Purification and kinetic properties of human recombinant dihydrofolate reductase produced in Bombyx mori chrysalides.
Recent reports describe the inhibition of human dihydrofolate reductase (hDHFR) by natural tea polyphenols. This finding could explain the epidemiologic data on their prophylactic effects for certain forms of cancer, and it raises the possibility that natural and synthetic polyphenols could be used in cancer chemotherapy. In order to obtain larger quantities of hDHFR to support structural studies, we established and validated a baculovirus system for the expression of this protein in Bombyx mori chrysalides (pupae of the silkworm enclosed in a cocoon). To isolate the expressed protein, whole infected pupae were homogenized, and the expressed protein was purified by affinity chromatography. Here, we demonstrate the efficient expression of recombinant hDHFR in this model and report that this newly expressed protein has high enzymatic activity and kinetic properties similar to those previously reported for recombinant hDHFR expressed in Escherichia coli. The purified protein showed dissociation constants for the binding of natural polyphenols similar to that expressed in E. coli, which ensures its usage as a new tool for further structural studies. Although the hDHFR yield per individual was found to be lower in the chrysalides than in the larvae of B. mori, the former system was optimized as a model for the scaled-up production of recombinant proteins. Expression of proteins in chrysalides (instead of larvae) could offer important advantages from both economic and biosecurity aspects.
Chazarra
Soledad
S
CEEIM, Universitario de Espinardo, Murcia, Spain.
Aznar-Cervantes
Salvador
S
Sánchez-del-Campo
Luis
L
Cabezas-Herrera
Juan
J
Xiaofeng
Wu
W
Cenis
José Luis
JL
Rodríguez-López
José Neptuno
JN
D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000502:physiology; D012831:Bombyx / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism / Q000821:virology; D005419:Flavonoids / Q000378:metabolism; D005818:Genetic Engineering; D005822:Genetic Vectors / Q000235:genetics / Q000502:physiology; D006801:Humans; D007700:Kinetics; D007814:Larva / Q000235:genetics / Q000254:growth & development / Q000378:metabolism / Q000821:virology; D010636:Phenols / Q000378:metabolism; D059808:Polyphenols; D011679:Pupa / Q000235:genetics / Q000254:growth & development / Q000378:metabolism / Q000821:virology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D005419:Flavonoids; D010636:Phenols; D059808:Polyphenols; D011994:Recombinant Proteins; D013762:Tetrahydrofolate Dehydrogenase