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Soluble expression in Escherichia coli, one-step purification, and characterization of Chinese hamster dihydrofolate reductase.

Abstract：

: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.

journal_name

Protein Expr Purif

journal_title

Protein expression and purification

authors

Fan YX,Wang B,Jing GZ

doi

10.1006/prep.1996.0676

subject

Has Abstract

pub_date

1997-03-01 00:00:00

pages

246-52

issue

2

eissn

1046-5928

issn

1096-0279

pii

S1046-5928(96)90676-2

journal_volume

9

pub_type

杂志文章

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