Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

The following protocol describes protein expression using the methyl atrophic yeast opus. The preparation of electro competent yeast cells and transformation of the vector with the gene of interest into PPAs are shown, followed by yeast DNA purification step to check for proper integration of your gene of interest into the yeast genome. At the end, we'll perform the expression of the recombinant protein.

Hi, I'm Maria from the laboratory of Steven Hallam at the Department of Microbiology and Immunology at the University of British Columbia. I'm Marcus also from the Helm Lab. Today we will show you a procedure to express recombinant proteins in the TRO yeast PQ.Past stories.

We use this procedure in our laboratory to study proteins from unceded microorganisms where environmental DNA has been extracted and archived and meta atomic libraries. So let's get started. The expression of recombinant proteins in the methyl atrophic yeast PIAAs Taurus requires CLO your gene of interest in frame in a PPA parent vector.

In our experiment, we use the vector PPIC Z alpha A.This vector contains the AO X one promoter for tightly regulated methanol induced expression of the gene of interest, the alpha factor secretion signal for secretion of the recombinant protein, zaas and resistance gene for selection in both e coli and pic, a C terminal peptide containing acemic epitope and a poly histamine tag for detection and purification of the recombinant protein Prior to transformation. Linearize the vector containing your gene of interest by restriction digest. We use the enzyme PME one, but other restriction sites are possible as long as your insert does not contain that restriction site.

To obtain sufficient linearized vector DNA, set up three to four 50 microliter restriction, digest reactions in separate tubes after the digest, combine all the solutions and concentrate your construct. Using an AmCon centrifugal filter unit and a MicroCon centrifugal filter unit, you will need five to 20 micrograms of linearized DNA in five to 10 microliters of sterile water for transformation into PPA dous. To prepare electro competent PPA dous streak out cells on A-Y-P-D-S plate without SN four days prior to the intended transformation, grow at 30 degrees Celsius for one to two days or until single colonies form two days before transformation.

Grow five milliliters of the PPA or strain in YPD medium in a 50 milliliter Falcon tube at 30 degrees Celsius overnight. Place the Falcon tube in a flask to fix it in the shaker one day before transformation. Use 0.25 milliliters of the overnight culture to inoculate 500 milliliters of fresh YPD medium in a two liter flask.

Grow overnight again in a shaker at 30 degrees Celsius to an OD 600 of 1.3 to 1.5. On the day of the transformation, have ice, cold, sterile water and one molar sorbitol on hand. Centrifuge the cells at 1500 Gs for five minutes at four degrees Celsius.

After centrifugation, we suspend the pellet with 500 milliliters of ice, cold, sterile water. Depending on the capacity of the centrifuge tubes, you may have to split the volume into two separate tubes or do multiple runs. Centrifuge the cells again and resuspend the pellet with 250 milliliters of ice, cold, sterile water.

Depending on the capacity of the centrifuge tubes, you can combine the separate cell pellets in one tube. When Resus suspending centrifuge, the cells again this time resuspend the pellet. In 20 milliliters of ice cold one molar sorbitol transfer the resuspended cells to a 50 milliliter falcon tube and centrifuge.

After a final centrifugation, resus suspend the pellet in one milliliter of ice cold, one molar sorbitol to a final volume of about 1.5 milliliters. The resuspended cells in micro centrifuge tubes store cells on ice or at four degrees Celsius until they're used for transformation. Prior to transformation of the concentrated linearized construct into PPA stor cells, prepare the following reagents and devices.

Fill a micro centrifuge tube with one milliliter of one molar sorbitol and place it on ice. Place a 0.2 centimeter electroporation. Vet on ice.

Label a sterile 15 milliliter glass tube and have a sterile glass past your pipette on hand. Transfer 80 microliters of the previously prepared electro competent cells to the ice cold. Point two centimeter electroporation vete.

Add the plasma DNA solution to the cells and mix by moving the pipette tip from side to side. In the electroporation vete. Incubate the vete with the cells on ice for five minutes.

Next, wipe the outside of the vete with the tissue and pulse the cells according to the parameters for budding yeast as suggested by the manufacturer of your specific electroporation device. A BioRad gene Pulser is used in this demonstration with the following conditions. Charging voltage of 1500 volts, capacitance of 25 micro ferres and resistance of 200 ohms immediately after pulsing.

Add one milliliter of ice cold, one molar sorbitol to the vet. Transfer the vete content to a sterile 15 milliliter tube using a sterile glass past your pipette. Incubate the tube at 30 degrees Celsius without shaking For 1.5 hours.

After 1.5 hours, the electroporated cells are ready for plating. Add five to 10 sterilized glass beads to each of four labeled YPTS plates containing 100 micrograms per milliliter of Zain. Spread 250 microliters of the electroporation.

Mix on each plate and shake the plate horizontally to spread out the cells evenly. Let the plates dry for 15 minutes and then remove the beads from the agro by inverting the plates. Wrap the plates in a black plastic to prevent degradation of the light sensitive Zain and incubate plates upside down for two to three days at 30 degrees Celsius until colonies form after colonies have formed.

Pick 12 colonies. Purify them by streaking the clones on fresh YPDS plates containing 100 micrograms per milliliter of zein. To prepare PPA cells for protein expression, pick a single colony from the purified pichia colonies and inoculate in 25 milliliters of VMGY medium in a sterile 250 milliliter flask grow at 30 degrees Celsius in a shaking incubator until the culture reaches an OD 600 of two to six.

When the cells have reached the appropriate OD 600, prepare a glycerol stock of the cells for storage. Transfer 800 microliters of the cell culture to a two milliliter cryogenic vial and add 200 microliters of sterilized glycerol. Store the glycerol stock at minus 80 degrees Celsius.

In addition, transfer 1.5 milliliters of the cell culture to a micro centrifuge tube. Harvest the cells by centrifuging at 1300 GS for one minute at room temperature. Store the pellet at four degrees Celsius.

These cells will be used for yeast DNA purification to analyze the pichia Irans after PCR with insert specific primers on an groose gel to confirm that the gene of interest has integrated into the pichia genome. Transfer the rest of the 25 milliliter culture to a 50 milliliter Falcon tube and harvest the cells by centrifuging at 3000 Gs for five minutes At room temperature, decant the supernatant and place the tube upside down on a tissue to remove any residual media to wash the cell pellet resus. Suspend it by vortexing or pipetting up and down in 20 milliliters of BMMY medium centrifuge.

The cell suspension again after decanting, the supernatant resuspend the cell pellet by vortexing or pipetting up and down to an OD 600 of 1.0 in BMMY. Medium to induce expression. Transfer the culture to a one liter baffled flask and cover the flask with a 200 milliliter peaker.

We highly recommend the use of baffled flasks as they introduce more oxygen in the culture. Media for efficient protein expression during methanol induction, incubate the flask in the incubator to continue growth at 30 degrees Celsius. It is important that the temperature does not exceed 30 degrees Celsius if the temperature of your incubator fluctuates.

Set the temperature at 28 degrees Celsius. Add sterilized pure methanol to a final concentration of 0.5%methanol every 24 hours to maintain induction at certain time points after the start of the expression, transfer one milliliter of the culture to a 1.5 milliliter micro centrifuge tube centrifuge at 1300 Gs for 2.5 minutes. At room temperature, transfer the supernatant to a separate 1.5 milliliter micro centrifuge tube.

Store the supernatant and cell pellets at minus 80 degrees Celsius until further analysis. The samples from different time points will be analyzed to establish the optimal time period for protein expression after induction. Now we show some representative results of successfully expressing recombinant proteins using PIA Pastis as a host system.

This western blot image shows four expressed proteins after 24 hours of expression. The antibody used for immuno blotting was acemic HRP antibody. The second lane is a negative control and was loaded supernatant from cells that do not express recombinant protein because they were transformed with the parent vector.

We've just shown you how to express recombinant proteins using the eukaryotic host PQ.Plus, When doing this procedure is important to have your construct sequenced to confirm that your gene of interest is clone and framed, and to have all your plates and reagents ready before you start. It is also important that you prepare a sufficient amount of linearized plasmid to obtain a high number of transformers before protein expression by methanol induction. Make sure to wash your cell padded well to remove any remaining traces of glycerol that can inhibit your protein expression.

So that's it. Thanks for watching and good luck with the experiments.