Secretion of a target protein is a common expression strategy in yeast. This approach is used to produce recombinant extracellular eukaryotic proteins that often do not express well in bacteria. One technical challenge is that a secreted target protein becomes diluted in large volumes of growth medium complicating its recovery. Additionally, many common tags do not perform well, either being limited in their scalability (e.g. antibody resins) or often suffering from interference by growth medium components (e.g. nickel affinity resins).
The pKLCF-series vectors offer the ability to secrete a target protein fused to a chitin binding domain (CBD) tag in the yeast Kluyveromyces lactis. The CBD tag permits rapid recovery of secreted fusion proteins using chitin resin or chitin magnetic beads. The CBD tag tightly and selectively binds chitin without the need for concentrating or buffer exchanging the growth medium. Elution from chitin occurs sharply upon brief exposure to a high pH buffer.
Different pKLCF-series vectors permit construction of N- or C-terminal CBD tags on the target protein. A protease site can be engineered between the CBD and target protein. To further enhance the purity of purified CBD-tagged proteins, an engineered K. lactis strain that lacks endogenous chitin-binding proteins may be used as a host.
FAQs for Yeast Chitin Binding Domain Tag
Protocols for Yeast Chitin Binding Domain Tag
- Isolation of CBD-fusion protein using Chitin Magnetic Beads
- 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
- 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: Secretion of MBP
The pKLAC2 Expression Vector
pKLAC2 Multiple Cloning Site
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