Team:Mississippi State/On Pichia Pastoris

In doing a lot of reading and research on Pichia Pastoris, the yeast we'll be using for iGEM this year, I thought I should record what I learn and experience from both reading and my successes and failures in working with the organism. Though all of this info is from outside sources, I think it will be beneficial for future iGEMers to learn about this organism from the viewpoint of another iGEMer. So, here we go.

Pichia in a Nutshell
Pichia Pastoris is type of yeast. Its appeal arises from the fact that it blends the growth and management benefits of prokaryotes with the protein expression capabilities of eukaryotes. Cell density is very good, and it utilizes a very strong promoter. AOX1, for alcohol oxidase, is an enzyme used by the yeast to utilize methanol as a food source when glucose levels are low. The AOX1 promoter is very strong and, when glucose deprived, causes 30% of protein production to be alcohol oxidase. Therefore, one can induce expression by the addition of methanol. Additionally, yeast has a built in secretion system so that tagged proteins can be easily exported out of the cell into the media.

What does this mean? For our purposes, growing yeast has many of the benefits of growing bacteria. It usually takes a day or so longer and high cell densities are achieved. This means more protein production. On the other hand, prokaryotes sometimes have trouble with large proteins from higher organisms which results in dysfunctional protein production. Yeast has the capability of allowing these larger, more complex proteins to fold correctly. Note here that Pichia does not like expressing DNA with introns. So, cDNA must be cloned into the vector, not genomic DNA. The AOX1 promoter is ideal for our purposes because the main purpose of engineering microbes is usually to selectively boost protein production. Finally, the ultimate triumph of yeast, for our project, is its secretion system.

Pichia Secretion System
The first problem we have encountered has been with secretion systems. We ordered the pPIC6 Starter Kit from Invitrogen. We wanted the pPIC6alpha vector because it has the alpha-factor secretion system built in. After further reading, it appears that the alpha-factor vector has the alpha mating signal sequence built into the vector. The pPIC6 does not. Beware that Invitrogen's product descriptions are somewhat ambiguous about secretion systems. Apparently, all the pPIC6 vectors have an "É -factor secretion signal for efficient transport of proteins to the medium." I have yet to discover what this is.

Sreekrishna et al have shown that choice of a secretion signal is arbitrary in Pichia, provided the protein is normally secreted. In our case, Lignin Peroxidase is a normally secreted enzyme, so secretion should not pose a problem. In addition, they say that the native secretion signal works wonderfully. We will probably have to go with this choice due to the lack of a built in signal from Invitrogen. This great source on similar cloning done on the laccase genes in White Rot Fungi shows that using the native signal resulted in seven times higher secretion than the alpha-factor signal when the protein was expressed in Pichia. They also observed adverse affects of pH(below 4) on growth. Note that successful expression of the gene requires a buffered medium. This is due to inactivation of the lignolytic enzymes.

Notes from the pPIC6alpha Manual
I'll note first of all that information regarding recombinant protein expression in Pichia is found in another manual. Secondly, there is a list of required supplies of which I will refer the reader to the manual. The plasmid and Blasticidin are to be stored at -20C, and the X-33 stab is to be stored at +4C. The polyhistidine (6xHis) is a tag built into the vector to allow for easy purification of the expressed protein. Next, is listed steps for successful expression and cloning of a gene in the vector: The Meaning of Being in Frame
 * 1) Propagate pPIC6α A, B, and C by transformation into a recA, endA1 E. coli strain such as TOP10, DH5α, or JM109.
 * 2) Develop a cloning strategy and ligate your gene into one of the pPIC6α vectors in frame with the α-factor secretion signal and the C-terminal tag.
 * 3) Transform into E. coli and select transformants on Low Salt LB plates containing 100 μg/ml blasticidin.
 * 4) Analyze 10-20 transformants by restriction mapping or sequencing to confirm in-frame fusion of your gene with the α-factor secretion signal and the C-terminal tag.
 * 5) Purify and linearize the recombinant plasmid for transformation into Pichia pastoris.
 * 6) Transform X-33 or your Pichia strain and plate onto YPDS plates containing the appropriate concentration of blasticidin.
 * 7) Select for blasticidin-resistant transformants.
 * 8) Optimize expression of your gene.
 * 9) Purify your fusion protein on metal-chelating resin (e.g. ProBond).

Premature termination of AT rich regions has been observed in Pichia. We also need to prepare a Glycerol Stock to store the cultures:
 * 1) Streak the original colony out on an Low Salt LB plate containing 100 μg/ml blasticidin. Incubate the plate at 37°C overnight.
 * 2) Isolate a single colony and inoculate into 1-2 ml of Low Salt LB containing 100 μg/ml blasticidin.
 * 3) Grow the culture to mid-log phase (OD600 = 0.5-0.7).
 * 4) Mix 0.85 ml of culture with 0.15 ml of sterile glycerol and transfer to a cryovial.
 * 5) Store at -80°C.