Hey,
in my old lab we developed a system to express two proteins from one
plasmid using the before mentioned GAL1-10 Promoter. Giving the use of
different plasmids with different selection markers, coexpression of
multiple proteins is possible in yeast. The system was used in a study
published in Structure last year and you could contact my old lab to
request plasmids. We used a TRP1 and a LEU2 plasmid and coexpressed
three proteins in this study, but it is easily extendable to at least 4
proteins.
You can find the paper here:
http://www.ncbi.nlm.nih.gov/pubmed/23954503
To receive plasmids from the study, please contact Ed Hurt, the
corresponding author at the BZH in Heidelberg/Germany.
I stumbled across a similar system recently, which is also based on the
GAL promoter, but don't find the reference now...
Good luck,
Karsten
Am 13.08.2014 15:31, schrieb Chris Putnam:
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.
--
Karsten Thierbach, Dr. rer. nat.
California Institute of Technology
Division of Chemistry & Chemical Engineering
Hoelz laboratory
1200 E. California Blvd., M/C 147-75
Pasadena, CA 91125, U.S.A.
