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K. lactis Protein Expression Kit |
 | | | | K. lactis Protein Expression Kit |
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Prices are in US dollars and valid only for US orders.
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- Supplied with competent K. lactis cells
- Easy-to-use protocols for those inexperienced with yeast systems
- Attractive commercial sublicensing
- Clone and express genes toxic to E. coli
- No expensive antibiotics or methanol required
Description:
The K. lactis Expression Kit provides an easy method for expressing a gene of interest in the yeast Kluyveromyces lactis (Figure 1). Proteins may be produced intracellularly or be secreted using the supplied integrative expression vector pKLAC2 (Figure 3). To achieve protein secretion, a gene of interest is cloned downstream of the K. lactis α-mating factor secretion domain (α-MF; Figure 4) which is eventually processed in the Golgi resulting in secretion of the desired protein (Figure 1).
The K. lactis expression system offers several advantages over other yeast and bacterial protein expression systems. First, K. lactis has been used to produce proteins at industrial scale in the food industry for over a decade due to its ability to rapidly achieve high culture densities and abundantly produce recombinant proteins (Figure 2). Second, yeast expression is driven by a variant of the strong LAC4 promoter that has been modified to lack background expression in E. coli (1). Therefore, genes toxic to E. coli can be cloned into pKLAC2 in bacteria prior to their expression in yeast. Third, the kit includes highly competent K. lactis cells making the technology easy-to-use for those not accustomed to working with yeast. Their high transformation efficiency makes the system suitable for methods that require large numbers of transformants, for example, expression cloning using cDNA libraries. Selection of yeast transformants uses a unique antibiotic-free method in which acetamidase (amdS) expressed from pKLAC2 permits transformed cells to utilize acetamide as a sole nitrogen source on defined medium. Acetamide selection promotes formation of cells containing multiple integrations of pKLAC2 which results in higher yields of protein. Finally, proteins expressed in K. lactis have access to eukaryotic protein folding and glycosylation machinery that E. coli cells do not possess, making it an important alternative to bacterial expression systems.
Figure 1: Secreted protein processing. 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.
Figure 2: SDS-polyacrylamide gel electrophoresis separation of secreted recombinant maltose binding protein (MBP) and detection by Coomassie staining. Lane 1: Protein Molecular Weight Markers. 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.
Advantages:- High yield protein expression
- Rapid high cell density growth
- Supplied with competent K. lactis cells
- Attractive commercial sublicensing
- Easy-to-use protocols for those inexperienced with yeast systems
Kit Components:
K. lactis GG799 Competent Cells
Integration Primer Set
NEB Yeast Transformation Reagent
NEBuffer 4 (10X)
pKLAC1-malE Control Plasmid
pKLAC2 Vector
SacII
Yeast Medium Pack
Storage Conditions
Storage Temperature:
-80°C
FAQs
- Where can I find many more detailed FAQs for K. lactis Protein Expression Kit?
- What type of proteins express well in K. lactis?
- What promoter drives heterologous gene expression in K. lactis?
- Is PLAC4-PBI an inducible/repressible promoter?
- Does pKLAC1 replicate as an episomal plasmid in K. lactis?
- Can pKLAC1 be used for cytosolic protein expression?
- How does acetamide selection work?
Figure 3: The pKLAC2 expression vector. pKLAC2 (9107 bp) contains the 5´ and 3´ ends of the LAC4 promoter (PLAC4-PBI) separated by DNA encoding β-lactamase (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 ADH2 promoter (PADH2) 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.
Figure 4: 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.
References
- Colussi, P.A. and Taron, C.H. (2005) Appl. Environ. Microbiol., 71, 7092-7098.
Reagents Sold Separately
K. lactis GG799 Competent Cells
Integration Primer Set
NEBuffer 4
pKLAC1-malE Control Plasmid
pKLAC2 Vector
SacII
Yeast Medium Pack
Companion Products
α-Mating Factor Sequencing Primer (18-mer)
Taq DNA Polymerase with Standard Taq Buffer
Taq DNA Polymerase with ThermoPol Buffer
BstXI
Deoxynucleotide Solution Mix
LAC4mt Promoter Sequencing Primer (18-mer)
pKLAC1 Reverse Sequencing Primer (18-mer)
pKLAC2 Vector
Yeast Medium Pack
Legal
Licenses/Patents/Disclaimers:
Notice to Buyer/User: The K. lactis Protein Expression Kit was developed from basic research at New England Biolabs, Inc. and DSM Biologics Company B.V. The buyer and user has a non-exclusive sublicense to use this system or any component thereof, including the K. lactis GG799 Competent Cells, for RESEARCH PURPOSES ONLY. A license to use this system for manufacture of clinical grade material or commercial purposes is available from New England Biolabs, Inc., or DSM Biologics Company B.V.
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