As you can see the protein of interest in supernatant only by western blot but not through coomasie indicates that the expression levels are too low either to be seen on coomasie stain or through Ni-affinity chromatography. Please try to optimise the conditions for expression and I guess that your cloning is ok.

Thank you Dhatchana,

I tried to check the expression at 1, 2, 3 and 4 day post induction. What conditions should I try to change for expression of my protein?

Was the WB done with an anti-His antibody or something else?

If the second, one possible reason would be clipping of the C-terminal His tag by proteases in the supernatant. Thus, you'd see the protein by WB but wouldn't be able to pull it down via IMAC. One possible way out is to try somehting else, e.g. ion exchange.

Another possibility would be that you failed to properly condition your sample for IMAC. Nickel affinity columns depend on the protonation status of His residues, hence, they work only at neutral-basic pH. However, Pichia is usually grown in relatively acid (pH5-6) media. You need to change the pH of your culture supernatant before you can expect IMAC to work.

Hope it helps

Thank you Alejandro,

I am doing Western blot with anti-His antibody.

We have adjusted the pH of supernatant to 7.9 before IMAC work.

Then perhaps the His tag is masked somehow by the structure of the native protein (but is exposed by partial denaturation in SDS-PAGE/WB). Never had such a case myself, but I have seen variants of the same C-terminally His-tagged protein exhibiting larger than 10-fold differences in their Kd for an anti-His mAb (and something similar is reported in the literature, see e.g. http://www.ncbi.nlm.nih.gov/pubmed/17081490). Don't see why the same thing cannot happen with coordinated metal groups in IMAC gels.

Hi Alejandro,

Do you always see the protein of your interest in supernatant (if secreted) by Coomassie Brilliant Blue staining? I am suspecting my protein to be in lower expression range. I can only able to see the protein in WB (5 uL supernatant). What is your experience?

Oh yes, unfortunately your case is more common than the sales reps at Invitrogen would like to admit. The rate-limiting step in Pichia is often passage through the secretory pathway, so as you can imagine the solutions are not easy (trying different signal peptides, optimizing cleavage at KEX2 sites if using alfa-factor pre-propeptides, looking for potential ER retention signals, providing folding partners in trans, etc). If you just need the protein for an experiment and this is not going to be a long-term project, I would try to concentrate the protein by whatever means at your disposal (Even ultrafiltration, if you have adequate cartridges/hardware for them) from large volumes of culture medium, and purify from there.

Monkeying with culture parameters is another possibility, but you'd be lucky if you get more than a 10-fold increase in yield, and it's best done in a fermentor.

Sorry for adding my ask here: We have positive overexpresion in flasks but we cannot see a protein in supernatant after batch in bioreactor. Everything looks that it is going ok (level o biomas, using of H+/OH- reagents etc...) but yeld of proteins are even smaller in bioreactor then ths which we have in flasks.... Anyone "figt" with binding overexpressed protein to surface of the yeasts or sthg like that after overexpression? Thanks for all suggestions.

Hi Nirajkumar Makadiya, well I am here to ask you a question...actually I am also tryng to ecpress one 36 kDa protein in P.pastoris X-33. I tried to concentrate the supernatant with acetone but SD-PAGE is not clear..A smear appears rather than clearly resolved bands...could you suggest some alternative to concentrate the supernatant....besides, I tried to blot it biut it could not be detected with anti-myc..Kindly help

Hi Sonalika,

I understand that your protein of interest (POI) is secreted in the media. Did you confirmed the junction of secretion signal and your gene of interest for the reading frame? In how much volume do you express the induction? Can you attach the image of your SDS-PAGE?

Hope to hear from you soon.

Sir,

I have cloned my gene in pPICzalphaA. I transformed in JM109. i have sequenced the plasmid for my insert. thereafter i retransformed the plasmid in P.Pastoris. i screen 8 Zeo transformant colonies for insert using 5"AOX and 3'AOX primer by colony PCR. I induce the colonie positive by insert-screening PCR in 50 ml culture. i made collections at 0, 24, 48, 72 and 120 h. I have concentrated the supernatant by TCA method and reconstituted in 1/20 volume of STB (Laemmeli buffer). For. e.g. I have concentrated 4 ml of supernatant collected at 120 h and reconstituted in 200 microlieter of LAemmeli buffer. I will send the SDS PAge image tommmorow

Hi Pawel Filipkowski,

Invitrogen says in their manual that the acidic proteases (particularly pep4) can cleave the protein of interest in secreted fraction. We have experienced this situation. Invitrogen provide different strains of the yeast to overcome this problem. They have pep4 deficient strain. But, in our case it didn't made any difference. We have used protease inhibitor aprotinin at 1 ug/mL concentration. Which didn't stopped the cleavage but it appears that we got some full length protein too. Hope this will help you.

Are you able to detect your full length protein of interest (POI) by western blot? Do you know the molecular weight of your POI?

Hope to hear from you soon.

Hi Sonalika,

Do you perform the Western blot for detecting the expression of your POI?

Dear Nirajkumar Makadiya ,

We tryied for example lowering the temperature after induction with methanol....

Now we are at stage: testing some detergents added into medium.

In my opinion our protein is on the surface or it is digested with proteases (as You suggested). Everything during the broth going fine: we've got enough biomas, still some base is used durning the overexpression/induction etc...(if we loose control of pO2 very fast acid is taken etc..., if we try to gave them some other surce of carbon they consume it very fast etc...)- i mean pichia and overexpression works.... but to the hell ;-) where is our protein???

Our protein is something about 30kDa and yes it is present with Western.

What is interesting that we have higher lever of overexpression in shaked flasks then in bioreactor... and it doesnt depend (up tp now) on time, temperature, level of methanol, level of biomas....

Btw someone ask about a method for concentrating the supernatant/medium - i recommend liophylisation.

Hi Nirajkumar Makadiya Sir,

i have done western blotting with anti-myc as well as anti-His but no band. But in the gel one band can be seen at 36kDa position. however in negative control there is no band visible below 42 kDa. may be its not that concentrated. Besides, I was using BMGY medium for inoculation and BMMY medium for induction. Is it a possibility that my protein could be in pellet or membrane bound pr is degraded by proteases. I dont know wats going wrong even after concentrating to1/50 of original volume, bands are not that clear.

Dear Pawel,

Proteins with a high pI can bind to the negatively charged pichia cell surface. I have washed cell pellets with 0.5 M NaCl and increased yield, but not by much (~5%) and my protein (lysozyme) had pI of 11. If most of your protein is binding to cell membrane you should see it in SDS gel/ WB of cell pellet.

It could be that something about your bioreactor protocol is causing significant cell lysis leading to proteolytic degradation of your protein. Invitrogen protocols use high salt media which can stress cells especially when switching to methanol (see these papers:

http://www.ncbi.nlm.nih.gov/pubmed/16447173

http://www.ncbi.nlm.nih.gov/pubmed/11722185

http://www.ncbi.nlm.nih.gov/pubmed/17137292)

I found low salt media based on the Brady protocol (2nd ref) to improve yield and simplify purification.

Thanks Fergal,

Our protein has pI something about 4-5. Actually that was our firs idea: (in shaked flasks pH i changable in bioreator we using constans pH 6) moeby our protein just agregating/precipitating in so close to their pI medium broth with pH 6. But when we stop to control pH after induction with methanol, or just to change it durning all period of bioreactor batch nothing change - still level of observed protein was lower then that observed in shaked flasks...

Therefore we start to thinking about binding to Pichia cell or egzo-proteases...

Now i know that it (our protein of interests) is not visible on SDS-PAGE of cell pellet :-(

This what You say about negatively charged Pichia surface (I almost forget about it!-thaks one more time) actually is a vote to proteses way to....

p.s.we are not using high salt at second stage....at rising biomass in matter of fact there is not a lot of salt too. I mean not here with Pichia X33. (in other application like this with Pichia pink - yes)

Hi Pawel and all,

I have related questions for you.

1.) How much protein yield do you get per litre of BMGY used? I am afraid to tell that we are getting around 150-200 micrograms after inducing the pellet in 200 mL of BMMY. I tried three proteins and all of these are around the same range.

2.) Do you use baffled flasks during growth and induction? I am using the normal glass conical flasks. Do you think this might be an issue for low yield?

Do you have any idea/tips/tricks to increase the protein yield in small scale induction and purifications of secreted proteins?

Hi Sonalika,

I am using the protocols from Intivtogen manual MAN0000717

"Express recombinant PichiaPink™ strains, continued" P46

"Scale-up expression" P53

Did you performed the expression of more than one clones? Did you tried to express the other proteins in Pichia?

I also work with Pichia pastoris and I have a few questions for you. Is your protein a membrane protein.

I am also working on Pichia pastoris and my desired protein will be secretory. I wanted to know what will be the ideal process for protein purification from supernatant of Pichia?

And also if elution will be done with 500mM Imidazole does it have any effect on protein structure and bioactivity??