Purification and Kinetic Properties of Human Recombinant Dihydrofolate Reductase Produced in Bombyx mori Chrysalides

Abstract

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.

Rotavirus A-specific single-domain antibodies produced in baculovirus-infected insect larvae are protective in vivo

“…Various kinds of protein have been successfully expressed in larvae, including diagnostic reagents [13-17], vaccines [14,18-26,29], therapeutic proteins [27], enzymes [32,33], and conventional complete or fragmented antibodies [34,35]. However, to the best of our knowledge, this is the first description that evaluates the efficiency of insects as living biofactories to express recombinant single-domain antibodies (VHH) derived from llama.…”

Student design and characterization of visible DHFR fusions for biochemistry tools to improve learning during lab exercises

“…We had students determine the K M app and k cat app parameters of DHF for their enzymes in the presence of saturating NADPH. One challenge with the wild type hDHFR and EcDHFR enzymes is the low K M value they have for DHF 18,19 . This causes difficulty for students in measuring initial velocity under the pseudo first‐order conditions of low substrate concentration.…”

“…One challenge with the wild type hDHFR and EcDHFR enzymes is the low K M value they have for DHF. 18,19 This causes difficulty for students in measuring initial velocity under the pseudo first-order conditions of low substrate concentration. For this reason, we prefer the EcDHFR mutant as a learning tool because it has a higher K M value which allows for easily detected differences in initial velocity of progress curves at sub K M concentrations of DHF.…”

Molecular Characteristics and Serodiagnostic Potential of Dihydrofolate Reductase from Echinococcus granulosus

“…DHFR has been studied extensively in a variety of organisms, including mammals 37 , protozoa 38 , bacteria 39 and several insects 27, 40 . Moreover, DHFR can be used as a drug target in the treatment of cancer and bacterial and parasitic infections 25 .…”