Expression of recombinant proteins is essential for almost any life science institution, be it academic, or a for-profit organization, such as biotech and pharmaceutical companies. Many companies market various expression systems and the numerous options make decisions difficult. Although the most studied and most efficient expression system for foreign proteins is the one that uses the bacterium
E. coli, in many instances these systems will not be suitable if, for example, the target protein requires post-translational modifications. Also, some foreign proteins will not correctly fold when expressed in
E. coli and will form insoluble inclusion bodies from which it may be difficult to rescue the expressed protein. I have gained extensive experience with the production of recombinant proteins in
E. coli and have been pretty successful most of the time. However, for my current project, I was asked to produce several recombinant proteins in the yeast
S. cerevisiae.
After searching the market, I decided to purchase and work with Invitrogen’s S. cerevisiae yeast expression system which includes competent, wild-type yeast INVSc1 (catalog number C810-00) and a collection of pYES plasmids: I purchased the plasmid pYES2/CT (catalog number V8251-20). The INVSc1 strain is a fast-growing diploid strain ideal for expression. It has the following genotype: MATá his3D1 leu2 trp1-289 ura3-52 MAT his3D1 leu2 trp1-289 ura3-52. Yeast INVSc1 is not appropriate for yeast genetic studies because it does not sporulate well (according to Invitrogen’s online catalog). INVSc1 cells are provided as chemically competent cells. The pYES2 family of plasmids are designed for different purposes, such as native protein expression (pYES2), different cloning strategies with subsequent expression (pYES2-DEST52, pYES2.1, pYES2.1-E and pYC2-E), protein expression with N- and C-terminal tag epitopes for detection and affinity purification (pYES2/NT and pYES2/CT) and pYES3/CT and pYES6/CT for the same purpose, but these latter vectors include selection markers different from the rest. In addition, some plasmids contain the á-factor signal sequence for expression of secreted proteins, but the secretion efficiency is greatly affected by the chimera of the protein and the á-factor signal sequence. All plasmids vary in size between 4.6 and 6 kb except for pYES2-DEST52, which is 7.8 kb. All plasmids feature the following elements: A multiple cloning site (MCS) that contains 8-9 unique restriction sites, URA3 (for selection in yeast), ampicillin resistance (for selection in bacteria), PGAL1 (for induction in yeast) and 2µ origin for high copy maintenance. The vectors pYES3/CT and pYES6/CT are different in that they contain either TRP1 or Blasticidin selection markers instead of URA3 and Ampicillin.
The experimental procedure involved first sub-cloning the gene encoding my protein of interest into the pYES2/CT vector, then propagating the plasmid in E. coli, transforming yeast INVSc1 with the purified plasmid DNA, expressing the recombinant protein and finally, detecting the expressed protein. Although not experienced in working with S. cerevisiae, I found this system very reliable and all the steps from cloning to detection of the expressed protein went flawlessly. The manual clearly describes each step and if you stick to the protocol, there is a good chance you will be successful. One big advantage is that the plasmid is compatible with both S. cerevisiae and E. coli, which saves time because the same plasmid is used for everything: Cloning, DNA preparation, transformation and expression.
Because each protein behaves differently, it is important to optimize the growth and expression conditions. The protocol recommends using glucose/galactose selective media and inducing expression at an OD600=0.4. My recommendation is to try different growth conditions such as Raffinose and Casamino acid supplements, as well as to start induction at a higher OD600, 0.7-1.0. Another point that may improve expression is to add a wash step of the starter culture from glucose; this will help to suppress repression more quickly. In parallel to the induced culture, you may grow an un-induced culture to serve as a negative control. In my experiments I also deviated from the suggested volumes and used higher volumes.
Overall, this expression system is highly recommended. As an inexperienced user of yeast expression systems, I found the INVSc1/pYES2 system reliable, with an easy-to-follow protocol; the system may also save you time.
Research Scientist
R&D and Product Development
ProSci Incorporated