Expression cloning and radiotracer uptakes in Xenopus laevis oocytes.
This protocol describes the method of expression cloning of heterologous proteins using Xenopus laevis oocytes and the functional characterization of membrane proteins using radiotracer assays. It can be used to isolate proteins for which sequence data is unavailable and to characterize the functions of proteins. A cDNA library is generated, that is, translated into proteins in Xenopus oocytes, and the function of these proteins is assessed by a radiotracer assay. Their large size, high degree of expression and ease of handling makes Xenopus oocytes an optimal tool for the expression and characterization of protein function when compared with traditional expression systems, such as Escherichia coli, yeast or eukaryotic cell lines. The expected results of this technique include the following: functional identification of novel proteins; molecular (kinetic) characterization of protein function; and determination of functionally relevant residues and domains of membrane proteins, including transporters, ion channels and receptors. The identification of novel proteins can take several months, whereas functional characterization in Xenopus oocytes can take 1 week.
Markovich
Daniel
D
School of Biomedical Sciences, University of Queensland, St Lucia 4072, Queensland, Australia. d.markovich@uq.edu.au
D000818:Animals; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005786:Gene Expression Regulation / Q000502:physiology*; D007553:Isotope Labeling; D009865:Oocytes / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis
D018076:DNA, Complementary; D011994:Recombinant Proteins