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.
Overview of Protein Secretion in
K. lactis 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.