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.
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