I think after extraction you should maintain an optimum temperature not to degrade your protein of interest. It happens with highly sensitive protein. Aina Azazi

Dear Dr.

I send you the procedure to clone, express and Recombinant Protein in Pichia pastoris that was performed in LIP-MB laboratory at INSAT and described and published in:

I have already sent this protocol in Research Gate site

1) Over-expression of recombinant camel hepcidin cDNA in Pichia pastoris. Purification and characterization of the polyHis-tagged peptide HepcD-His

Mohamed Boumaiza, Haifa Chahed, Aymen Ezzine, Maryse Jaouan, Alessandra Gianoncelli, Giovanna Longhi, Paolo Arosio, Marie-Agnès Sari and M. Nejib Marzouki (2016)

Journal of Molecular Recognition DOI 10.1002/jmr.2561

2) Heterologous expression and biochemical characterization of a novel thermostable Sclerotinia sclerotiorum GH45 endoglucanase in Pichia pastoris.

Haifa Chahed, Mohamed Boumaiza, Aymen Ezzine and M. Nejib Marzouki (2017) International Journal of Biological Macromolecules DOI: 10.1016/j.ijbiomac.2017.08.062

Transformation and selection of P pastoris clones

Transformation of P pastoris X‐33 strain was performed by electroporation: Briefly, to prepare Pichia cells for electroporation, a single yeast colony was inoculated in 5 mL of YPD medium (1% yeast extract, 2% peptone, and 2% dextrose) and incubated overnight to saturation at 30°C. The night before transformation, a 2‐L sterile flask containing 500 mL YPD medium was inoculated with an appropriate amount of the saturated culture and grown overnight with vigorous shaking at 30°C to 1.108 cells/mL Optical Density (OD ~ 1.3‐1.5). Culture was harvested by centrifugation at 4000g (5000 rpm) at 4°C and resuspended vigorously in 80 mL sterile H2O. The final volume of resuspended yeast should be 1.0 to 1.5 mL, and the final OD should be approximately 200.

The yeast cells were diluted to 500 mL with water. Cells were washed and concentrated 3 times by centrifugation at 6000g: the first time with 250 mL ice‐cold water, the second with 20 to 30 mL ice‐cold 1M sorbitol, and the last time with 0.5 mL ice‐cold 1M sorbitol.

Transformation of competent cells was performed using the BRL Cell‐Porator. Parameters used were 400 V/cm, 10 μF, and low resistance.

In general, transformation is performed using 50 ml of competent cells with 3 yg of linearized recombinant plasmid DNA.

The expression cassette pPICZ A-Recombinant gene was linearized using the endonuclease SacI to target its integration into the AOX1 locus of P. pastoris strain X-33.

After transformation the selection of recombinant P pastoris was achieved on YPD agar plates containing 100μg/mL Zeocin (Invitrogen) during 3 days of incubation culture at 30°C.

The integration of the recombinant plasmid into the Pichia genome was verified by PCR analysis using the recombinant Pichia genomic DNA.

First, the yeast genomic DNA extraction was extracted using the classic phenol/chloroform method, then it served as matrix for the polymerase chain reaction. This latter was carried out using the 5_Forward-gene primer and the 3_Reverse AOX1 primer in order to identify the integration of the target gene at the AOX1 locus in the P pastoris genome.

The amplified PCR product was visualized on a 1% agarose gel stained with Ethidium bromide.

DNA from of the recombinant Pichia clones obtained on YPD-Agar plates containing 100 yg/ml Zeocin was used as positive test.

DNA from A P.pastoris colony transformed with the empty pPICZA vector was used as a negative control. Also a DNA from P pastoris none transformed was used as a second negative control

Expression and purification of the recombinant protein:

Positive PCR P. pastoris transformants were grown in BMGY medium (buffered glycerol-complex medium (1% yeast extract, 2% pep-tone, 0.1 M potassium phosphate buffer pH 6.0, 1.34% YNB,4 × 10−5% biotin, and 1% glycerol) at 30◦C, with vigorous shaking(250-300 rpm), until culture reaches OD600= 2-6 (approximately16-18 h).

The BMGY pre-culture was then used to inoculate 50 ml BMGY at 30◦C with vigorous shaking (250 rpm) until culture reaches logarithmic growth phase OD600= 2-6. After centrifugation at 3000g for 5 min, cell pellet was re-suspended in BMMY (1% methanol instead of glycerol), until OD600= 1, to induce expression of the gene of interest. After 4 days at 28◦C, using a 500 ml flask, with daily addition of methanol (1% v/v), 1 ml of the supernatant was collected, every 24 h, to analyze total protein expression level and activity.

My propositions to optimize and verify protein expression:

1) You may also induce AOX promoter and gene expression by varying Methanol inductor at 1%, 1.5% and 2% v/v.

2) Add protease inhibitor as PMSF in the supernatant at 0.2 to 1mM to ovoid a possible proteolysis

3) In the case that your recombinant protein is glycosylated in Pichia, you may obtain a highest and different MW than the expected one

4) Instead of SDS PAGE, You could test the native activity (Enzymatic activity for example) of your recombinant protein in the supernatant.

5) Final, you may use immunological approach to detect your recombinant protein: Elisa or Western blot Analysis.

Good Work and success.

You need to do three things:

1) check mRNA to see if it is transcribed. If not, fix.

2) check whole cell lysates to see if translated, but not secreted. If not, fix.

and finally,

3) secreted proteins, especially effector proteins, are extremely difficult to obtain from culture supernatants due to their small size and extreme hydrophilic nature. For that, you need a better protocol and so I give you this:

Article Simple and Reliable Method To Precipitate Proteins from Bact...

I agree with Randolph, you should check if your protein is produced but stuck in the cell. Use a specific antibody on a cell crude extract (ideally TCA) to detect it. I had a similar problem and could not see my protein in the medium because it was all intracellular.

If this is the case, Randolph suggests to fix this. I don't know how to do that (in my case I had to change expression system) but if you manage to do it let me know ;-)

Marco Geymonat depending on what is being expressed, it could be as simple as a missing/incomplete secretion signal sequence or the system's expression levels are too low to support normal secretion.

Dear all, thank you for all the suggestions.

I tried to optimize the protein expression by varying methanol inductor at 1%, 2% and 3% v/v as suggested by Prof Mohamed Nejib Marzouki . Unfortunately, my protein still could not be secreted and detected by SDS-PAGE. I did check the pellet in case my protein expressed as insoluble protein (inclusion body) by protein extraction but still negative results.

For your information, the proteins size that I tried to express are 42 kda, 31 kda, 26 kda, 19 kda and 15 kda (they were derived from the same structure ectodomain). I successfully expressed the 42kda protein. Unexpectedly, the other 4 proteins were failed to expressed using the same protocol/procedure. Sir Marco Geymonat

I also did analyze 5 recombinants for the correctly sized PCR product to rule out the possibility that colonies that i picked to express was problematic.

Thank you very much for the protocol Sir Randolph B Caldwell