On 8/13/14 2:29 PM, Theresa Hsu wrote:
Dear all
One of my human membrane proteins have been described to interact with
additional subunits for its activity. To obtain functional form in yeast
(Saccharomyces), I can think of two approach of either cloning all the subunits
under one promoter or reconstitute in vitro.
For the first option, what is the length of base pairs between the stop codon
of one gene and the start of Kozak sequence for the next one? Is there any
preference for the order so that only one subunit is His tagged?
Second option will need multiple purification steps and some trials with
protein ratios. Is this better?
Natively, Saccharomyces does not have operons. And I am unaware of
operon-like constructs (single promoter with multiple genes oriented in
the same direction) working in Saccharomyces. (The GAL1-10 divergent
promoter is probably the closest analog, but this involves transcription
of the GAL1 and GAL10 genes that are encoded on opposite strands in
opposite orientations.)
For multiprotein complexes, one strategy for in vivo complex assembly is
to encode each protein (with its own promoter) on separate plasmid (with
distinct selectable markers) and transform them all into the same
strain. This avoids the problems of in vitro reconstitution of the
complex as well as the problem of subunits that cannot be stably
expressed alone.
Chris.
