K. lactis Expression Kit – NEW ENGLAND BIOLAB

Neb KLAKTIS KIT

 K. lactis Expression Kit provides an easy method for expressing a gene of interest in the yeast Kluyveromyces lactis. Proteins may be produced intracellularly or be secreted and 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. 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

The K. lactis Protein Expression Kit provides a method for cloning and express-
ing a gene of interest in the yeast Kluyveromyces lactis (Figures 1 and 2) .
Proteins may be expressed intracellularly or be secreted from the cell using the
supplied expression vector pKLAC2 (Figure 3) . Secretion of proteins is the most common approach to
K. lactis
protein
expression (1) . To achieve protein secretion, the gene of interest is cloned into
pKLAC2 downstream of the
K. lactis
a
-mating factor domain (
a
-MF; Figure 4),
resulting in expression of an
a
-MF fusion protein . The
a
-MF domain directs the
fusion protein to be efficiently transported through the yeast secretory pathway
.
An
a
-MF fusion protein undergoes sequential processing by signal peptidase in
the endoplasmic reticulum (ER) and the Kex protease in the Golgi, resulting in
the secretion of a native form of the protein of interest into the growth medium
(Figure 1) .
Expression in yeast is driven by a mutant form of the strong
K. lactis LAC4
promoter (P
LAC4-PBI
) that has been engineered to lack background
E. coli
tran-
scriptional activity (2) . Therefore, genes encoding products toxic to
E. coli
can
be cloned into pKLAC2 in
E. coli
prior to their introduction into yeast cells . To
achieve expression in yeast, pKLAC2 containing a cloned gene of interest is
linearized by either SacII or BstXI to produce an expression cassette that can
integrate into the
K. lactis
genome at the
LAC4
locus by homologous recom-
bination . A fungal acetamidase gene (
amdS
) in pKLAC2 provides for selection
of yeast containing an integrated expression cassette by allowing their growth
on nitrogen-free minimal medium containing acetamide . Only cells expressing
amdS
can break down acetamide to ammonia for use as a nitrogen source . An
advantage of this selection method is that it enriches transformant populations
for cells that have integrated multiple tandem copies of the expression cassette
and therefore produce more recombinant protein . Finally, the supplied
K. lactis
GG799 strain is an industrial isolate that has no auxotrophies, rapidly grows to
high cell density, and efficiently secretes heterologous proteins .
Secretion of both eukaryotic (2-7) and prokayotic proteins (8,9) from
K. lactis
has been achieved . Typically, proteins that are normally secreted from cells
(e .g ., cytokines, serum albumins, antibody fragments and glycosidases) pro
duce the highest yields . In these cases, 10–50 mg of recombinant protein per
liter can often be achieved in shake flasks, and yields can be further enhanced
by high density cell fermentation . Secretion of other types of proteins is also
possible . Secreted recombinant proteins are routinely detected in the growth
medium of saturated cultures via SDS-PAGE and protein staining, Western
analysis or enzyme assay
. Secreted proteins may also bear post-translational
modifications (e .g ., asparagine-linked glycosylation) that can be removed by
treatment with Endo H (NEB #P0702) or PNGase F (NEB #P0704)
 

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