Two of the most frequent applications that use cell-free expression are the characterization of protein:protein interactions and the characterization of protein:nucleic acid interactions. Due to the convenience of expressing functional protein in few hours, cell-free expression is also a viable alternative to cell-based expression for other applications. Recent examples include:
It has been suggested that ERp57 plays a role in the folding of LDL-R protein. To investigate if this is the case, the LDL-R protein was expressed using cell-free expression. To determine if proper folding occurred, semi-intact cells (HT1080 cells +/- ERp57) protein were added to translation mixture. Samples were taken at various time points, and folding was measured by differential protein mobility via gel analysis.
Ahlen, G. et al. (2009) Inter. J. Gast. Hept. 58, 560–69.
HCV effectively establishes persistent infection in human livers. Many HCV proteins seem to interfere with host cell signaling. NS 3/4A complex participates in this process by cleavage of interferon promoter stimulator -1 (IPS-1 which inhibits the cell responses to viral double stranded RNA). To determine the mechanism of cleavage, IPS-1 was expressed using cell-free expression and various mutations of the NS 3/4A complex were added. Cleavage of IPS-1 was measured by gel analysis.
Kim, N-G. et al. (2009) Proc. Natl. Acad. Sci. USA 106, 5165–70.
Only a few proteins have been characterized to be regulated by the Wnt/GSK3 destruction complex. To evaluate if other proteins are regulated via this pathway, Xenopus cDNA pools (50–100 clones) were synthesized using cell-free expression and then incubated with Xenopus egg extract for three hours. Protein bands that exhibited mobility shift or degradation after the reaction were considered putative targets and characterized further.
Inhibition of Translation
Zhang, Y. and Inouye, M. (2009) J. Biol. Chem. 284 6627–38.
The effect on YoeB toxin was evaluated using both prokaryotic and eukaryotic cell-free expression systems. Various amounts of YoeB toxin were added to each of these reactions containing clones expressing individual proteins (MaxG for the prokaryotic system and luciferase for the eukaryotic system). Reactions were incubated for 1.5 hours, and aliquots assayed via gel analysis.
Methylalpinumisoflavone and alpinumisoflavone are compounds known to inhibit hypoxia inducible factor, an important transcription factor related to tumor development. Both cell-based and cell-free expression were used in the characterization of these inhibitors. Potential inhibition of protein expression was examined by using clone containing the luciferase gene that was expressed in cell-free expression in the presence of various concentrations of each compound. Inhibition was observed by reduced luciferase activity.
Latest posts by Gary Kobs (see all)
- Characterizing Compound Binding in Cell-Free Systems - April 17, 2019
- Mutation Analysis Using HaloTag Fusion Proteins - March 11, 2019
- Optimizing Pressure Cycling Sample Preparation for Bottom-Up Proteomics - February 11, 2019