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

Abstract

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 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 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 wellcharacterized 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.

Redox regulation of the yeast voltage-gated Ca2+ channel homolog Cch1p by glutathionylation of specific cysteine residues

“…One of the reasons for the relatively slow progress in studies on these channels has been the toxicity and instability of their cDNAs when expressed in E. coli (Clare, 2008). The yeast homolog is also toxic in E. coli, and thus in this work with Cch1p, the different mutants and constructs were created using the more arduous in vivo gap repair strategy which involves homologous recombination in yeast (Iida et al, 2004;Vu et al, 2009). This enabled a detailed alanine scanning of all the 18 exposed cysteine residues followed by functional analysis.…”

“…Owing to the toxicity of CCH1 in Escherichia coli, we used in vivo recombination as already described (Iida et al, 2004;Vu et al, 2009) with some modifications to clone CCH1 and its cysteine mutants in yeast. Briefly, CCH1 was amplified from S. cerevisiae chromosomal DNA using primers that possess a part of the CCH1 non-coding region and vector sequence.…”

“…The other exposed cysteine residues were C690, C696, C798, C1169, C1318, C1581, C1707, C1894, C1915 and C1955 and were also included in the study. Owing to the toxicity of this gene product in E.coli, the cloning was carried out in yeast using the previously defined homologous recombination strategy (Iida et al, 2004;Vu et al, 2009) as described in the Materials and Methods. The WT and mutants were functionally assayed by determining the oxidative stress (H 2 O 2 ) sensitivity.…”

Cch1 and Mid1 Are Functionally Required for Vegetative Growth under Low-Calcium Conditions in the Phytopathogenic Ascomycete Botrytis cinerea

“…Since Cch1 was suggested to interact physically with Mid1, we tried to clone a similar cch1-gfp construct in order to study the subcellular localization of Cch1 in B. cinerea. However, reamplification of the vector in E. coli never resulted in positive clones, as shown for the S. cerevisiae and Cryptococcus neoformans cch1 genes (22,57).…”

Activity of the Calcium Channel Pore Cch1 Is Dependent on a Modulatory Region of the Subunit Mid1 in Cryptococcus neoformans

“…grubii strains (H99 MATa serotype A) and S. cerevisiae strains (W303 MATa, mid1⌬) were recovered from 15% glycerol stocks at Ϫ80°C prior to use in these experiments. The mid1⌬, cch1⌬, and mid1⌬ cch1⌬ mutant C. neoformans strains were constructed as previously described (2)(3)(4)25). Strains were maintained on YPD (1% yeast extract, 2% peptone, and 2% dextrose) medium.…”