Pichia pastoris is a species of yeast which expresses recombinant proteins at high levels. As a eukaryote, the expressed proteins are glycosylated. Proteins are secreted into the culture media, which simplifies the purification process.
The Pichia expression vector, pPICZ, has his-tag and c-myc epitopes at the 3’ end of the multiple cloning region (MCR). These tags allow for detection and purification with commonly used methods. At the 5’ end of the MCR, is an alpha factor which increases expression and can be cleaved out automatically after expression. Only two amino acids will be added to your target protein if you clone into the EcoRI site. During cloning, the pPICZ vector requires zeocin for selection. Methanol is used to activate target gene expression (which is cheaper than IPTG which is commonly used in bacterial expression systems).
Briefly, the first step is vector construction. There are plenty of cloning sites to choose from. There are also three differently framed vectors. The second step is transformation of linearized DNA into Pichia pastoris. The EasySelect Pichia Expression Kit offers three different strains: X33, GS115 and KM71H. I prefer X33 because it has higher transformation efficiency and higher expression levels. If you have an electroporator, you can use it. If not, this kit includes chemical transformation reagents; they called it EasyComp Transformation. (I prefer electroporation, which has much higher efficiency.)
The third step is clone selection. The protocol uses a lot paper to teach you how to determine the Mut phenotype clones by plating them in different plates, then observing their growth rates. This takes 3 to 5 days. I just use PCR with AOX primers and get the result within 4 hours. The fourth step is expression. After your selected clone has grown in YPD media and reached an OD600 of 6.0, centrifuge to pellet the cells. Resuspend the cell pellet in expression media, such as BMMY and incubate at 30ºC overnight. The fifth step is purification. I used HisTrap Columns from GE Healthcare and obtained 90 percent purity with a single purification.
I expressed a 45 kDa glycoprotein that forms an antiparallel homodimer, covalently linked by two disulfide bridges. After purification, I checked its bioactivity with a cell proliferation assay. The expressed protein had full activity in vitro. I also analyzed its binding activity with its receptor in vitro. It seems the his-tag has little negative effect on binding as compared to untagged proteins purified from bacterial and Bacular cells.
This system is easy to manipulate, I have expressed more than fifty analogs. For each of them, I have obtained more than 1 mg of purified protein from about 1 liter of expression media.
Xiang Yang
Research Scientist
Georgetown University
Lombardi Cancer Center