Hello there,
I am writing to ask for insight as to how best to express my heterologous gene of interest extracellularly from Saccharomyces cerevisiae. The recombinant strain is then required to express the gene of interest in non-selective medium, without impairing said strain's ethanol production.
In fulfilling the requirements above, I have been made to understand that I need a plasmid vector that contains (a) a high expression constitutive promoter for expression of my gene, (b) positive marker allowing for growth only from transformants and (c) contains signal sequence coding for secretion of expressed protein
Additionally, on point (b), I understand that the best scenario would be the use of growth markers, of which only positive transformants would be able to grow under said non-selective conditions. This would therefore necessitate use of Saccharomyces strains lacking in the growth factor, of which I currently have no knowledge of, and no access to as yet.
The second option would be the use of native Saccharomyces strains, given the ability to grow in the presence of antibiotics such as hygromycin and G418. However, as stated, the above option remains best.
As such, I'd like to ask for suggestions on suitable plasmids and corresponding yeast strains for use, along with how to obtain them. Additional suggestions for my work are also welcomed. Thank you!
I used pPICZ-Alpha A expression vector and P.pastoris X-33 strain for my studies.
It is zeocin resistant, alpha signal helps is excretion of expressed protein in supernatant and also has his tag for purification. Hope it helps.
i used pPICZ-Alpha A vector and P.pastoris X-33 strain for my work.
http://tools.thermofisher.com/content/sfs/manuals/ppiczalpha_man.pdf
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