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Optimized condition for enhanced soluble-expression of recombinant mutant anabaena variabilis phenylalanine ammonia lyase.
[phenylketonuria]
Recently
discovered
Anabaena
variabilis
phenylalanine
ammonia
lyase
(
AvPAL
)
proved
to
be
a
good
candidate
for
enzyme
replacement
therapy
of
phenylketonuria
.
Outstanding
stability
properties
of
a
mutant
version
of
this
enzyme
,
produced
already
in
our
laboratory
,
have
led
us
to
the
idea
of
culture
conditions
optimization
for
soluble
expression
of
this
therapeutically
valuable
enzyme
in
E
.
coli
.
In
the
present
study
,
the
gene
encoding
mutant
version
of
AvPAL
was
cloned
into
the
pET
28
a
expression
vector
.
Different
concentrations
of
IPTG
,
induction
period
,
growth
temperature
,
shaking
speed
,
as
well
as
different
types
of
culture
media
were
examined
with
respect
to
the
amount
of
recombinant
protein
produced
and
specific
activity
of
the
enzyme
.
B
ased
upon
our
findings
,
maximum
amount
of
active
mutant
enzyme
was
attained
by
addition
of
0
.
5
mM
IPTG
at
150
rpm
to
the
TB
culture
media
.
The
yield
of
active
enzyme
at
cluture
tempreature
of
25
°
C
and
induction
period
of
18
hour
was
the
highest
.
The
results
of
this
study
indicated
that
the
yield
of
mutant
AvPAL
production
in
E
.
coli
can
be
affected
mainly
by
culture
temperature
and
inducer
concentration
.
