Functional reconstitution of human telomerase expressed in Saccharomyces cerevisiae.
Telomerase is a ribonucleoprotein enzyme complex that adds DNA repeats at the ends of chromosomes. In an effort to establish an in vivo heterologous expression system for active human telomerase, we expressed human telomerase reverse transcriptase (hTERT) in Saccharomyces cerevisiae and affinity-purified the protein as a fusion with glutathione S-transferase (GST). Addition of the GST moiety to the N terminus of hTERT did not interfere with telomerase activity when GST-hTERT was expressed in rabbit reticulocyte lysate (RRL) in the presence of the human telomerase RNA (hTR). Active human telomerase was immunoprecipitated from yeast lysates that co-expressed GST-hTERT and hTR. In addition, telomerase activity could be reconstituted in vitro by the addition of hTR to GST-hTERT that was immunoprecipitated from either RRL or S. cerevisiae lysates. The expression and reconstitution of human telomerase activity in yeast will provide powerful biochemical and genetic tools to study the various components required for the assembly and function of this enzyme.
Bachand
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Department of Anatomy, McGill University, Montréal, Québec H3A 2B2, Canada.
Autexier
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C
D001483:Base Sequence; D020134:Catalytic Domain; D002846:Chromatography, Affinity; D017931:DNA Primers; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D019098:Telomerase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D019098:Telomerase