Biomedical Engineering Reference
In-Depth Information
The ideal promoter is one that is tightly regulated
so that the growth phase can be separated from
the induction phase. This minimizes the selection of
non-expressing cells and can permit the expression
of proteins normally toxic to the cell. The ideal
promoter will also have a high induction ratio. One
promoter which has these characteristics and which
is now the most widely used is that from the
GAL1
gene. Galactose regulation in yeast is now extremely
well studied and has become a model system for
eukaryotic transcriptional regulation (see Box 9.1).
Following addition of galactose, GAL1 mRNA is
rapidly induced over 1000-fold and can reach 1%
of total mRNA. However, the promoter is strongly
repressed by glucose and so in glucose-grown cul-
tures this induction only occurs following depletion
of glucose. To facilitate galactose induction in the
presence of glucose, mutants have been isolated which
are insensitive to glucose repression (Matsumoto
et al
. 1983, Horland
et al
. 1989). The
trans
-activator
GAL4 protein is present in only one or two molecules
per cell and so GAL1 transcription is limited. With
multicopy expression vectors, GAL4 limitation is
exacerbated. However, GAL4 expression can be made
autocatalytic by fusing the GAL4 gene to a GAL10
promoter (Schultz
et al
. 1987), i.e. GAL4 expression
is now regulated (induced) by galactose.
In recent years, methylotrophic yeasts, such as
Pichia pastoris
, have proved extremely popular as
hosts for the overexpression of heterologous pro-
teins. There are a number of reasons for this. First,
the alcohol oxidase (AOX1) promoter is one of the
strongest and most regulatable promoters known.
Second, it is possible to stably integrate expression
plasmids at specific sites in the genome in either
single or multiple copies. Third, the strains can be
cultivated to very high density. To date, over 300
foreign proteins have been produced in
P. pastoris
(Cereghino & Cregg 1999, 2000). Promoters for use
in other yeasts are shown in Table 9.2.
purification fusion, etc. Some representative exam-
ples are shown in Fig. 9.9. Two aspects of these
vectors warrant further discussion: secretion and
surface display.
In yeast, proteins destined for the cell surface or
for export from the cell are synthesized on and
translocated into the endoplasmic reticulum. From
there they are transported to the Golgi body for
processing and packaging into secretory vesicles.
Fusion of the secretory vesicles with the plasma
membrane then occurs constitutively or in response
to an external signal (reviewed by Rothman & Orci
1992). Of the proteins naturally synthesized and
secreted by yeast, only a few end up in the growth
medium, e.g. the mating pheromone
factor and
the killer toxin. The remainder, such as invertase
and acid phosphatase, cross the plasma membrane
but remain within the periplasmic space or become
associated with the cell wall.
Polypeptides destined for secretion have a
hydrophobic amino-terminal extension, which is
responsible for translocation to the endoplasmic
reticulum (Blobel & Dobberstein 1975). The exten-
sion is usually composed of about 20 amino acids
and is cleaved from the mature protein within the
endoplasmic reticulum. Such signal sequences pre-
cede the mature yeast invertase and acid phos-
phatase sequences. Rather longer leader sequences
precede the mature forms of the
α
mating factor
and the killer toxin (Kurjan & Herskowitz 1982,
Bostian
et al
. 1984). The initial 20 amino acids or so
are similar to the conventional hydrophobic signal
sequences, but cleavage does not occur in the endo-
plasmic reticulum. In the case of
α
factor, which has
an 89 amino acid leader sequence, the first cleavage
occurs after a Lys-Arg sequence at positions 84 and
85 and happens in the Golgi body ( Julius
et al
. 1983,
1984).
To date, a large number of non-yeast polypeptides
have been secreted from yeast cells containing the
appropriate recombinant plasmid and in almost all
cases the
α
-factor signal sequence has been used.
There is a perception that
S. cerevisiae
has a lower
secretory capacity than
P. pastoris
and other yeasts
(Muller
et al
. 1998), but the real issue may be the
type of vector used. For example, Parekh
et al
.
(1996) found that
S. cerevisiae
strains containing
one stably integrated copy of an expression cassette
α
Specialist vectors
Many different specialist yeast vectors have been
developed which incorporate the useful features
found in the corresponding
E. coli
vectors (see
p. 70), e.g. an f1 origin to permit sequencing of
inserts production of the cloned gene product as a
