The development of a system to facilitate the stable expression of mammalian proteins in the filamentous fungus 'Aspergillus oryzae'
Abstract
Human interleukin 6 (hIL6) is
a multifunctional cytokine effecting the
function and proliferation of many cell types. The further
understanding of
hIL6
and
its
possible medical applications rely on the availability of this
protein.
The filamentous fungus Aspergillus oryzae
is
an
important industrial
organism and
is
used
for the large
scale production of many enzymes.
As this
fungus has
an
impressive
secretory output
it
was
decided to attempt
to
produce
hIL6 in this organism.
The initial
work was
based
on the development
of a gene transfer
system
for A.
oryzae
in
order that the hIL6
gene could
be introduced to the
organism.
A transformation system
based
on the homologous
nitrate
reductase gene
is described. This
system yielded up to 800 transformants per µg
plasmid
DNA. Additionally, the adaptation of the transformation system
based
on the A.
nidulans anuiS gene and the use of other transformation
systems
is
reported.
In
order to ensure that the hIL6
gene was efficiently transcribed it
was
considered
important that homologous
control regions
from highly
produced
and regulated
A.
oryzae genes were
linked to the hIL6
gene.
Therefore the
genes encoding glucoamylase and a-amylase were
isolated from A.
oryzae.
A
method
for the purification of
A.
oryzae
Si
nuclease
is described
and the
amino acid sequence of the N terminus is
reported.
A.
oryzae produces
large
amounts of extracellular proteases, a
feature
unlikely to be
attractive
in
a
heterologous host. Therefore the production of
protease production
in A.
oryzae was studied.
A
method
is described for the
selection of protease
deficient
mutants.
Using this method two protease
mutants were
isolated
and these have been
characterized.
One
mutation
designated
prtA2 protects against the degradation
of
hJL6 in
vitro.
The A.
oryzae alkaline protease gene was
isolated
and mutagenised and the attempts
made to produce specific protease mutant
by
reverse genetics are
described.
A
system
is described
where
by A.
oryzae can
be
engineered to produce
relatively
high levels
of
hIL6. Using
gene
fusion
constructs transformants
producing
in the range of
1
mg per
Litre have been isolated. This
system
is
heterologous,
recommendations
for increasing hIL6
production are
included.
Type
Thesis, PhD Doctor of Philosophy
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