Using the BacMam Baculovirus System to Study Expression and Function of Recombinant Efflux Drug Transporters in Polarized Epithelial Cell Monolayers

Fung, King Leung; Kapoor, Khyati; Pixley, Jessica N.; Talbert, Darrell J.; Kwit, Alexandra D.T.; Ambudkar, Suresh V.; Gottesman, Michael M.

doi:10.1124/dmd.115.066506

Cited by 8 publications

(5 citation statements)

References 27 publications

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“…To address the glycosylation propensity of the Pfs48/45 molecule during insect cell production, two approaches were undertaken. In an effort to minimize modifications (e.g., mutations) to the native gene sequence, the primary approach was to include tunicamycin, an antibiotic, in the expression culture as it has been demonstrated to effectively inhibit N-glycosylation in the insect/baculovirus expression system 28,29 . The secondary approach to eliminate N-glycosylation in the 6C fragment was to mutate the two high probability N-glycosylation sites at positions N299 and N303 in the 6C construct (termed 6C-Mut).…”

Section: Resultsmentioning

confidence: 99%

A C-terminal Pfs48/45 malaria transmission-blocking vaccine candidate produced in the baculovirus expression system

Lee

Hickey

Miura

et al. 2020

Sci Rep

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the Plasmodium falciparum gametocyte surface protein, Pfs48/45, is a potential target for malaria transmission-blocking vaccines. However, due to its size and complexity, expression of the fulllength protein has been difficult, leading to focus on the C-terminal six cysteine domain (6C) with the use of fusion proteins to facilitate expression and folding. in this study, we utilized the baculovirus system to evaluate the expression of three Pfs48/45 proteins including the full-length protein, the 6C domain fragment and the 6C domain mutant to prevent glycosylation. Expression of the recombinant Pfs48/45 proteins was conducted in super Sf9 cells combined with the use of tunicamycin to prevent Nglycosylation. the proteins were then evaluated as immunogens in mice to demonstrate the induction of functionally active polyclonal antibody responses as measured in the standard membrane feeding assay (SMFA). Only the 6C protein was found to exhibit significant transmission-reducing activity. Further characterization of the biologically active 6C protein demonstrated it was homogeneous in terms of size, charge, conformation, absence of glycosylation, and containing proper disulfide bond pairings. this study presents an alternative expression system, without the need of a fusion protein partner, for the Pfs48/45 6C protein fragment including further evaluation as a potential transmissionblocking vaccine candidate.Malaria transmission-blocking vaccines (TBVs) are being evaluated for their potential to serve as a supplemental tool to accelerate the elimination of malaria. TBVs function via the induction of antibodies that block the fertilization or the penetration of the mosquito midgut by sexual stage parasite, thereby breaking the cycle of parasite transmission between human and mosquito hosts 1-3 . Several sexual stage antigens (e.g., Pfs25, Pfs230 and Pfs48/45) have been identified as promising TBV targets for research or early stage clinical development. In clinical testing, Pfs25, when delivered as nanoparticles 4 , induced functional antibodies. However, the responses were of low magnitude and short-lived 5,6 . In recent years, development attention has shifted to the TBV candidates Pfs230 and Pfs48/45 in view of the poor outcomes associated with Pfs25-based constructs and the fact that sera from Plasmodium infected individuals are associated with transmission-blocking antibodies attributable to these gametocyte surface antigens 7 . Moreover, affinity purified anti-Pfs48/45 and anti-Pfs230 antibodies from naturally exposed individuals can prevent the transmission of cultured P. falciparum gametocytes 8 , when concentrated nine times the physiological concentration, thereby demonstrating the functionality of these natural antibody responses and the potential for these antigens in TBV development.While the preclinical 9-11 and clinical (NCT 02942277) development of Pfs230 is in progress, the development of Pfs48/45 as a TBV candidate has remained challenging. The Pfs48/45 protein, expressed on the surface of gametocyte, s...

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Section: Resultsmentioning

confidence: 99%

A C-terminal Pfs48/45 malaria transmission-blocking vaccine candidate produced in the baculovirus expression system

Lee

Hickey

Miura

et al. 2020

Sci Rep

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“…Transwell assays using Madin-Darby canine kidney (MDCK) or pig kidney (LLC-PK1) cells transfected with P-gp have routinely been used to identify substrates of P-gp. MDCK and LLC-PK1 cells have also been transfected to express both Pgp and ABCG2 to explore the effects of their coexpression [35][36][37] . Caco-2 colorectal adenocarcinoma cells also form tight junctions and express relatively high endogenous levels of P-gp and ABCG2.…”

Section: Transwell Assaysmentioning

confidence: 99%

ATP-binding cassette transporters at the zebrafish blood-brain barrier and the potential utility of the zebrafish as an in vivo model

Hotz

Thomas

Katz

et al. 2021

CDR

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The brain is protected from toxins by a tightly regulated network of specialized cells, including endothelial cells, pericytes, astrocyes, and neurons, known collectively as the blood-brain barrier (BBB). This selectively permeable barrier permits only the most crucial molecules essential for brain function to enter and employs a number of different mechanisms to prevent the entry of potentially harmful toxins and pathogens. In addition to a physical barrier comprised of endothelial cells that form tight junctions to restrict paracellular transport, there is an active protective mechanism made up of energy-dependent transporters that efflux compounds back into the bloodstream. Two of these ATP-binding cassette (ABC) transporters are highly expressed at the BBB: Pglycoprotein (P-gp, encoded by the ABCB1 gene) and ABCG2 (encoded by the ABCG2 gene). Although a number of in vitro and in vivo systems have been developed to examine the role that ABC transporters play in keeping compounds out of the brain, all have inherent advantages and disadvantages. Zebrafish (Danio rerio) have become a model of interest for studies of the BBB due to the similarities between the zebrafish and mammalian BBB systems. In this review, we discuss what is known about ABC transporters in zebrafish and what information is still needed before the zebrafish can be recommended as a model to elucidate the role of ABC transporters at the BBB.

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“…However, it is also a significant hindrance to the transportation of medications from circulation into the CNS. Currently, there are few therapeutics effectively used in treating CNS diseases, in addition, the efficacy of the current drug is restricted for insufficient drug transportation via the BBB ( Fung et al, 2016 ). Drug transit via BBB is largely determined by drug characteristics, such as molecular size, dissociation degree, and hydrophilicity ( Bharadwaj et al, 2016 ).…”

Section: Natural Products Based Nose To Brain Drug Delivery (Nbdd)mentioning

confidence: 99%

Natural products for the treatment of chemotherapy-related cognitive impairment and prospects of nose-to-brain drug delivery

He,

Zhou,

Jiang

et al. 2024

Front. Pharmacol.

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Chemotherapy-related cognitive deficits (CRCI) as one of the common adverse drug reactions during chemotherapy that manifest as memory, attention, and executive function impairments. However, there are still no effective pharmacological therapies for the treatment of CRCI. Natural compounds have always inspired drug development and numerous natural products have shown potential therapeutic effects on CRCI. Nevertheless, improving the brain targeting of natural compounds in the treatment of CRCI is still a problem to be overcome at present and in the future. Accumulated evidence shows that nose-to-brain drug delivery may be an excellent carrier for natural compounds. Therefore, we reviewed natural products with potential anti-CRCI, focusing on the signaling pathway of these drugs’ anti-CRCI effects, as well as the possibility and prospect of treating CRCI with natural compounds based on nose-to-brain drug delivery in the future. In conclusion, this review provides new insights to further explore natural products in the treatment of CRCI.

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Using the BacMam Baculovirus System to Study Expression and Function of Recombinant Efflux Drug Transporters in Polarized Epithelial Cell Monolayers

Cited by 8 publications

References 27 publications

A C-terminal Pfs48/45 malaria transmission-blocking vaccine candidate produced in the baculovirus expression system

A C-terminal Pfs48/45 malaria transmission-blocking vaccine candidate produced in the baculovirus expression system

ATP-binding cassette transporters at the zebrafish blood-brain barrier and the potential utility of the zebrafish as an in vivo model

Natural products for the treatment of chemotherapy-related cognitive impairment and prospects of nose-to-brain drug delivery

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