Alternative Applications for Cell-Free Expression #3

Protein location: outer mitochondrial membrane (Yeast in vitro import assay)

Curado, S. (2010) Dis.Mod. Mech. 3, 486-95. PubMed ID 20483998.
Chemically mutagenized zebra fish were assayed for liver defects in their F3 progeny.This screen led to the identification of mutant called oliver. Oliver mutants have an o-shaped liver of a much deprived size due to the depletion of most of the hepatocytes. This mutation maps to the Tomm22gene which encodes a translocase of the outer membrane and thought to play an important role in protein import into mitochondria. Various Tomm22 mutants were expressed and used in a yeast in vitro import systemto determine if correct inserted into the yeast outer mitochondrial membrane.

Protein modification: hydroxylation

Serchov, T. (2010) J. Biol. Chem. 285, 21223-232. PubMed ID 20418372 .

Proline hydroxylation is also a vital component of hypoxia via hyposxia inducible factors. The cellular response to hypoxia involves the induction of the hypoxia-inducible factor considered to be the major transcription factor involved in gene regulation of hypoxia. This factor is hydroxylated by prolyl-hydroxase dolman proteins (PHDs). To investigate if a newly identified component of the hypoxia pathway (Elk3) is also hydroxylated, proteins were expressed +/- PHDs cofactors and protein mobility was measured via gel analysis.

Gene Experession: Programmed Ribosomal Frameshift

Kobayashi, Y. (2010) J. Biol. Chem. 285, 19776-784. PubMed ID 20427288.

Programmed -1 ribosomal frameshifting (PRF) is a distinctive mode of gene expression utilized by some viruses (HIV-1 for example). Recently a genome-wide screen demonstrated that down regulation of eukaryotic release factor (eRF1) inhibited HIV-1 replication. In order to characterize the dose dependent effect of eRF1, increasing amounts were expressed in the presence of dual luciferase reporter vectors harboring a HIV-1 PRF signal

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