K. lactis Protein Expression Kit

Catalog #	Concentration	Size
E1000S	Not Applicable	1 set
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Product Information

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.

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.

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.

Figure 4:
pKLAC2 (9107 bp) contains the K. lactis a-mating factor secretion leader sequence (blue background) and a polylinker immediately downstream of the P_LAC4-PBI promoter. Unique polylinker restriction sites are indicated. Arrows indicate the positions of pKLAC-series vector-specific sequencing primers that you can have synthesized to confirm your target gene is properly cloned.

Highlights

Kit Components

The following reagents are supplied with this product:

	Store at (°C)	Concentration
CutSmart® Buffer	-20	10 X
Yeast Carbon Base Medium Powder	4
Acetamide solution (sterile) (10 ml)	4	100 X
K. lactis GG799 Competent Cells	-80	5 reactions
NEB® Yeast Transformation Reagent (5 ml)	4	1 X
pKLAC1-malE Control Plasmid	-20	1,000 µg/ml
pKLAC2 Vector	-20	1,000 µg/ml
SacII	-20	20,000 units/ml
Integration Primer 1	-20	10 X
Integration Primer 2	-20	10 X
Integration Primer 3	-20	10 X

Product Categories:: Yeast Products

Applications:: Gaussia Luciferase,; Yeast Chitin Binding Domain Tag,; Yeast Protein Expression

Advantages and Features
Features
- High level and scalable expression of recombinant proteins
- Rapid high cell density growth
- Simultaneous expression of multiple proteins possible
- No background gene expression during E.coli cloning steps
- Easy and fast cell transformation procedure
- Antibiotic-free selection
Properties & Usage
Storage Temperature
-80°C
Shipping Notes
- Ships on dry ice
Related Products
Companion Products
Product Notes
1. NEB 5-alpha Competent E. coli (High Efficiency) (NEB #C2987), NEB 5-alpha Electrocompetent E. coli (NEB #C2989) and NEB 5-alpha Competent E. coli (Subcloning Efficiency) (NEB #C2988) are all recommended for propagation and subcloning pKLAC1 control plasmid and pKLAC2 vector.
References
1. Colussi, P.A. and Taron, C.H. (2005). Appl. Environ. Microbiol.. 71,
2. van Ooyen, A.J.J.et al. (2006). FEMS Yeast Research . 6, 381-392.
3. Spohner, S.C. et.al (2016). J Biotechnol. 222, 104-116.
4. Chuzel, L., Ganatra M.B. et. al (2017). Genome Announc. 5, e00623-17.
5. Read, J.D.et al. (2007). Appl. Environ. Microbiol.. 73, 5088-5096.
6. Platko, J.D.et al. (2008). Protein Expr. Purif.. 57, 57-62.
7. Ekborg, N.A.et al. (2007). Appl. Environ. Microbiol.. 73, 7758-7788.
8. Foster, J.M. et. al (2008). Parasitol Res.. 104(5), 1047.
9. Vainauskas, S., Taron, C.H. (2016). New England Biolabs Application Note.
10. Looke, M. et. al (2011). Biotechniques. 50, 325-328.

Protocols, Manuals & Usage
Tools & Resources
- Selection Charts
  Protein Expression and Purification Selection Chart
FAQs & Troubleshooting
- FAQs
  What systems does NEB offer for protein expression and purification?
  
  I received my kit and don’t see any competent cells or transformation reagent. Where are they?
  
  I’ve expressed my protein, now how do I purify it?
  
  I’ve noticed untransformed K. lactis will grow when streaked on the selection media. Is the selection not working?
- Tech Tips
  
  NEBuilder HiFi DNA Assembly Cloning Kit (NEB #E5520) or the Gibson Assembly Cloning Kit (NEB #E5510) are excellent ways to assemble a linear yeast expression cassette. (See "Improved method for assembly of linear yeast expression cassettes using NEBuilder® HiFi DNA Assembly Master Mix" in the Application Notes section under the Other Tools and Resources tab)
Citations & Technical Literature
- Citations
  
  Product Citation Tool
  
  Powered by Bioz See more details on Bioz
- Publications
  Chuzel, L., Ganatra, M.B., Schermerhorn, K.M., Gardner, A.F., Anton, B.P., Taron, C.H. (2017) . Complete genome sequence of Kluyveromyces lactis strain GG799, a common yeast host for heterologous protein expression Genome Announcements.; 5(30), PubMedID: 28751387
  
  Sakhtah, H., Behler, J., Ali-Reynolds, A., Causey, T.B., Vainauskas, S., Taron, C.H. (2019) . A novel regulated hybrid promoter that permits autoinduction of heterologous protein expression in Kluyveromyces lactos Appl. Environ. Microbiol..; pii: e00542-19. PubMedID: 31053583
Quality, Safety & Legal
- Quality Control Assays
  
  Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.
- Specifications
  The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]
  
  E1000S_v1
- Certificate of Analysis
  The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]
  
  E1000-1_v0_10028164
  
  E1000-1_v0_10048655
- Safety Data Sheets
  The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.
  
  CutSmart® Buffer
  
  Acetamide solution (sterile) (10 ml)
  
  K. lactis GG799 Competent Cells
  
  NEB® Yeast Transformation Reagent (5 ml)
  
  pKLAC2 Vector
  
  SacII
- 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.
  Licenses
  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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Applications

Categories

You’ll be Thrilled to Pieces

Tools

Resources

K. lactis Protein Expression Kit

Highlights

Kit Components

Features

Storage Temperature

Shipping Notes

Companion Products

Product Citation Tool

Licenses

K. lactis Protein Expression Kit

Highlights

Kit Components

Features

Storage Temperature

Shipping Notes

Companion Products

Product Citation Tool

Licenses

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