromatography on a methotrexate-Sepharose column. The purified enzyme migrates as a single protein band on SDS-polyacrylamide gel electrophoresis with approximate mass of 23 kDa, in accord with that calculated from the DNA sequence. The initiation methionine residue at the N-terminus of the enzyme is completely removed by E. coli methionine aminopeptidase as judged by amino-terminal analysis. The steady-state kinetic parameters, dissociation constants for binary complexes of dihydrofolate, NADPH, and methotrexate with ch-DHFR, and the inhibitor constant of methotrexate have also been determined. The enzyme is activated about 4-fold in 3 M urea and about 2.5-fold in 0.5 M guanidine hydrochloride.
Fan
Y X
YX
National Laboratory of Biomacromolecules, Academia Sinica, Beijing, China.
Wang
B
B
Jing
G Z
GZ
D000818:Animals; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005453:Fluorescence; D010957:Plasmids / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012995:Solubility; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D017186:Titrimetry
D011994:Recombinant Proteins; D013762:Tetrahydrofolate Dehydrogenase