Development of an efficient transient expression system for Siraitia grosvenorii fruit and functional characterization of two NADPH-cytochrome P450 reductases

2021 ◽  
Vol 189 ◽  
pp. 112824
Author(s):  
Jingjing Liao ◽  
Lei Xie ◽  
Hongwu Shi ◽  
Shengrong Cui ◽  
Fusheng Lan ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Transient Expression ◽  
Expression System ◽  
Functional Characterization ◽  
Nadph Cytochrome ◽  
Transient Expression System ◽  
Siraitia Grosvenorii

Functional characterization of a stilbene synthase gene using a transient expression system in planta

2008 ◽  
Vol 28 (4) ◽  
pp. 589-599 ◽  
Author(s):  
Jose Condori ◽  
Giuliana Medrano ◽  
Ganapathy Sivakumar ◽  
Vipin Nair ◽  
Carole Cramer ◽  
...  
Keyword(s):  
Transient Expression ◽  
Expression System ◽  
Functional Characterization ◽  
Stilbene Synthase ◽  
In Planta ◽  
Transient Expression System ◽  
Stilbene Synthase Gene

scholarly journals Harnessing a Transient Gene Expression System in Nicotiana benthamiana to Explore Plant Agrochemical Transporters

Plants ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 524
Author(s):  
Bingqi Wu ◽  
Zhiting Chen ◽  
Xiaohui Xu ◽  
Ronghua Chen ◽  
Siwei Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transient Expression ◽  
Nicotiana Benthamiana ◽  
Heterologous Gene Expression ◽  
Expression System ◽  
Functional Characterization ◽  
Transient Gene Expression ◽  
High Mobility ◽  
Gene Expression System

Functional characterization of plant agrichemical transporters provided an opportunity to discover molecules that have a high mobility in plants and have the potential to increase the amount of pesticides reaching damage sites. Agrobacterium-mediated transient expression in tobacco is simple and fast, and its protein expression efficiency is high; this system is generally used to mediate heterologous gene expression. In this article, transient expression of tobacco nicotine uptake permease (NtNUP1) and rice polyamine uptake transporter 1 (OsPUT1) in Nicotiana benthamiana was performed to investigate whether this system is useful as a platform for studying the interactions between plant transporters and pesticides. The results showed that NtNUP1 increases nicotine uptake in N. benthamiana foliar discs and protoplasts, indicating that this transient gene expression system is feasible for studying gene function. Moreover, yeast expression of OsPUT1 apparently increases methomyl uptake. Overall, this method of constructing a transient gene expression system is useful for improving the efficiency of analyzing the functions of plant heterologous transporter-encoding genes and revealed that this system can be further used to study the functions of transporters and pesticides, especially their interactions.

scholarly journals Functional characterization of a glycine 185-to-valine substitution in human P-glycoprotein by using a vaccinia-based transient expression system.

1996 ◽  
Vol 7 (10) ◽  
pp. 1485-1498 ◽  
Author(s):  
M Ramachandra ◽  
S V Ambudkar ◽  
M M Gottesman ◽  
I Pastan ◽  
C A Hrycyna
Keyword(s):  
Drug Resistance ◽  
Atpase Activity ◽  
Cyclosporin A ◽  
Transient Expression ◽  
Expression System ◽  
Rhodamine 123 ◽  
Wild Type ◽  
P Glycoprotein ◽  
Transient Expression System

Human P-glycoprotein (Pgp) is a 170-kDa plasma membrane protein that confers multidrug resistance to otherwise sensitive cells. A mutation in Pgp, G185-->V, originally identified as a spontaneous mutation, was shown previously to alter the drug resistance profiles in cell lines that are stably transfected with the mutant MDR1 cDNA and selected with cytotoxic agents. To understand the mechanism by which the V185 mutation leads to an altered drug resistance profile, we used a transient expression system that eliminates the need for drug selection to attain high expression levels and allows for the rapid characterization of many aspects of Pgp function and biosynthesis. The mutant and wild-type proteins were expressed at similar levels after 24-48 h in human osteosarcoma (HOS) cells by infection with a recombinant vaccinia virus encoding T7 RNA polymerase and simultaneous transfection with a plasmid containing MDR1 cDNA controlled by the T7 promoter. For both mutant and wild-type proteins, photolabeling with [3H]azidopine and [125I]iodoarylazidoprazosin, drug-stimulated ATPase activity, efflux of rhodamine 123, and accumulation of radiolabeled vinblastine and colchicine were evaluated. In crude membrane preparations from HOS cells, a higher level of basal Pgp-ATPase activity was observed for the V185 variant than for the wild-type, suggesting partial uncoupling of drug-dependent ATP hydrolysis by the mutant. Several compounds, including verapamil, nicardipine, tetraphenylphosphonium, and prazosin, stimulated ATPase activities of both the wild-type and mutant similarly, whereas cyclosporin A inhibited the ATPase activity of the mutant more efficiently than that of the wild-type. This latter observation explains the enhanced potency of cyclosporin A as an inhibitor of the mutant Pgp. No differences were seen in verapamil-inhibited rhodamine 123 efflux, but the rate of accumulation was slower for colchicine and faster for vinblastine in cells expressing the mutant protein, as compared with those expressing wild-type Pgp. We conclude that the G185-->V mutation confers pleiotropic alterations on Pgp, including an altered basal ATPase activity and altered interaction with substrates and the inhibitor cyclosporin A.

Characterization of transient expression system for retroviral vector production

2006 ◽  
Vol 101 (4) ◽  
pp. 361-368 ◽  
Author(s):  
Akitsu Hotta ◽  
Yoshikazu Saito ◽  
Kenji Kyogoku ◽  
Yoshinori Kawabe ◽  
Ken-ichi Nishijima ◽  
...  
Keyword(s):  
Transient Expression ◽  
Expression System ◽  
Retroviral Vector ◽  
Transient Expression System

scholarly journals Biochemical and Functional Characterization of a Novel Mutation in the NADPH Cytochrome P450 Oxidoreductase

2019 ◽  
Vol 33 (S1) ◽  
Author(s):  
Amit V Pandey ◽  
Sara Benito‐Sanz ◽  
Shaheena Parween ◽  
Juan‐Pedro López‐Siguero ◽  
Núria Camats ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Novel Mutation ◽  
Functional Characterization ◽  
P450 Oxidoreductase ◽  
Nadph Cytochrome ◽  
Cytochrome P450 Oxidoreductase
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