The functional expression of toxic genes: lessons learned from molecular cloning of CCH1, a high-affinity Ca2+...

Some genes cannot be cloned by conventional methods because in most cases the genes or gene products are toxic to Escherichia coli. CCH1 is a high-affinity Ca(2+) channel present in the plasma membrane of Cryptococcus neoformans and other fungi. Like many toxic genes, the molecular cloning of CCH1 has been a major challenge; consequently, direct studies of CCH1 channel activity in heterologous expression systems have been impossible. We have devised a straightforward approach that resulted in the molecular cloning and functional expression of CCH1 by exploiting homologous recombination both in vitro and in vivo. This approach precluded the standard enzyme digestion-mediated ligation reactions and the subsequent isolation of plasmids from E. coli. The shuttle plasmid carrying CCH1-GFP, which was prepared in vitro and propagated in yeast, was successfully expressed in the mammalian cell line HEK293 (human embryonic kidney 293). CCH1 transcripts were detected only in HEK293 cells transfected with the plasmid DNA. Fluorescence microscopy studies revealed the expression of CCH1-GFP fusion protein on the cell surface of HEK293 cells, similar to the localization pattern of a well-characterized plasma membrane-associated K(+) channel. This approach will be particularly useful for genes that encode ion channels and transporters that cannot be cloned by conventional techniques requiring E. coli.