TNT® Eukarayotic Cell-free Protein Expression Systems - Promega

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TNT® Eukarayotic Cell-free Protein Expression Systems - Promega

CELL-FREE PROTEIN EXPRESSION

Plant-Related Applications

TnT® in vitro Transcription/Translation Systems

References #3

Cell-free protein expression can be utilized for the analysis of: protein:protein interactions, protein:nucleic acid

interactions, analysis of post-translational modifications and many other applications. The majority of these

references are based on the characterization of mammalian proteins. However there are several references

using TnT®-based systems (either rabbit reticulocyte lysate or wheat germ-based) for the analysis of proteins

from plants. Examples include:

1. Zhang, Z. et al. (2011) The Plant Cell 23, 273-88.

Using the Beet serve curly top virus C2 protein as bait

in a yeast–two hybrid screen, a C2-interacting protein

S-adenosyl-methionine decarboxylase1 (SAMDC1) was

identified from an Arabidopsis thaliana cDNA library. In

conjunction with other techniques the interaction was

confirmed and characterized using TnT in conjunction

with GST pull-down assays.

2. Gao, M-J. et al. (2009) The Plant Cell 21, 54–71.

The seed maturation program is repressed during germination

and seedling development so that embryonic

genes are not expressed in vegetative organs. Noted in

this reference was evidence that ASIL1 functions as a negative

regulator of a large subset of Arabidopsis embryonic

and seed maturation genes in seedlings. Electrophoretic

mobility shift assays using protein expressed in the TnT

system and labeled DNA oligos corresponding the various

regions of the 2S3 promoter were one of the techniques

used to characterize the specific binding to this

promoter region.

3. Huizinga, D. et al. (2010) Molecular Plant 3, 143–55.

In the characterization of the Arabidopsis farnesycyteine

lysase a protein larger than predicted molecular mass was

observed via gel analysis. In order to determine if this was

due to N-glycosylation of the protein, the FC lyase protein

was synthesized using the TnT system in the presence or

absence of canine pancreatic microsomal membranes.

4. Tanz, S. et al. (2009) Plant Physiology 150, 1515-29.

Carbonic anhydrase catalyzes the reversible hydration of

CO2 and is involved in both C(3) and C(4) photosynthesis,

however its role and intercellular and intracellular locations

differ between C(3) and C(4) plants. Three different

cDNA encoding distinct β-carbonic anhydrases were

isolated from leaves of the C(3) plant Flaveria pringlei. To

determine if either of the proteins encoded by the cDNA

clones are targeted for chloroplasts. Labeled proteins

were expressed using the TnT system and subjected to

chloroplast protein import analysis.

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