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High-level expression of Escherichia coli and Bacillus subtilis thymidylate synthases.

Abstract：

:Procedures are described for the preparation of highly purified thymidylate synthases from Escherichia coli and Bacillus subtilis. The yields in each case are quite high with about 350 mg of pure protein obtained from 1 liter of cells. Basically all that is required to obtain pure enzyme is an induction step from a high-expression vector, followed by a DE-52 column elution. Both enzymes appeared to be fairly stable in that incubation at 43 degrees C for 10 min resulted in the loss of 50% of the E. coli thymidylate synthase activity, while 50 degrees C for 10 min was required to obtain the same effect with the B. subtilis enzyme. In the presence of the substrate, dUMP, each protein was stabilized further by 6 to 7 degrees C, which was increased to 9 to 10 degrees C on addition of dihydrofolate. It was shown also that the E. coli thymidylate synthase could be maintained at 4 degrees C for at least 4 months with little or no loss in activity provided that mercaptoethanol was not present. The presence of the latter led to a progressive loss in activity until little activity could be detected after 18 weeks, which was due, in part, to the formation of a disulfide bond with the active site cysteine. Addition of dithiothreitol restored the enzyme activity to its original state.

journal_name

Protein Expr Purif

journal_title

Protein expression and purification

authors

Changchien LM,Garibian A,Frasca V,Lobo A,Maley GF,Maley F

doi

10.1006/prep.2000.1245

keywords:

subject

Has Abstract

pub_date

2000-07-01 00:00:00

pages

265-70

issue

2

eissn

1046-5928

issn

1096-0279

pii

S1046-5928(00)91245-2

journal_volume

19

pub_type

杂志文章

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