Alternative Applications Using Cell-Free Protein Expression: #2

As noted in a previous posting the primary use of cell free expression as been the characterization of protein interaction. Due to the convenience of expressing functional protein in a few hours, cell-free expression is also a viable alternative to cell-based expression for other applications. Recent examples include:

Chloroplast import assay

Tanz, S. et al. (2009) Loss of the transit peptide and an increase in gene expression of an ancestral chloroplastic carbonic anhydrase were instrumental in the evolution of the cytosolic C4 carbonic anhydrase in Flaveria. Plant Physiology, 150, 1515–29

Carbonic anhydrase catalyzes the reversible hydration of CO2 and is involved in both C3 and C4 photosynthesis, however its role and intercellular and intracellular locations differ between C3 and C4 plants. Three different cDNA encoding distinct β-carbonic anhydrases were isolated from leaves of the C3 plant Flaveria pringle. To determine if either of the proteins encoded by the cDNA clones are targeted for chloroplasts, [35S]Met labeled proteins were expressed using cell free expression and subjected to chloroplast protein import analysis.

Translational regulation

Carra, S et al. (2009) HspB8 participates in protein quality control by a non-chaperone-like mechanism that requires eIF2{alpha} phosphorylation. J. Biol. Chem. 284, 5523–32.

The HspB8-Bag 3 protein complex suppresses mutated Huntington aggregation via autophagy and may involve translational arrest via eIF2 phosphorylation. To evaluate this theory, cell free expression was used to express a clone containing the Huntington exon 1 fragment in the presence of different amounts of recombinant HspB8 protein. To determine if HspB8 induces elF2(αlpha) phosphorylation, recombinant HspB8 was added (in addition to controls) and the extent of phosphor-elF2 levels were determined by Western blotting.

In vitro Compartmentalization

Chen, Y. et al. (2008) Cell-free selection of RNA-binding proteins using in vitro compartmentalization. Nuc. Acids. Res. 36, e128.

Using In vitro compartmentalization individual genes can be expressed in minute droplets of emulsified cell-free expression system. Formation of the droplet results in co-compartmentalization of the gene and its product, making it possible to select larger gene libraries compared to phage display. This reference illustrates a modification of this technique developed to link genotype and phenotype by fusing the target RNA binding protein to zinc finger proteins that bind to a cognate DNA sequence inserted upstream of the promoter.

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Gary Kobs

Strategic Marketing Manager at Promega Corporation
Gary earned his B.S. in Bacteriology, UW-Madison in 1982. From 1982–1986 he served as Research Tech at UW-Madison. From 1986 to the present Gary has been with Promega Corporation serving in many capacities including as the very first editor of Promega Notes. He was also Manager Tech Services and Training, Product Manager Restriction/Modifying Enzymes, Product Manager Protein Analysis, and is now Marketing Manager Protein Analysis.

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