• 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.
Description:
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 yeastKluyveromyces 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. colireplication 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.
Source:
pKLCF-n is isolated from E. coli strain ER2268 by a standard DNA purification procedure.
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