Expression of proteins in yeast is a common alternative to prokaryotic and higher eukaryotic expression. Yeast cells offer many of the advantages of producing proteins in microbes (growth speed, easy genetic manipulation, low cost media) while offering some of the attributes of higher eukaryotic systems (post translational modifications, secretory expression). Several yeast protein expression systems exist in organisms from the genera Saccharomyces, Pichia, Kluyveromyces, Hansenula and Yarrowia.
At the heart of any yeast expression system is the expression vector. Vectors that integrate into the host chromosome are most widely used because of their mitotic stability in the absence of a selection. However, episomal expression vectors exist for some yeast systems. Expression vectors typically contain a strong yeast promoter/terminator and a yeast selectable marker cassette. Most yeast vectors can be propagated and amplified in E. coli to facilitate cloning and as such, also contain an E. coli replication origin and ampicillin selectable marker. Finally, many yeast expression vectors include the ability to optionally clone a gene downstream of an efficient secretion leader (usually that of mating factor) that efficiently directs a heterologous protein to become secreted from the cell.
A growing number of engineered yeast strains are becoming available for protein expression. Strains have been described that increase yield of secreted proteins, improve the performance of certain affinity tags, reduce proteolysis, define the composition of N-glycans, and permit non-native amino acids (e.g. selenomethionine) into proteins have been described.
One yeast system that is commonly used for protein expression is Kluyveromyces lactis. K. lactis was one of the first yeasts to be used as an expression host in the early 1980’s. It was developed for use in the food industry and was harnessed for industrial production of bovine prochymosin, an important cheese manufacturing enzyme. In addition, K. lactis and S. cerevisiae are the only two yeasts classified by the NIH as Host-Vector I systems, an important biosafety designation, making it an attractive host for use in regulated industries. The system was adapted for life sciences research by NEB and a variety of vectors and host strains in a frozen competent format suitable are offered.
In the nucleus, an integrated expression vector encoding a fusion between the α-MF domain (blue) and a desired protein (black) is expressed. A signal peptide in the α-MF domain directs entry of the fusion protein into the endoplasmic reticulum (ER) and is removed by signal peptidase (SP). The fusion protein is transported to the Golgi where the Kex protease removes the α-MF domain. The protein of interest is then secreted from the cell.
FAQs for Yeast Protein Expression
Protocols for Yeast Protein Expression
- Protein expression using the K. lactis Protein Expression Kit - Cloning a PCR fragment into pKLAC2 (E1000).
- Protein Expression using the K. lactis Protein Expression Kit - Growth of strains for detection of secreted protein
- Protein Expression using the K. lactis Protein Expression Kit - Identification of Multi-copy Integrants
- Protein Expression using the K. lactis Protein Expression Kit - Identification of properly integrated cells
- Protein expression using the K. lactis Protein Expression Kit - Linearization of pKLAC2 for integrative transformation of K. lactis.
- Protein Expression using the K. lactis Protein Expression Kit - Simultaneous Expression of Multiple Proteins
- Protein Expression using the K. lactis Protein Expression Kit - Transformation of K. lactis GG799 cells
- Protocol I: Yeast Carbon Base Medium Powder Agar Medium with 5 mM acetamide solution (500 ml)
- Protocol II: 1 M Tris-HCl Buffer Stock Solution (1 liter)
- Transformation Protocol for K. lactis GG799 Competent Cells (C1001)
- Co-expression of Multiple Proteins in Kluyveromyces lactis
Protein Expression & Purification Brochure
The Protein Expression and Purification brochure provides information on the advanced tools for protein expression and purification offered by NEB.
Bypassing Common Obstacles in Protein Expression
Why Choose the K. lactis Protein Expression Kit?
- Protein Expression and Purification Selection Chart
Other Tools & Resources
Example of K. lactis Expression
The pKLAC2 Expression Vector
pKLAC2 Multiple Cloning Site
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