Increased solubility of trimethoprim-resistant type S1 DHFR from Staphylococcus aureus in Escherichia coli cells overproducing the chaperonins GroEL and GroES.
The production of the trimethoprim-resistant type S1 dihydrofolate reductase (DHFR) from Staphylococcus aureus in Escherichia coli cells overproducing the chaperonins GroEL and GroES is described. The simultaneous overproduction of the chaperonins with DHFR results in an increased solubility of the enzyme. We compare the time course of production of active type S1 DHFR by measuring enzyme activity in cells overproducing or not overproducing the chaperonins. Although co-overproduction of the chaperonins reduces the total production level of type S1 DHFR, the amount of soluble and active DHFR is increased several-fold in comparison with cells producing only DHFR. Thus, the higher concentrations of GroES and GroEL in cells overproducing the chaperonins partially protect DHFR from aggregation, resulting in higher concentrations of soluble and active DHFR in the cell. Furthermore, we also demonstrate that the chaperonins can improve in vitro refolding yields of type S1 DHFR. These results suggest that it is possible to purify suitable amounts of trimethoprim-resistant type S1 DHFR for X-ray crystallographic studies.
Department of Biology, Pharmaceutical Research New Technologies, F. Hoffmann-La Roche Ltd, Basel, Switzerland.
G E
GE
Dale
H J
HJ
Schönfeld
H
H
Langen
M
M
Stieger
D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry*; D018835:Chaperonin 10 / Q000096:biosynthesis / Q000502:physiology*; D018834:Chaperonin 60 / Q000096:biosynthesis / Q000502:physiology*; D004926:Escherichia coli / Q000378:metabolism*; D005493:Folic Acid Antagonists; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012995:Solubility; D013211:Staphylococcus aureus / Q000187:drug effects / Q000201:enzymology*; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry*; D014295:Trimethoprim / Q000494:pharmacology; D014296:Trimethoprim Resistance
D001426:Bacterial Proteins; D018835:Chaperonin 10; D018834:Chaperonin 60; D005493:Folic Acid Antagonists; D011993:Recombinant Fusion Proteins; D014295:Trimethoprim; D013762:Tetrahydrofolate Dehydrogenase