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Many types of protein can be secreted from
yeast cells. As a general rule, those that tend to
secrete best are proteins that are also secreted
by their native host (e.g. glycosidases, serum
albumins, cytokines, etc). However, there
are numerous examples in the literature of
normally non-secreted proteins that have been
successfully secreted from various yeasts.
When in doubt, it is always best to try secreted
expression. Intracellular protein expression
in yeast is also possible for a wide range of
proteins and is a great alternative to bacterial
protein expression. |
| K. lactis strain GG799 (as
supplied in the NEB Kit) is a haploid (α) wild-type industrial
isolate
that has no genetic markers. It was originally
chosen as a host strain in the food industry
because of its ability to grow to very high cell
density and to efficiently secrete heterologous
proteins. |
| Avoid toxicity problems in E. coli |
| The K. lactis Protein
Expression Kit vector (pKLAC1) contains a variant of the strong
K. lactis LAC4 promoter (PLAC4-PBI) for
expression of a desired gene in K. lactis. The
major advantage of the PLAC4-PBI promoter is
that it is transcriptionally silent while in E. coli.
In contrast, the wild-type PLAC4 promoter
shows background transcriptional activity in
E. coli which can be detrimental to the process of assembling
or amplifying expression constructs in E. coli prior
to their introduction into yeast cells. This is especially problematic if the cloned
gene of interest encodes a translated product that is toxic to E. coli cells.
Therefore, pKLAC1 is well-suited for the cloning and yeast
expression of genes encoding proteins that are
toxic or otherwise detrimental to bacteria. |
| High yield, lower cost protein expression |
| Integrative expression with no auxotrophic
markers: pKLAC1 is an integrative expression
vector that inserts into the promoter region
of the LAC4 locus of the locus of the K. lactis
genome upon its introduction into K. lactis
cells. While K. lactis
episomal plasmids do exist, they can present some problems for large-scale protein
production. For example, plasmids are easily
lost by cells in the absence of a selection. For
large-scale fermentation, plasmid selection
using antibiotics can be too costly, and selection
using an auxotrophic marker can reduce yields.
While auxotrophic markers have historically
been used for genetic manipulation of yeasts,
they are not always desirable to achieve maximum protein expression. In some cases, an
auxotrophy (e.g. uracil) can cause a significant
reduction in the strain’s ability to produce a
heterologous protein even if exogenous uracil
or uridine is provided in the growth medium.
Integrative expression vectors are attractive
because they insert into the genome, thus
becoming part of the host chromosome, and
are therefore quite stable even in the absence of
selection. |
| Acetamide selection: There are two main
advantages to acetamide selection: cost and
selection of multiple integrants. Acetamide is
significantly less expensive than antibiotics.
Additionally, acetamide selection enriches
transformant populations for cells that
have integrated multiple tandem copies of
the pKLAC1 expression vector. Multi-copy
integrants are desirable because they often
produce more recombinant protein than single
integrants. Acetamide acts as a nitrogen source.
A cell transformation mixture (containing a
population of cells that are either transformed
or untransformed by vector pKLAC1) is spread
onto yeast carbon base (YCB) medium agar
containing 5 mM acetamide. YCB medium
contains all of the nutrients and carbon source
required for K. lactis
cells to grow, but lacks a nitrogen source. The acetamide provided in the
medium can be utilized as a source of nitrogen
only if it is broken down to ammonia by the
enzyme acetamidase (expressed from the amdS
gene present on pKLAC1). Therefore, only
transformed cells are able to grow into colonies. |
| Flexible cloning strategies |
| For cytosolic protein expression: The gene of
interest can be cloned into pKLAC1 in a manner
that places it downstream of PLAC4-PBI without
the α-mating factor secretion domain being
present. Genes expressed in this manner must
include a methionine as their first codon, to
initiate translation. |
| For tagged proteins: A PCR method for the
addition of a carboxy-terminal HA tag to a
secreted protein is described in the kit manual.
This example can be adapted for the addition of
other antibody epitope tags e.g. FLAG, c-myc
or His tag. Additionally, a chitin-binding domain
has been used as a tag for capture of secreted
proteins onto inexpensive chitin beads directly
in spent medium (1). |
| For an amino-terminal antibody epitope tag: A new forward PCR primer
is used that contains an XhoI restriction site, the Kex protease
cleavage site, the desired tag’s sequence and
DNA homologous to the 5´ end of the desired
gene or cDNA. After Kex protease processing
of the expressed protein in the Golgi, a protein
bearing the desired tag at its amino-terminus is
produced and secreted. |
 |
| Genomic integration of a linear expression cassette. Vector
pKLAC1 containing the gene of interest is digested with either SacII or BstXI (SacII shown) and introduced into K.
lactis cells. The 5´ PLAC4 and 3´ PLAC4 sequences direct insertion of the cassette into
the promoter region of the LAC4 locus in
the K. lactis genome. |
| Reference |
| 1. 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. 71:2862–2869. |
