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Yeast Chitin Binding Domain Tag
Product Listing
Application Overview
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.
Featured Products
K. lactis Protein Expression Kit
K. lactis YCT284 Competent Cells
Chitin Resin
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FAQs for Yeast Chitin Binding Domain Tag
FAQs
Protocols
Example of K. lactis
Expression: Secretion
of MBP
The pKLAC2
Expression Vector
pKLAC2 Multiple
Cloning Site
Legal Information
What systems does NEB offer for protein expression and purification?
Where can I find sequence and restriction maps of NEB's expression vectors?
What are the advantages of magnetic affinity matrices?
K. lactis Protein Expression FAQs
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
Legal and Disclaimers
This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New
England Biolabs, Inc (NEB).
While NEB develops and validates its products for various applications, the use of this product may require
the buyer to obtain additional third party intellectual property rights for certain applications.
For more information about commercial rights, please contact NEB's Global Business Development team at
[email protected].
This product is intended for research purposes only. This product is not intended to be used for therapeutic or
diagnostic purposes in humans or animals.
Example of K. lactis Expression: Secretion of
MBP
Lane 1: Protein Marker, Broad Range (NEB #P7702)
Lane 2: spent culture medium (15 µl) from wild-type K. lactis cells.
Lane 3: spent culture medium (15 µl) from K. lactis cells harboring an integrated expression cassette containing
the E. coli malE gene.
The pKLAC2 Expression Vector
pKLAC2 (9107 bp) contains the 5´ and 3´ ends of the LAC4 promoter (PLAC4-PBI) separated by DNA encoding βlactamase (ApR) and the pMB1 origin (ori) to allow for its propagation in E. coli. The K. lactis α-mating factor
secretion leader sequence (α-MF), multiple cloning site (MCS), and the LAC4 transcription terminator (TT) lie
immediately downstream of 3´ PLAC4-PBI. The yeast ADH1 promoter (PADH1) drives expression of an acetamidase
selectable marker gene (amdS). The vector can be linearized by digestion with SacII or BstXI to create a linear DNA
fragment capable of inserting into the native LAC4 promoter region of the K. lactis genome.
pKLAC2 Multiple Cloning Site
pKLAC2 (9107 bp) contains the K. lactis α-mating factor secretion leader sequence (blue background) and a
polylinker immediately downstream of the PLAC4-PBI promoter. Unique polylinker restriction sites are indicated.
Half arrows show the positions of pKLAC-series vector-specific sequencing primers available from New England
Biolabs.
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