Recombinant Protein Production Using the Baculovirus Expression Vector System (BEVS).

The baculovirus expression vector system (BEVS) is one of the most popular eukaryotic systems for recombinant protein production. The focus of our protein production platform is the provision of recombinant proteins for research use, where generally only small quantities are required, in the range of tens of micrograms to a few hundred milligrams. Here, we present methods that reflect our standard operating procedures and setup to be able to frequently, and often repeatedly, produce many different types of proteins.

New Insights into the Interaction of Class II Dihydroorotate Dehydrogenases with Ubiquinone in Lipid Bilayers as a Function of Lipid Composition.

The fourth enzymatic reaction in the de novo pyrimidine biosynthesis, the oxidation of dihydroorotate to orotate, is catalyzed by dihydroorotate dehydrogenase (DHODH). Enzymes belonging to the DHODH Class II are membrane-bound proteins that use ubiquinones as their electron acceptors. We have designed this study to understand the interaction of an N-terminally truncated human DHODH (HsΔ29DHODH) and the DHODH from Escherichia coli (EcDHODH) with ubiquinone (Q10) in supported lipid membranes using neutron reflectometry (NR). NR has allowed us to determine in situ, under solution conditions, how the enzymes bind to lipid membranes and to unambiguously resolve the location of Q10. Q10 is exclusively located at the center of all of the lipid bilayers investigated, and upon binding, both of the DHODHs penetrate into the hydrophobic region of the outer lipid leaflet towards the Q10. We therefore show that the interaction between the soluble enzymes and the membrane-embedded Q10 is mediated by enzyme penetration. We can also show that EcDHODH binds more efficiently to the surface of simple bilayers consisting of 1-palmitoyl, 2-oleoyl phosphatidylcholine, and tetraoleoyl cardiolipin than HsΔ29DHODH, but does not penetrate into the lipids to the same degree. Our results also highlight the importance of Q10, as well as lipid composition, on enzyme binding.

Protein-lipid interactions of human dihydroorotate dehydrogenase and three mutants associated with Miller syndrome.

Human dihydroorotate dehydrogenase (DHODH) catalyzes the fourth step of the de novo pyrimidine biosynthesis pathway and uses ubiquinone Q10, a lipophilic molecule located in the inner mitochondrial membrane (IMM), as its co-substrate. DHODH is anchored to the IMM by a single transmembrane helix located at its N-terminus. Nevertheless, how DHODH function is determined by its surrounding membrane environment and protein-lipid interactions, as well as the mechanism by which ubiquinone Q10 accesses the active site of DHODH from within the membrane are still largely unknown. Here, we describe the interaction between wild-type DHODH and three DHODH mutants associated with Miller syndrome and lipids using enzymatic assays, thermal stability assays and Quartz Crystal Microbalance with Dissipation monitoring (QCM-D). Our results provide evidence indicating that the N-terminal part of human DHODH is not only a structural element for mitochondrial import and location of DHODH, but also influences enzymatic activity and utilization of ubiquinone Q10 and ubiquinone analogues in in vitro assays. They also support the role of tetraoleoyl cardiolipin as a lipid interacting with DHODH. Additionally, the results from QCM-D show that the Miller syndrome mutants studied differ in their interactions with supported lipid bilayers compared to wild-type DHODH. These altered interactions add another dimension to the effects of mutations found in Miller syndrome. To the best of our knowledge, this is the first investigation of the protein-lipid interactions of DHODH variants associated with Miller syndrome.

Protein production, kinetic and biophysical characterization of three human dihydroorotate dehydrogenase mutants associated with Miller syndrome.

Miller syndrome is a rare Mendelian disorder caused by mutations in the gene encoding human dihydroorotate dehydrogenase (DHODH). Human DHODH, a Class II DHODH, is an integral protein of the inner mitochondrial membrane (IMM) catalyzing the fourth step of the de novo pyrimidine biosynthesis pathway. Here we present a summary of the state of knowledge regarding Miller syndrome in the absence of any current review on the topic. We then describe the production and characterization of three distinct DHODH missense mutations (G19E, E52G, R135C) associated with Miller syndrome by means of enzyme kinetics and biophysical techniques. These human DHODH mutants were produced both in E. coli and in insect cells using the baculovirus expression vector system. We can show that the effects of these mutations differ from each other and the wild-type enzyme with respect to decreased enzymatic activity, decreased protein stability and probably disturbance of the correct import into the IMM. In addition, our results show that the N-terminus of human DHODH is not only a structural element necessary for correct mitochondrial import and location of DHODH on the outer side of the IMM, but also influences thermal stability, enzymatic activity and affects the kinetic parameters.Supplemental data for this article is available online at https://doi.org/10.1080/15257770.2021.2023749 .

Expression of tomato thymidine kinase 1 by means of the baculovirus expression vector system.

Tomato thymidine kinase 1 (ToTK1) is a deoxyribonucleoside kinase (dNK) that has been subject to study because of its potential to phosphorylate the nucleoside analogue 3-azido-2,3-dideoxythymidine (azidothymidine, AZT) equally well as its natural substrate thymidine (dThd). The combination of ToTK1 and AZT has been tested in two animal studies for its efficiency and use in suicide gene therapy for malignant glioma. The determination of the 3D structure of ToTK1 might shed light on the structure-function relationships of nucleoside activation by this enzyme and thereby show routes toward further improvement of ToTK1 and other TK1-like dNKs for suicide gene therapy. Here we report the successful expression of both full-length ToTK1 and a C-terminal truncated ToTK1 in Spodoptera frugiperda and Trichoplusia ni insect cells using the baculovirus expression vector system. This constitutes a further step on the road to determine the 3D structure of the first TK1 of plant origin, but also an enzyme with great potential for dNK-mediated suicide gene therapy.