Hello All,
I am writing this post as I don't have any prior knowledge in Protein Biochemistry and Structural Biology. I am trying to express a sub-cellular membrane protein in Pichia pastoris (strain SMD1163 his4). I have two constructs which have N-terminal and C-terminal EGFP tagged along with the protein. I have transformed the Pichia with the two constructs (linearized) and after induction with methanol, I have failed to express the protein in small volume/large volume cultures. I have following queries and any leads would be appreciated:
1. What might have gone wrong that the protein is not getting expressed?
2. Would it help to try a different strain of Pichia Pastoris (it seems Pichia is a very robust yeast which produces huge quantities of recombinant protein)
3. Can anyone have a look at the plasmid map of the constructs and suggest if I am missing something in it which is preventing its expression?
Any other ideas or ways that one can pursue to get the protein expressed would be highly appreciated.
Thank you
Dear Tom Masi,
1. Yes I have verified the protein coding region of the construct and they are in frame.
2. I had only one integration site (single site restriction digestion) in the plasmid and also I had increasing antibiotic concentration ranging from 0.1mg/ml to 1.5mg/ml.
3. I induce the culture with methanol for approx 48 hours with a replenishment of 0.5% methanol after first 24 hours.
Perhaps I need to clarify what I mean by multiple integration sites. When the linearized plasmid is transformed into the cells, it will integrate at the AOX locus in the genome but you can get multiple integrations of the plasmid (more than 1 copy) at this site. Since the antibiotic marker is also integrated, it will confer resistance to higher and higher concentrations of antibiotic, therefore you can select for colonies which have many integrations. Typically the more integrations into the genome the higher the expression level of your protein of interest, but this is not always true. You might want to try selecting your colonies on 5 or 10mg/ml antibiotic to screen for potentially high expressing clones. Don't waste your time with anything less than 5mg/ml. You may want to extend your expression time to 72 or 96 hours in methanol. From my experience, if you don't see anything at 96 hours, you probably will not see any. Another trick is to add 1% methanol and/or add casamino acids to the induction media which may help with expression.
In regards to trying a different strain of Pichia, SMD1163 is a double protease deficient strain and is more sickly compared to X-33 and GS115 (his4) which are both wild-type. You could try either one of those but do not do both.
There are many reasons for membrane proteins not to be expressed by pichia. First, the protein needs to be properly folded by the expression system, but pichia is unable to fold your POI. Second, the protein was degraded by pichia. You can do the codon optimization for your GOI. You can add a GFP tag to the c-terminus to see if there is fluorescence.
Hai Li Thanks for your suggestion. I have tried to express the construct with EGFP in the C-terminus as well as N-terminus but failed.
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