I am trying to express a 63kDa protein in Pichia pastoris intracellularly. I have confirmed gene insert by doing colony PCR, did RT-PCR from the culture pellet after 24 hours of induction. Got bands corresponding to the expected size, on agarose gel, controls being valid. (Negative control-no Reverse transcriptase, Positive control- House keeping gene primers). But, when I check on western blot, there is no protein band! All I am getting are some non specific bands which could be seen in the control lane as well. (control- empty vector transformed and induced). I have used 0.5% Methanol induction, strain being Mut+.
I have tried different induction OD's, Methanol concentrations - 0.5% and 1%, temperature- 290 C and 200 C.I have tried induction for up-to 7 days. But no luck!
Please suggest me some ways to get the protein!
You may need to look in to the sequences of the clone, make sure that there are no mutations and stop codons.
The other possibility is the protein degradation during cell lysis OR incomplete cell lysis.
May be you need to try with efficient lysis buffers like Yesat buster!
Best
Can you provide more details i.e. the identity of your target protein? Many things can potentially go wrong, but the devil is always in the details, so more information would be needed.
Cheers Markus
Markus Sack It's a plant enzyme, which is normally secreted. I have optimized the sequence for Pichia expression. It's monomeric, pI is 6.1, literature says it's sensitive to oxidation.
okay, still only a few bits and pieces of information here... But since you mentioned that it is a secreted plant enzyme the obvious question is why do you want to express it intracellularly? In particular since pichia is good host for protein secretion? When you say intracellularly does that mean in the cytosol? What about the signal peptide? What about PTMs such as disulfide bridges and N-glycosylation? If you provide the identity of your protein and details of your expression construct we can quickly check these issues and identify potential problems.
Markus Sack Yes, it's in the cytosol. first 28 aminoacids code for a signal peptide. There is one disulphide bridge and several N-linked glycosylations. It's an oxidoreductase enzyme. It's cloned into pPICZa vector(no secretory signal) within the restriction sites SfiI and KpnI. Pichia pastoris X-33 is the strain used.
Since I'm new to this field, I may not be giving right information to your questions. Apologies for that. Please correct if I go wrong in giving the details.
Since I didn't see any protein bands on SDS gel, I went ahead with NI-NTA purification. I am getting a 37kDa protein in the elute fraction, which is almost pure. expected band size is 63kDa.
lane 3 &4-washes with 25mM imidazole,
5,6,7,8 and 9-elute fractions with 250 mM Imidazole.
The pPICZalpha plasmid provides an N-terminal signal peptide from the alpha-factor. Please check your construct again, it is possible that your target protein is secreted (unless you use another signal/tag to retain it inside the cell).
If you post gels etc. please label the lanes so that people can understand what was loaded.
Regarding your 37 kDa protein please read:
Article The ubiquitous 38 kDa contaminant in glutamic acid decarboxy...
Cheers Markus
Markus Sack It's not pPICZalpha, it's pPICZ-A plasmid which is used specifically for intracellular expression. Sure, I will label the gels.
From the article, it's pretty clear that it's not my protein of interest. Thank you so much for sharing the article. I will try to load the culture media and see if it's getting secreted by any chance!
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