Protein Expression in Yeast

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.

 

(1) Colussi, P.A. and Taron, C.H. (2005) Appl. Environ. Microbiol., 71, 7092–7098. PMID: 16269745
(2) Read, J.D. et al. (2007) Appl. Environ. Microbiol., 73, 5088–5096. PMID: 17586678


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    Publications related to Protein Expression in Yeast
    • 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, 168-178. PubMedID: 21166768
    • 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
    • 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(3), 381-92. PubMedID: 16630278
    • 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 Announc; 5(30), PubMedID: 28751387
    • 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(16), PubMedID: 17586678
Expression of MBP with K. lactis

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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 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 a-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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