Expression Cloning in the Test Tube

摘要

Traditional biochemical techniques such as protein purification are powerful tools for identifying proteins on the basis of their function. Yet, isolating the genes that encode these proteins is often cumbersome, requiring multiple steps of purification, peptide sequencing, and subsequent isolation of complementary DNA (cDNA). An alternative approach to functional gene identification, which perhaps could be called "reverse" biochemistry, is expression cloning, in which cDNAs are first translated in prokaryotic or eukaryotic cells and then assayed for a specific biologic activity. This approach circumvents the requirement for protein purification, yet it retains the use of a functional assay as the basis of cDNA identification. Cell-based expression cloning strategies, which use mammalian cell lines or Xenopus laevis oocytes to express encoded proteins, have identified genes encoding membrane receptors, transmembrane channels, and secreted growth factors, but they have not been widely used to identify intracellular activities. Here we discuss a new cell-free expression cloning approach that substantially expands the range of biochemical assays that can be used to identify a cDNA on the basis of its function.