- This product was discontinued on January 05, 2015
The vector pKLCF-n permits secreted expression of a recombinant protein having a chitin-binding domain (CBD) affinity tag fused to its amino-terminus in the yeast Kluyveromyces lactis. It is compatible with the K. lactis Protein Expression Kit (NEB #E1000). CBD fusion proteins expressed from pKLCF-n can be affinity purified directly from untreated culture medium using Chitin Beads (NEB #S6651) or Chitin Magnetic Beads (NEB #E8036).
Vector pKLCF-n contains the strong K. lactis PLAC4-PBI promoter (1), DNA encoding the K. lactis Cts1p chitin-binding domain (2), a universal multiple cloning site (MCS), the K. lactis LAC4 transcription terminator (TT), and a fungal acetamidase selectable marker gene (amdS) expressed from the yeast ADH1 promoter (PADH1).An E. coli replication origin (ori) and ampicillin resistance gene (ApR) is present for propagation of pKLCF-n in E. coli. SacII or BstXI linearized pKLCF-n integrates into the LAC4 locus of the K. lactis genome upon transformation of K. lactis competent cells.
The sequence of the pKLCF-n vector (GenBank HQ214066) and additional pKLCF-n information are available here.
DNASU is a central repository for plasmid clones and collections that may also be helpful.
- PLAC4-PBI promoter does not express in E. coli, allowing toxic genes to be cloned prior to their expression in yeast.
- Universal MCS lies downstream of DNA encoding CBD and PLAC4-PBI promoter.
- Acetamidase expression for non-antibiotic selection in K. lactis.
- Ampicillin resistance for propagation in E. coli.
- Permits expression of CBD-tagged fusion proteins and their one-step purification directly from growth medium.
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