The functional expression of toxic genes: lessons learned from molecular cloning of CCH1, a high-affinity Ca2+ channel.
The functional expression of toxic genes: lessons learned from molecular cloning of CCH1, a high-affinity Ca2+ channel.
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DOI：
10.1016/j.ab.2009.06.039
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发表时间：
2009-10-15
影响因子：
2.9
通讯作者：
Gelli A
中科院分区：
文献类型：
--
作者：
Vu K;Bautos J;Hong MP;Gelli A
Some genes cannot be cloned by conventional methods because in most cases the genes or gene products are toxic to E. coli. CCH1 is a high-affinity Ca2+ 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 and consequently direct studies of CCH1 channel activity in heterologous expression systems have been impossible. We 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 a mammalian cell line (HEK293). 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.
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