Protocols for Studying Protein Stability in an Arabidopsis Protoplast Transient Expression System

2016 ◽  
pp. 175-194 ◽  
Author(s):  
Séverine Planchais ◽  
Laurent Camborde ◽  
Isabelle Jupin
Keyword(s):  
Protein Stability ◽  
Transient Expression ◽  
Expression System ◽  
Transient Expression System ◽  
Arabidopsis Protoplast

Isolation of Mesophyll Protoplast and Establishment of Gene Transient Expression System in Cotton

2014 ◽  
Vol 40 (2) ◽  
pp. 231 ◽  
Author(s):  
Ni-Na LI ◽  
Lin-Yun DING ◽  
Zhi-Yuan ZHANG ◽  
Wang-Zhen GUO
Keyword(s):  
Transient Expression ◽  
Expression System ◽  
Mesophyll Protoplast ◽  
Transient Expression System

scholarly journals Agroinfiltration of Strawberry Fruit — A Powerful Transient Expression System for Gene Validation

2016 ◽  
Vol 6 ◽  
pp. 19-37 ◽  
Author(s):  
Raquel F. Carvalho ◽  
Sofia D. Carvalho ◽  
Kevin O’Grady ◽  
Kevin M. Folta
Keyword(s):  
Transient Expression ◽  
Expression System ◽  
Strawberry Fruit ◽  
Transient Expression System

scholarly journals A Transient Expression System with Stably Integrated CRISPR-dCas9 Fusions for Regulation of Genes Involved in Immunoglobulin G Glycosylation

2022 ◽  
Author(s):  
Anika Mijakovac ◽  
Karlo Miškec ◽  
Jasminka Krištić ◽  
Vedrana Vičić Bočkor ◽  
Vanja Tadić ◽  
...  
Keyword(s):  
Transient Expression ◽  
Immunoglobulin G ◽  
Expression System ◽  
Transient Expression System

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.

Protoplast Transformation as a Plant-Transferable Transient Expression System

2016 ◽  
pp. 137-148 ◽  
Author(s):  
Patrícia Duarte ◽  
Diana Ribeiro ◽  
Inês Carqueijeiro ◽  
Sara Bettencourt ◽  
Mariana Sottomayor
Keyword(s):  
Transient Expression ◽  
Expression System ◽  
Protoplast Transformation ◽  
Transient Expression System

scholarly journals Identification and Characterization of Plasmodiophora brassicae Primary Infection Effector Candidates that Suppress or Induce Cell Death in Host and Nonhost Plants

2019 ◽  
Vol 109 (10) ◽  
pp. 1689-1697 ◽  
Author(s):  
Wang Chen ◽  
Yan Li ◽  
Ruibin Yan ◽  
Li Xu ◽  
Li Ren ◽  
...  
Keyword(s):  
Cell Death ◽  
Transient Expression ◽  
Primary Infection ◽  
Plasmodiophora Brassicae ◽  
Signal Sequence ◽  
Expression System ◽  
Secretory Protein ◽  
Secretory Proteins ◽  
Induce Cell Death ◽  
Transient Expression System

Clubroot caused by Plasmodiophora brassicaeis one of the most important diseases in cruciferous crops. The recognition of P. brassicae by host plants is thought to occur at the primary infection stage, but the underlying mechanism remains unclear. Secretory proteins as effector candidates play critical roles in the recognition of pathogens and the interactions between pathogens and hosts. In this study, 33 P. brassicae secretory proteins expressed during primary infection were identified through transcriptome, secretory protein prediction, and yeast signal sequence trap analyses. Furthermore, the proteins that could suppress or induce cell death were screened through an Agrobacterium-mediated plant virus transient expression system and a protoplast transient expression system. Two secretory proteins, PBCN_002550 and PBCN_005499, were found to be capable of inducing cell death associated with H2O2 accumulation and electrolyte leakage in Nicotiana benthamiana. Moreover, PBCN_002550 could also induce cell death in Chinese cabbage. In addition, 24 of the remaining 31 tested secretory proteins could suppress mouse Bcl-2-associated X protein-induced cell death, and 28 proteins could suppress PBCN_002550-induced cell death.

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