The K. lactis Expression Kit (NEB #E1000) provides an easy method for expressing a gene of interest in the yeast Kluyveromyces lactis. Proteins may be produced intracellularly or be secreted using the supplied integrative expression vector pKLAC2. To achieve protein secretion, a gene of interest is cloned downstream of the K. lactis α-mating factor secretion domain (α-MF) which is eventually processed in the Golgi resulting in secretion of the desired protein.
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. 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.
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.
FAQs for Yeast
Protocols for Yeast
- 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 - 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 - Simultaneous Expression of Multiple Proteins
- 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 - Transformation of K. lactis GG799 cells
- Transformation Protocol for K. lactis GG799 Competent Cells (C1001)
- Protein Expression Using BL21(DE3) (C2527)
- Transformation Protocol for BL21(DE3) Competent Cells (C2527)
- Protocol I: Yeast Carbon Base Medium Powder Agar Medium with 5 mM acetamide solution (500 ml)
- Protocol II: 1 M Tris-HCl Buffer Stock Solution (1 liter)
- 5 Minute Transformation Protocol (C2527)
- Co-expression of Multiple Proteins in Kluyveromyces lactis
Protein Expression & Purification Brochure
The Protein Expression and Purification brochure provides information on the advanced tools for protein expression and purification offered by NEB.
Bypassing Common Obstacles in Protein Expression
Why Choose the K. lactis Protein Expression Kit?
- Protein Expression and Purification Selection Chart
Other Tools & Resources
- Rocha S.N., et al. 2011. Heterologous expression of a thermophilic esterase in Kluyveromyces yeasts Appl. Microbiol. Biotechnol.. 89, PubMedID: 20862582, DOI:
- Ganatra, M.B., Rainauskas, S., Hong, J.M., Taylor, T.E., Denson, J.P.M., Esposito, D., Read, J.D., Schmeisser, H., Zoon, K.C., Hartley, J.L. and Taron, C.H. 2011. A set of aspartyl protease-deficient strains for improved expression of heterologous proteins in Kluyveromyces lactis FEMS Yeast Res.. 11, PubMedID: 21166768, DOI:
- Feng, Z., et al. 2011. Disruption of PMR1 in Kluyveromyces lactis improves secretion of calf prochymosin J. Sci. Food Agric.. 15, PubMedID: , DOI:
- Zhou, X., et al. 2011. Expression of heparin sulfate sulfotransferases in Kluyveromyces lactisand preparation of PAPS Glycobiology Advance Access. , PubMedID: , DOI:
- Van Ooyen, A.J.J., Dekker, P., Huang, M., Olsthoorn, M.M.A., Jacobs, D.I., Colussi P.A., and Taron, C.H. 2006. Heterologous protein production in the yeast Kluyveromyces lactis FEMS Yeast Res.. 6, PubMedID: 16630278, DOI:
- Colussi, P.A. and Taron, C.H. 2005. Kluyveromyces lactis LAC4 promoter variants that lack function in bacteria but retain full function in K. lactis Appl. Environ. Microbiol.. 71, PubMedID: 16269745, DOI:
- Colussi, P.A., Specht, C.A. and Taron, C.H. 2005. Characterization of a nucleus-encoded chitinase from the yeast Kluyveromyces lactis Appl. Environ. Microbiol.. 71, PubMedID: 15932978, DOI:
- Feng, Z., et al. 2010. Codon optimization of the calf prochymosin gene and its expression in Kluyveromyces lactis World J. Microbiol. Biotechnol.. 26, PubMedID: , DOI:
- Yuan W, et al. 2010. Gene synthesis of the bovine prochymosin gene and high-level expression in Kluyvermyces lactis Sheng Wu Gong Cheng Xue Bao. 26, PubMedID: 21141120, DOI:
- Rocha, S.N., et al. 2010. Heterologous expression of glucose oxidase in the yeastKluyveromyces marxianus Microb. Cell Fact.. 21, PubMedID: 20092622, DOI:
- Kuo, D., et al. 2010. Evolutionary divergence in the fungal response to fluconazole revealed by soft clustering Gen. Bio. . 11, PubMedID: 20653936, DOI:
- Wamalwa, B.M., et al. 2007. High-Level Heterologous Expression of Bacillus halodurans Putative Xylanase Xyn11A (BH0899) in Kluyveromyces lactis Biosci. Biotechnol. Biochem.. 71, PubMedID: 17341817, DOI:
- Zhao, H.L., et al. 2007. Circumventing the heterogeneity and instability of human serum albumin-interferon-_2b fusion protein by altering its orientation J. Biotech. . 131, PubMedID: 17698234, DOI:
- Read, J.D., Colussi, P.A., Ganatra, M.B. and Taron, C.H. 2007. Acetamide selection of Kluyveromyces lactis cells transformed with an integrative vector leads to high frequency formation of multicopy strains Appl. Environ. Microbiol.. 73, PubMedID: 17586678, DOI:
- Madinger, C.L., Sharma, S.S., Anton, B.P., Fields, L.G., Cushing, M.L., Canovas, J., Taron, C.H. and Benner, J.S. 2009. The effect of carbon source on the secretome of Kluyveromyces lactis Proteomics. 9, PubMedID: 19743416, DOI:
- Feng, Z., et al 2009. Effect of temperature-shift strategy on recombinat chymosin of Kluyveromyces Lactis Heilongjiang Animal Science and Veterinary Medicine. , PubMedID: , DOI:
- Swaim, C.L., et al. 2008. Physical and computational analysis of the yeast Kluyveromyces lactissecreted proteome Proteomics. 8, PubMedID: 18601269, DOI:
- Takakuwa, N., Oshnishi, M., and Oda, Y. 2008. Significance of the KLAC1 gene in glucosylceramide production by Kluyveromyces lactis FEMS Yeast Res. 9, PubMedID: 18631186, DOI:
- Foster, J.M., Raverdy, S., Ganatra, M.B., Colussi, P.A., Taron, C.H. and Carlow, C.K.S. 2008. The Wolbachia endosymbiont of Brugia malayi has an active phosphoglycerate mutase: a candidate target for anti-filarial therapies Parasitol. Res. . , PubMedID: 19043737, DOI:
- Sugiki, T. Shimada, I. and Takahashi H. 2008. Stable isotope labeling of protein byKluyveromyces lactis for NMR study J. Biomol NMR.. 42, PubMedID: 18827973, DOI:
- Platko, J.D., et al. 2008. Heterologous expression of Mytilus californianus foot protein three (Mcfp-3) in Kluyveromyces lactis Platko, J.D., et al.. 57, PubMedID: 17923416, DOI:
Publications related to Yeast
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