Overexpression of a Modified Amaranth Protein in Escherichia coli with Minimal Media and Lactose as Inducer

Abstract

In this research it was attempted to overexpress the acidic subunit, from the 11S amaranth seed globulin termed amarantin, modified with antihypertensive peptides in Escherichia coli Rosetta (DE3) by manipulating some factors in batch fermenter such as growth medium composition, inducer (isopropyl β-D-thiogalactopyranoside [IPTG] or lactose), air flow, cultivation temperature, agitation speed and induction time. It was investigated the possibility of using several minimal media and lactose as inducer to increase yields of the recombinant protein. Previous fermentations at flask level showed that two minimal culture media (A6 and A7) and 0.5% (w/v) lactose presented high yields of the engineered protein expression. Thus, the latter two media were tested at fermenter level, the lactose inducer, and different environmental conditions. Factors with significant effects were identified by Plackett-Burman design with center points and were adjusted at the level suggested and the yields of the recombinant protein were increased from 303.2 to 1,531 mg L-1 in A6 and from 363.4 to 1,681 mg L-1 in A7. Unlike some patents where the highest productivity was achieved at 24 h or afterwards, in this research the best productivity of the recombinant acidic subunit was attained at 4 and 6 h of induction using both media, respectively.

Full text

Journal
of
Biotechnology
158 (2012) 59–
67
Contents
lists
available
at
SciVerse
ScienceDirect
Journal
of
Biotechnology
jou
rn
al
hom
epage:
www.elsevier.com/locate/jbiotec
Overexpression
of
a
modified
protein
from
amaranth
seed
in
Escherichia
coli
and
effect
of
environmental
conditions
on
the
protein
expression
Claudia
Castro-Martíneza,1,
Silvia
Luna-Suárezb,1,
Octavio
Paredes-Lópezc,∗
aCentro
de
Investigación
para
el
Desarrollo
Integral
Regional,
CIDIIR-IPN,
Sinaloa,
Mexico
bCentro
de
Investigación
en
Biotecnología
Aplicada,
CIBA
-
IPN,
Mexico
cDepartamento
de
Biotecnología
y
Bioquímica,
Centro
de
Investigación
y
de
Estudios
Avanzados
del
IPN,
Apdo.
Postal
629,
36821
Irapuato,
Gto.,
Mexico
a
r
t
i
c
l
e
i
n
f
o
Article
history:
Received
27
October
2011
Received
in
revised
form
12
December
2011
Accepted
14
December
2011
Available online 9 January 2012
Keywords:
Amaranth
seed
Antihypertensive
peptides
11S
acidic
subunit
ACE
inhibitory
activity
a
b
s
t
r
a
c
t
Amaranth
seeds
are
considered
as
an
excellent
complementary
source
of
food
protein
due
to
their
balanced
amino
acid
composition.
Amarantin
acidic
subunit
has
the
potential
as
a
functional
and
nutraceutical
protein,
and
it
is
structurally
a
good
candidate
for
modification.
The
aim
of
this
work
was
to
improve
its
functionality,
then
the
primary
structure
was
modified
into
the
third
variable
region
of
11S
globulins,
by
inserting
antihypertensive
peptides:
four
Val-Tyr
in
tandem
and
Arg-Ile-Pro-Pro
in
the
C-terminal
region.
Modified
protein
was
expressed
in
Escherichia
coli
Origami
(DE3)
and
was
purified.
The
culture
conditions,
including
the
culture
media,
temperature,
agitation
speed
and
air
flow
were
tested
in
order
to
obtain
an
increased
expression
levels
of
the
modified
protein.
A
23factorial
design
was
used
for
evaluate
the
effect
of
environmental
conditions
on
modified
protein
production.
The
results
indicated
that
the
yield
of
modified
protein
could
be
increased
by
up
3-fold
in
bioreactor
as
compared
with
flask.
In
addition,
the
temperature,
the
agitation
speed
and
the
oxygen
were
significant
factors
on
the
expres-
sion
of
the
antihypertensive
protein.
The
maximum
production
was
99
mg
protein-L−1.
The
hydrolyzed
protein
showed
a
high
inhibitory
activity
of
the
angiotensin
converting
enzyme
(IC50 =
0.047
mg
mL−1).
© 2012 Elsevier B.V. All rights reserved.
1.
Introduction
The
modern
diet
together
with
a
sedentary
lifestyle
has
pro-
duced
an
epidemic
of
nutritionally
related
diseases.
Hypertension
is
one
of
these
important
diseases
in
our
society,
given
its
high
preva-
lence
and
its
role
in
cardiovascular
diseases,
including
coronary
heart
disease,
peripheral
arterial
disease,
end-stage
renal
disease
and
stroke
(Glasser,
2001;
Seppo
et
al.,
2003;
Madureira
et
al.,
2010).
About
one
billion
people
are
now
suffering
from
hyperten-
sion
and
it
is
expected
to
increase
60%
by
2025
worldwide
(Akama
et
al.,
2009;
Nakahara
et
al.,
2010).
Drugs
that
inhibit
the
renin-angiotensin
system
(important
regulator
of
blood
pressure),
either
by
inhibiting
angiotensin-
converting
enzyme
(ACE)
or
by
blocking
angiotensin
(AT1)
receptors,
are
widely
used
in
the
treatment
of
hypertension.
ACE
inhibitors
have
a
dual
effect
on
this
system:
they
inhibit
the
pro-
duction
of
the
vasoconstrictor
angiotensisn
II
and
they
inhibit
the
degradation
of
the
vasolidator
bradykinin
(Seppo
et
al.,
2003;
∗Corresponding
author
at:
Km
9.6
Libramiento
Norte
Carr.
Irapuato-León,
Apdo.
Postal
629,
C.P.
36821,
Irapuato,
Guanajuato,
Mexico.
Tel.:
+52
462
623
96
41;
fax:
+52
462
624
59
96.
E-mail
address:
oparedes@ira.cinvestav.mx
(O.
Paredes-López).
1These
authors
contributed
equally
to
this
work.
Madureira
et
al.,
2010;
Chobanian
et
al.,
1990);
in
vitro
inhibition
of
angiotensin
II
formation
has
been
used
for
screening
therapeu-
tic
agents
such
as
ACE
inhibitors
against
hypertension.
Chemically
synthesized
hypotensive
drugs,
such
as
captopril,
propranolol,
and
losartan
are
still
broadly
used
to
treat
and
prevent
hyperten-
sion.
Nevertheless,
these
drugs
are
reported
to
have
many
side
effects
such
as
dry
cough,
taste
disturbances,
skin
rashes,
and
many
other
dysfunctions
of
human
organs
(Fitzgerald
and
Meisel,
2000).
In
this
regard,
the
influence
of
nutritive
compounds
on
pre-
vention
and
treatment
of
hypertension
has
considerably
attracted
the
attention
during
the
last
decade.
Among
these
compounds
are
peptides
derived
from
food
proteins
that
exert
antihypertensive
activity
(Hu
et
al.,
1999;
Vercruysse
et
al.,
2005;
Erdmann
et
al.,
2008).
For
example,
various
studies
on
bioactive
peptides
in
amino
acid
sequences
of
natural
proteins
such
as
milk,
egg,
fish,
soybean,
spinach,
and
many
other
sources
have
been
reported
(Akama
et
al.,
2009;
Prak
et
al.,
2006;
Murray
and
FitzGerald,
2007;
Hong
et
al.,
2008).
These
protein-derived
bioactive
peptides
are
inactive
within
the
sequences
of
the
parent
proteins
but
can
be
released
by
enzy-
matic
proteolysis
during
gastrointestinal
digestion.
Once
liberated
in
the
body,
bioactive
peptides
many
act
as
regulatory
compounds
with
hormone-like
activity;
they
usually
contain
2–20
amino
acids
residues
per
molecule,
but
in
some
cases
may
consist
of
more
than
20.
Because
of
their
health-enhancing
potential
and
safety
profiles
0168-1656/$
–
see
front
matter ©
2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.jbiotec.2011.12.012

60 C.
Castro-Martínez
et
al.
/
Journal
of
Biotechnology
158 (2012) 59–
67
bioactive
peptides
may
be
used
as
components
in
functional
foods
or
nutraceuticals
(Erdmann
et
al.,
2008).
Antihypertensive
peptides
can
be
introduced
into
food
proteins
and/or
concentrated
for
the
purpose
of
treatment
of
a
disease
or
disorder,
for
example
in
those
people
with
cardiac
or
renal
insuffi-
ciency
or
diabetes
(Seppo
et
al.,
2003).
An
antihypertensive
effect
of
bioactive
peptides
were
demonstrated
by
studies
in
vivo
in
sponta-
neously
hypertensive
rats
(Nakamura
et
al.,
1995;
Muguerza
et
al.,
2006;
Quirós
et
al.,
2007)
and
in
human
subjects
(Hata
et
al.,
1996;
Seppo
et
al.,
2002;
Tuomilehto
et
al.,
2004;
Bütikofer
et
al.,
2008).
In
particular,
the
dipeptide
Val-Tyr
(VY)
is
one
of
the
biopeptides
tested
in
vivo
that
has
hypotensive
effect
in
humans
and
it
easily
can
be
absorbed
into
the
human
circulatory
blood
system
(Kawasaki
et
al.,
2000;
Matsui
et
al.,
2002).
Moreover,
Matsui
et
al.
(2005)
showed
that
this
small
peptide
(VY)
exerts
an
antiproliferative
effect
on
vascular
smooth
muscle
cells,
which
suggests
the
possibil-
ity
of
developing
novel
medicinal
foods
containing
this
peptide
to
prevent
certain
diseases.
Other
researchers
have
been
focused
on
the
two
antihypertensive
peptides,
Val-Pro-Pro
(VPP)
and
Ile-Pro-
Pro
(IPP);
their
antihypertensive
effects
in
rats
(Nakamura
et
al.,
1995),
and
in
humans
(Aihara
et
al.,
2005;
Mizuno
et
al.,
2005)
have
been
evaluated
in
several
clinical
trials.
These
studies
suggested
that
continuous
intake
of
VPP
and
IPP
might
have
the
potential
to
improve
arterial
stiffness
as
well
as
central
blood
pressure
and
peripheral
brachial
blood
pressure
(Mizuno
et
al.,
2005;
Nakamura
et
al.,
2009).
Moreover,
in
the
last
years,
various
expression
systems
using
mammalian
cells,
yeast
cells
or
prokaryotic
cells
have
been
estab-
lished
for
the
production
of
recombinant
proteins
(Yokoyama,
2003;
Kyle
et
al.,
2009).
Moreover,
several
studies
have
reported
that
Escherichia
coli
is
an
attractive
host
for
large-scale
produc-
tion,
as
it
grows
in
low-cost
media,
offers
good
genetic
stability,
has
abundant
available
plasmids
and
permits
scale-up
of
the
pro-
duction
process
without
loss
of
yield
(Romanos,
1995;
Hartmann
et
al.,
2008;
Riley
et
al.,
2009;
Moers
et
al.,
2010).
On
the
other
hand,
amaranth
seeds
(Amaranthus
hypochon-
driacus)
have
been
considered
as
an
excellent
alternative
or
complementary
source
of
food
protein
due
to
their
balanced
amino
acid
composition.
However,
their
potential
as
a
source
of
bioactive
peptides
has
been
little
explored
(Gorinstein
et
al.,
2002;
Vecchi
and
A˜
nón,
2009).
Amarantin
is
an
11S
globulin,
and
it
is
the
most
predominant
storage
proteins
in
seeds
of
amaranth,
its
amino
acid
composition
is
close
to
the
optimum
amino
acid
balance
required
in
the
human
diet,
and
has
remarkable
heat
stability
and
emulsi-
fying
properties
(Romero-Zepeda
and
Paredes-López,
1996).
This
protein
is
a
homohexameric
molecule
with
a
molecular
mass
of
398
kDa
and
it
has
two
disulfide
liked
subunits:
acidic
(32–34
kDa)
and
basic
(22–24
kDa).
Research
of
our
group
has
been
focused
on
the
expression
of
recombinant
amarantin
in
different
expression
systems
(Medina-
Godoy
et
al.,
2004);
expression
in
maize
plants
and
in
seeds
of
transgenic
tobacco,
resulting
in
important
increases
of
seed
pro-
tein
content
(Rascón-Cruz
et
al.,
2004;
Valdez-Ortiz
et
al.,
2005).
Recently,
Luna-Suárez
et
al.
(2008)
expressed
in
E.
coli,
purified
and
characterized
a
His-tagged
version
of
the
acidic
subunit
from
11S
amaranth
seed
protein
and
they
showed
that
this
protein
was
as
stable
as
the
complete
amarantin.
In
addition,
the
results
obtained
suggest
that
the
acidic
subunit
has
the
potential
as
a
functional
and
nutraceutical
protein,
and
it
is
structurally
a
good
candidate
for
modification.
Moreover,
using
protein
engineering,
further
charac-
teristics
could
be
incorporated
into
this
high-nutritional
protein,
such
as
bioactives
peptides
or
modified
amino
acid
sequence,
to
enhance
functional
and
nutraceutical
properties.
Thus,
the
objective
of
the
present
study
was
to
improve
the
nutraceutical
properties
of
the
amarantin.
Therefore,
the
pri-
mary
structure
of
this
acidic
subunit
was
modified
with
two
antihypertensive
peptides
in
two
sites:
in
the
globulins
11S
III
vari-
able
region
were
inserted
two
amino
acids
in
tandem
of
four
(Val-
Try),
and
in
the
C-terminal
of
the
acidic
subunit
were
inserted
four
amino
acids
(Arg-Ile-Pro-Pro).
The
modified
protein
was
expressed
in
E.
coli.
We
also
purified
and
determined
the
ACE
inhibitory
activ-
ity
in
vitro
of
this
protein.
Moreover,
we
evaluated
the
influence
of
the
environmental
factors
(temperature,
agitation,
oxygen)
on
the
expression
of
the
modified
amarantin
acidic
subunit.
However,
an
efficient
production
system
in
a
bioreactor
was
necessary,
while
there
have
been
no
such
report
for
the
amarantin
production.
2.
Materials
and
methods
2.1.
Strains
and
plasmids
E.
coli
TOP10
(Invitrogen,
Carlsbad,
CA)
was
used
for
plas-
mid
routine
transformation
and
propagation.
E.
coli
Origami
(DE3)
(Novagem,
Markham,
Ontario,
Canada)
was
used
for
the
expres-
sion
of
the
modified
amarantin
acidic
subunit.
Origami
host
strains
are
K-12
derivates,
which
carries
mutations
in
both
the
thiore-
doxin
reductase
(trxB)
and
the
glutathione
reductase
(gor)
genes,
which
enhance
disulfide-bond
formation
in
the
cytoplasm
of
E.
coli
(Berrow
et
al.,
2006).
The
plasmid
pET-AC-M1
(Luna-Suárez
et
al.,
2010)
which
contains
the
encoding
sequence
of
the
amarantin
acidic
subunit
modified
in
the
third
variable
region
with
the
inser-
tion
of
the
codons
for
four
VY
biopeptides
in
tandem,
was
used
as
source
of
DNA
template
for
PCR.
Cloning
vector
pPCR®TOPO
2.1
(Invitrogen,
Carlsbad,
CA)
was
used
to
clone
the
PCR
prod-
ucts,
according
to
manufacturer’s
instructions.
Expression
plasmid
pET-32b(+)
(Novagen,
Markham,
Ontario,
Canada)
was
used
for
modified
amarantin
acidic
subunit
expression.
Expression
of
ama-
rantin
acidic
subunit
was
under
the
control
of
a
T7
promoter
induced
with
the
isopropyl-beta-d-thiogalactoside
(IPTG).
2.2.
Construction
of
the
modified
amarantin
acidic
subunit
expression
plasmid
The
expression
plasmid
pET-AC-M1
(Luna-Suárez
et
al.,
2010)
was
used
as
PCR
template
to
construct
plasmid
pET-
AC-M36H
plasmid,
specific
oligonucleotides
were
designed
for
PCR
amplification
that
include
the
region
of
the
acidic
sub-
unit
and
a
sequence
of
six
histidines
just
before
the
peptide
RIPP
on
the
COOH
terminal.
The
primers
for
amplification
were:
forward
5-GGGTGATTAATGGAAGGAAGGTTTAGAGAGTTTCAAC-3
(VspI
restriction
site
is
underlined
and
start
codon
is
in
italics)
and
reverse
5ATGGAGTGTGGTGGTGGTGGTGGTGTCTTAAGGAGGAATC
CTATTGGGAAGGTAC-3(EcoRI
restriction
site
is
in
italics,
stop
codon
is
underlined
in
italics,
the
6-His
encoding
sequence
inserted
is
underlined,
and
the
RIPP
peptide
encoding
sequence
inserted
is
in
bold
letter).
After
amplification,
the
PCR
product
was
ligated
into
pPCR®2.1
TOPO®.
E.
coli
cells
harboring
recombinant
plasmid
were
selected
on
LB
plates
containing
100
␮g
ml−1ampicillin
and
X-gal.
The
DNA
fragment
enconding
amarantin
acidic
subunit
M36His
was
released
from
pPCR®2.1
TOPO®vector
using
VspI
and
EcoRI
restriction
enzymes.
The
VspI/EcoRI
fragment
was
ligated
into
plas-
mid
pET-32b(+)
and
transformed
into
the
TOP
10
cloning
host.
E.
coli
transformants
were
selected
on
LB
plates
containing
100
␮g
ml−1
carbenicillin.
The
positive
clones
were
confirmed
by
PCR
amplifi-
cation,
restriction
analysis
and
DNA
sequencing.
2.3.
Transformation
of
expression
cells
E.
coli
Origami
(DE3)
cells
were
transformed
with
plasmid
pET-
AC-M36His
following
instructions
provided
by
the
manufacturers,

C.
Castro-Martínez
et
al.
/
Journal
of
Biotechnology
158 (2012) 59–
67 61
and
selected
on
LB
agar
plates
containing
100
␮g
ml−1penicillin,
15
␮g
ml−1kanamycin
and
12.5
␮g
ml−1tetracycline.
2.4.
Expression
and
detection
of
modified
amarantin
acidic
subunit
in
E.
coli
induced
cells
The
expression
and
detection
of
the
modified
amarantin
acidic
subunit
were
carried
out
according
to
Luna-Suárez
et
al.
(2008)
using
E.
coli
Origami
(DE3)
cells
harboring
the
plasmid
pET-AC-
M36His.
IPTG
was
added
at
four
different
concentrations
(0.1,
0.2,
0.3
and
0.4
mM),
when
the
cultures
reached
0.3–0.4
OD
at
600
nm
and
incubation
was
continued
after
IPTG
addition,
samples
were
taken
at
0,
3,
6
and
24
h.
In
order
to
determine
the
best
induction
time
for
the
modified
acidic
subunit
expression,
samples
of
1
mL
of
each
culture
were
transferred
to
a
1.5
mL
centrifuge
tube.
Sample
cells
were
harvested
by
centrifuging
at
12,000
×
g
for
3
min
at
room
temperature.
Supernatants
were
discharged
and
cell
pellets
were
stored
at
−20 ◦C
until
their
protein
content
could
be
determined.
Cell
pellets
were
resuspended
in
100
␮L
of
water
plus
100
␮L
of
loading
buffer
[62.5
mM
Tris–HCl,
pH
6.8,
2%
(w/v)
SDS,
10%
(v/v)
2-mercaptoethanol]
and
heated
at
95 ◦C
for
5
min.
Samples
were
analyzed
by
12%
SDS–PAGE
(Laemmli,
1970)
and
proteins
stained
with
Coomassie
brilliant
blue.
Modified
protein
was
identified
by
Western
blot,
using
as
primary
antibody
rabbit
polyclonal
anti-
bodies
against
amarantin
(1:60000
dilution),
and
goat
antirabbit
IgG
(H+L)
antibody
conjugated
to
alkaline
phosphatase
(Bio-Rad,
Hercules,
CA),
as
the
secondary
one
(1:3000
dilution).
As
negative
control,
transformed
E.
coli
Origami
(DE3)/pET-32b(+)
cells
were
cultured
and
sampled
as
described.
Quantitative
analyses
of
mod-
ified
amarantin
acidic
subunit
accumulation
in
this
and
further
sections
were
carried
out
densitometrically,
using
the
Quantity
One
Software
v.4.2.1
(Bio-Rad
laboratories,
Hercules,
CA).
2.5.
Cultivation
conditions
to
improve
the
expression
of
modified
amarantin
acidic
subunit
Shake
flask
cultures
were
performed
in
250
mL
Erlenmeyer
flasks
containing
50
mL
of
appropriate
medium
at
37 ◦C
on
an
orbital
shaker
(200
rpm).
Cultivations
in
fermentor
were
carried
out
in
a
5
L
bioreactor
(BIOFlo
II
C,
New
Brunswick
Scientific
Co.,
USA)
with
a
working
volume
of
3.5
L.
The
pH
was
measured
dur-
ing
the
fermentations
and
was
not
controlled.
Both
shake
flask
and
bioreactor
cultivations
were
inoculated
with
2.5%
(v/v)
with
over-
night
shake
flask
culture
made
from
the
LB
medium
and
incubated
at
37 ◦C
on
a
rotary
shaker
at
an
agitation
frequency
of
200
rpm.
The
growth
kinetic
was
done
following
the
absorbance
at
600
nm
of
each
culture
of
recombinant
E.
coli
cells
harboring
the
pET-AC-M36His
plasmids
with
and
without
inductor.
All
fermen-
tations
were
carried
out
in
duplicate.
2.6.
Expression
improvement
of
the
modified
amarantin
acidic
subunit
In
order
to
improve
the
accumulation
of
the
modified
amarantin
acidic
subunit,
first
we
tested
in
Erlenmyer
flasks
two
different
media
for
the
cultivations:
(1)
Luria-Bertain
(LB)
medium
and
(2)
Terrific
broth
(TB).
Luria-Bertain
medium
was
prepared
using
LB
broth
from
Sigma,
20
g
L−1.
Terrific
broth
medium
contained
(per
litre):
12.0
g
tryptone,
24.0
g
yeast
extract,
4
mL
glycerol,
KH2PO4
2.3
g,
K2HPO412.5
g.
The
cultures
were
incubated
at
37 ◦C
in
an
orbital
shaker.
The
best
of
these
media
was
then
employed
for
sub-
sequent
experiments.
The
second
step
for
the
over
accumulation
of
the
modified
amarantin
was
to
evaluate
the
effect
of
the
environ-
mental
conditions,
such
as:
temperature,
speed
of
agitation
and
air
flow
in
bioreactor
(Table
1).
The
effects
of
environmental
conditions
Table
1
Coded
factor
levels
and
real
values
for
the
experimental
design.
Factor
Low
value
High
value
(−1)
(+1)
Temperature
(◦C)
28 33
Speed
of
agitation
(rpm) 200
400
Air
flow,
oxygen
(vvm)
0
0.2
were
assessed
via
comparisons
of
the
yields
of
the
protein
of
inter-
est
(mg
mL−1)
and
volumetric
productivity
(mg
L−1h−1),
according
to
the
statistical
results.
Expression
of
modified
amarantin
acidic
subunit
started
when
the
culture
reached
0.3–0.4
OD
at
600
nm
by
adding
IPTG
at
0.3
mM
final
concentration;
the
incubation
was
continued
for
6
h.
2.7.
Experimental
design
A
simple
factorial
design
for
three
factors
with
replicates
at
the
centre
point
was
used
for
evaluate
modified
protein
production
in
E.
coli
Origami
(DE3).
The
variables
used
were
temperature
(T),
speed
of
agitation
(Ag)
and
air
flow
(O2)
each
at
three
coded
levels
(−1,
0,
1)
as
shown
in
(Table
1).
All
experiments
were
carried
out
in
the
bioreactor
containing
Terrific
broth
plus
antibiotics
and
IPTG
at
0.3
mM
final
concentrations.
The
statistical
software
package,
Statgraphics,
Centirion
XVI
(Statistical
Graphics
Corp,
Herndon,
USA)
was
used
for
the
anal-
ysis
of
experimental
data.
An
analysis
of
variance
(ANOVA)
was
used
to
estimate
the
statistical
parameters
for
improvement
of
the
modified
protein
production
and
fermentation
conditions.
2.8.
Analysis
of
soluble
and
insoluble
fraction
To
determine
in
which
fraction
(soluble
or
insoluble)
the
modi-
fied
amarantin
acidic
subunit
was
accumulated,
the
cultures
were
fractionated
according
to
Luna-Suárez
et
al.
(2010).
The
fractions
were
subjected
to
12%
SDS–PAGE.
Proteins
were
visualized
with
staining
solution
containing
Coomassie
Brilliant
Blue.
Modified
amarantin
acidic
subunit
was
detected
by
Western
blot
analysis.
2.9.
Protein
measurement
All
protein
concentrations
were
determined
using
the
Pierce
BCA
Protein
Assay,
a
bicinchoninic
acid
method
(Rockfrod,
IL),
using
BSA
(Sigma,
St.
Louis,
MO)
as
a
protein
standard.
2.10.
Purification
of
the
modified
acidic
subunit
from
amarantin
The
insoluble
crude
protein
extract
was
differentially
pre-
cipitated
by
the
addition
of
ammonium
sulfate
at
four
distinct
saturation
range:
0–10%,
10–20%,
20–30%
and
30–40%;
fractions
harboring
modified
amarantin
acidic
subunit
were
dialyzed
and
applied
to
an
electrophoresis
system
Protean®Plus
DodecaTM Cell
(Bio-Rad,
Hercules,
CA).
The
proteins
were
stained
by
copper,
distained
by
EDTA
and
were
electroeluted
using
the
Whole
Gel
Electroeluter
(Bio-Rad,
Hercules,
CA).
2.11.
Two-dimensional
electrophoresis
To
determine
the
isoelectric
point
of
the
modified
subunit,
5
␮g
of
protein
were
subjected
to
isoelectric
focusing
using
a
7
cm
immo-
bilized
strip
of
3–10
pH
gradient
(Bio-Rad,
Hercules
CA)
in
a
Protean
IEF
Cell
(Bio-Rad,
Hercules
CA).
After
focusing,
the
strip
was
applied
to
12%
SDS–PAGE,
and
then
the
amarantin
acidic
subunit
was
visualized
by
Coomassie
Brillant
Blue
staining
and
Western
blot
analysis.
Protein
standards
were
separated
in
the
same
way.

62 C.
Castro-Martínez
et
al.
/
Journal
of
Biotechnology
158 (2012) 59–
67
2.12.
In
vitro
digestion
of
the
modified
acidic
subunit
from
amarantin
The
modified
acidic
subunit
from
amarantin
(1
mg
mL−1)
was
adjusted
to
pH
8.0
and
digested
with
trypsin
and
chymotrypsin
(E/S
=
1/200
(w/w))
for
18
h
at
37 ◦C
(Mallikarjun
et
al.,
2006).
After
digestion,
the
solution
was
boiled
to
stop
the
reaction,
and
the
reac-
tion
mixture
was
centrifuged.
This
suite
of
peptides
was
used
as
the
source
of
ACE
inhibitory
peptides.
2.13.
ACE
inhibitory
activity
ACE-inhibitory
activity
was
measured
by
spectrophotometric
assay
(Hernández-Ledesma
et
al.,
2007)
with
some
modifications.
Summarized,
80
␮L
of
each
sample
was
added
to
200
␮L
of
0.1
M
sodium
borate
buffer
(pH
8.3)
containing
0.3
M
NaCl
and
5
mM
Hyp-
puryl
Histidyl
Leucine
(HHL).
ACE
(1
mU)
(EC
3.4.15.1;
5.1
U
mg−1)
was
added
and
the
reaction
mixture
was
incubated
at
37 ◦C
for
30
min.
The
reaction
was
terminated
by
the
addition
of
250
␮L
1
M
HCl.
The
hippuric
acid
formed
was
extracted
with
ethyl
acetate,
then
it
was
centrifuged
and
it
was
heat
evaporated
at
95 ◦C
for
10
min,
redissolved
in
distilled
water,
and
measured
spectropho-
tometrically
at
228
nm.
The
activity
of
each
sample
was
tested
in
triplicate.
The
ACE-inhibitory
activity
was
calculated
as
the
peptide
concentration
needed
to
cause
50%
inhibition
of
the
original
ACE
activity
(IC50).
The
results
were
analyzed
by
analysis
of
variance
(ANOVA)
followed
by
Fisher’s
tests
to
assess
differences
among
treatments.
3.
Results
and
discussion
3.1.
Introduction
of
IPP
peptide
into
amarantin
acidic
subunit
The
amarantin
acidic
subunit
harbors
four
hipervariable
regions
of
the
five
detected
in
the
11S
seed
globulins
(Wright,
1988;
Dickinson
et
al.,
1990;
Adachi
et
al.,
2003).
The
antihyperten-
sive
peptide
IPP
was
introduced
by
site
directed
mutagenesis
into
the
fourth
variable
region,
at
the
carboxi
terminal
region
of
the
amarantin
acidic
subunit
to
produce
the
expression
vector
pET-AC-
M3-6His
(Fig.
1).
The
constructed
plasmid
was
sequenced
to
verify
the
insertion
(data
not
shown).
The
six
histidine
tag
was
inserted
after
the
sequence
S
R
Y
L
P
N,
after
that,
was
inserted
an
R
aminoacid
in
order
to
release
IPP
peptide
by
trypsin,
wich
was
inserted
after
R
(Keil,
1992).
We
use
the
PSIPRED
protein
structure
prediction
server
(bioinf.cs.ucl.ac.uk/psipred/psiform.html)
to
assess
the
structural
changes
because
of
the
biopeptides
insertion;
we
found
that
the
insertion
did
not
produce
difference
in
the
secondary
structure
of
the
modified
protein
(data
not
shown).
3.2.
Expression
of
the
modified
amarantin
acidic
subunit
in
E.
coli
To
express
the
modified
amarantin
acidic
subunit,
the
plasmid
pET-AC-M3-6His
was
introduced
into
E.
coli
strain
Origami
(DE3),
then
cells
were
grown
in
LB
broth
at
37 ◦C
and
induced
at
different
IPTG
concentrations
(0.1,
0.2,
0.3,
0.4
mM).
Analysis
of
SDS–PAGE
gels
revealed
that
the
modified
amarantin
of
∼32
kDa
was
accumu-
lated
from
0.1
mM
of
IPTG
(Fig.
2).
As
can
been
seen
in
Fig.
2A
(lanes
1–4)
the
results
show
that
the
IPTG
concentration
used
for
induc-
tion
have
an
effect
on
the
modified
amarantin
accumulation,
being
0.3
mM
the
best
concentration
of
inductor.
Moreover,
Fig.
2B
shows
the
inmmunological
detection
of
the
modified
amarantin
(Western
blot)
of
the
total
protein
extracts
from
cell
culture
of
E.
coli
harbor-
ing
the
pET-AC-M3-6His
plasmid
at
different
induction
times.
The
level
of
protein
expression
observed
after
induction
(0.3
mM)
for
3
h
was
estimated
to
be
9.3
mg
of
modified
amarantin-L−1culture
(Table
2).
After
this
time,
protein
accumulation
decreased,
which
may
be
due
to
the
proteloltic
activity
as
deduced
by
the
appearance
of
a
low-molecular
weight
band
(Fig.
2B,
lanes
2
and
3).
The
level
of
expression
of
this
protein
was
less
than
that
obtained
by
our
lab-
oratory
group
in
the
same
E.
coli
strain
harboring
the
pET-AC-6His
Fig.
1.
Amarantin
acidic
subunit
expression
vector
pET-AC-M36His,
harboring
a
low
copy
number
pBR322
origin
and
ampicillin
resistance
gene
(AmpR).

C.
Castro-Martínez
et
al.
/
Journal
of
Biotechnology
158 (2012) 59–
67 63
Fig.
2.
SDS–PAGE
(A)
and
Western
blot
(B)
analysis
of
total
protein
extract
from
E.
coli
strain
Origami
(DE3).
(A)
lanes
1–4,
E.
coli/pET-AC-M3-6His
induced
with
0.1,
0.2,
0.3,
0.4
mM
IPTG
respectively
at
3
h
after
induction;
M,
protein
molecular
weight
marker.
(B)
Different
times
of
expression.
Lanes
1–3:
3,
6
and
24
h
after
induction
respectively;
lane
4:
E.
coli/pET-AC-M3-6His
uninduced;
M,
protein
molecular
weight
marker.
The
arrow
indicates
modified
amarantin
acidic
subunit.
Approximately
equal
amounts
of
total
cell
proteins
were
loaded
into
each
lane.
vector
(Luna-Suárez
et
al.,
2008)
was
also
less
than
that
obtained
with
the
complete
amarantin
using
a
richer
culture
broth
(Medina-
Godoy
et
al.,
2004)
and
the
level
was
more
than
the
6.2
mg
L−1
obtained
with
the
acidic
subunit
modified
only
in
the
third
region
by
insertion
of
four
VY
peptides
(Luna-Suárez
et
al.,
2010),
this
may
indicate
that
the
new
version
of
protein
is
more
stable
than
the
first
one.
3.3.
Improvement
of
the
expression
of
modified
amarantin
acidic
subunit
in
E.
coli
The
improvement
of
the
expression
of
modified
amarantin
in
E.
coli
was
evaluated
in
both
shake
flask
and
bioreactor
culture.
First
we
tested
the
effect
of
culture
medium
in
shake
flask.
After
selec-
tion
of
culture
medium
we
investigated
the
effect
of
environmental
conditions
in
bioreactor.
3.3.1.
Effect
of
culture
medium
In
the
shake
flask
culture
the
final
cell
density
(data
not
shown)
and
yield
were
found
to
depend
upon
the
media
used.
Terrific
broth
medium
achieved
yield
of
30.1
mg
of
modified
amarantin
acidic
subunit/L
of
culture
medium.
Up
to
3.2
fold
increase,
in
comparison
to
the
modified
amarantin
expression
under
standard
conditions
(Fig.
3,
Table
2),
and
a
similar
higher
value
for
volumetric
produc-
tivity
(Table
2).
This
result
indicates
that
Terrific
medium
is
best
than
LB
medium
for
overproduction
of
modified
amarantin
by
E.
coli
Origami
(DE3).
Beneficial
effects
of
complex
additives
have
been
earlier
reported
by
some
bioreactor
studies.
For
example,
Tsai
et
al.
(1987)
reported
a
10-fold
increase
in
intracellular
human
IGF-1
accumula-
tion
by
the
addition
of
yeast
extract
and
tryptone.
Terrific
medium
is
rich
in
yeast
extract
and
phosphate
salts,
additionally
is
sup-
plemented
with
glycerol.
Then,
yeast
extract
is
a
known
source
of
large
quantities
of
free
amino
acids,
short
peptides
and
growth
fac-
tors
that
improve
modified
amarantin
production
(Tripathi
et
al.,
2010).
Additionally,
terrific
medium
contain
glycerol
that
and
it
has
glycerol
which
is
used
to
increase
plasmid
yield
(Sambrook
et
al.,
1989).
On
the
other
hand,
our
modified
amarantin
yield
resulted
lower
than
that
obtained
with
the
non
modified
acidic
subunit
at
the
same
conditions
(Luna-Suárez
et
al.,
2008),
which
suggests
that
the
modified
protein
is
less
stable
than
the
non
modified
sample.
3.3.2.
Effect
of
environmental
conditions
In
order
to
improve
the
productivity
of
the
laboratory-scale
microbial
process
for
the
production
of
the
recombinant
amarantin,
Table
2
Comparison
of
modified
amarantin
expression
levels
in
recombinant
E.
coli
Origami
(DE3)
grown
using
different
culture
medium
and
environmental
conditions.
Conditions
Y
(mg
L−1)
P
(mg
L−1h−1)
Shake
flasks LB
9.3a3.1b
Terrific
30.1c10d
Bioreactor*Exp
1:
(28 ◦C,
200
rpm,
0
vvm)
69.7e23.2j
Exp
2:
(33 ◦C,
200
rpm,
0
vvm)
78.6f31.4k
Exp
3:
(28 ◦C,
400
rpm,
0
vvm)
73.2f29.2k
Exp
4:
(33 ◦C,
400
rpm,
0
vvm)
83.3g33.3k
Exp
5:
(28 ◦C,
200
rpm,
0.2
vvm)
93.6h37.4l
Exp
6:
(33 ◦C,
200
rpm,
0.2
vvm)
99.0i39.4l
Exp
7:
(28 ◦C,
400
rpm,
0.2
vvm)
74.3f37.2l
Exp
8:
(33 ◦C,
400
rpm,
0.2
vvm)
82.3g41.2l
Center
point:
(30.5 ◦C,
300
rpm,
0.1
vvm)
85.1g34.2k
Results
from
three
repetitions:
in
shake
flasks
different
letters
indicate
significant
difference
(p
<
0.01).
*An
analysis
of
variance
(ANOVA)
and
t-test
were
used
to
estimate
the
statistical
parameters
to
evaluate
the
effect
of
the
environmental
conditions
on
modified
amarantin
expression.
Y,
modified
amarantin
yield;
P,
modified
amarantin
volumetric
productivity.
Different
letters
indicate
significant
difference
(p
<
0.05).

64 C.
Castro-Martínez
et
al.
/
Journal
of
Biotechnology
158 (2012) 59–
67
Fig.
3.
SDS–PAGE
and
Western
blot
analyses,
effect
of
culture
medium.
Lane
M,
protein
molecular
weight
marker;
lane
1,
E.
coli/pET-AC-M3-6His
uninduced;
lane
2,
expression
of
modified
amarantin
using
LB
broth;
lane
3,
expression
of
modi-
fied
amarantin
using
Terrific
broth;
M,
protein
molecular
weight
marker.
The
arrow
indicates
modified
amarantin
acidic
subunit.
Approximately
equal
amounts
of
total
cell
proteins
were
loaded
into
each
lane.
we
investigated
the
effect
of
environmental
conditions
both
on
the
growth
of
Origami
(DE3)
E.
coli
cells
carrying
the
pET-AC-M3-6His,
and
on
their
modified
protein
yield
and
volumetric
productivity.
The
analysis
of
variance
(ANOVA)
of
Table
2
showed
that
the
yield
(mg
L−1)
and
the
volumetric
productivity
(mg
L−1h−1)
pre-
sented
the
same
high
correlation
coefficients
(>0.95).
From
ANOVA
analysis
for
modified
amarantin
yield,
linear
terms
T,
Ag,
O2
and
interaction
term
Ag*O2were
statistically
significant
(p
<
0.05)
(Fig.
4A).
The
ANOVA
analysis
for
volumetric
productivity
shown
the
same
behavior
that
modified
amarantin
yield
for
linear
terms
and
for
interaction
term,
all
interaction
terms
were
statistically
significant
(p
<
0.05)
except
interaction
term
T*Ag
(Fig.
4B).
The
sta-
tistical
significance
of
the
model
equation
was
evaluated
by
the
t-test
which
showed
that
the
regression
is
statistically
significant
at
95%
(p
<
0.05)
confidence
level.
Analysis
of
the
standardized
Pareto
chart
from
this
experiment
indicated
that
the
air
flow
is
the
condition
that
most
significantly
influences
modified
amarantin
expression,
followed
by
the
tem-
perature
and
the
speed
of
agitation.
High
temperature
and
high
air
flow
have
a
positive
effect
on
modified
amarantin
yield,
whereas
high
speed
of
agitation
diminishes
the
modified
protein
produc-
tion.
In
addition,
this
analysis
shows
that
the
speed
of
agitation
and
air
flow
has
a
negative
synergistic
effect
on
the
modified
ama-
rantin
expression
(Fig.
4A).
This
effect
is
different
than
the
reported
the
recombinant
malarial
antigen
(Yazdani
et
al.,
2004).
The
strong
influence
positive
of
air
flow
on
volumetric
productivity
is
also
observed
in
Fig.
4B.
In
addition,
an
increase
of
the
temperature
and
of
the
speed
of
agitation
favored
the
modified
amarantin
produc-
tivity.
However,
a
slightly
negative
effect
for
the
interaction
T–O2
and
Ag–O2was
observed
(Fig.
4B).
Maximum
modified
amarantin
expression
was
obtained
at
33 ◦C,
200
rpm
and
with
air
flow
of
the
0.2
vvm
(99
mg
mL−1)
(Table
2).
This
condition
in
bioreactor
gave
more
than
a
3-fold
Fig.
4.
Effect
of
environmental
conditions
on
modified
amarantin
expression
in
recombinant
E.
coli
cells
(see
Table
1).
Pareto
charts
showing
the
relative
effects
of
environmental
conditions
on
modified
amarantin
production
were
obtained
using
the
statistical
software
Statgraphics,
Centirion
XVI
(see
Table
2).
(A)
Effects
of
envi-
ronmental
conditions
on
modified
amarantin
yield.
(B)
Influence
of
environmental
conditions
on
modified
amarantin
volumetric
productivity.
Extension
of
the
bars
at
the
right
or
the
vertical
line
indicates
the
significant
factors
among
the
selected
ones
for
positive
values
and
vice
versa
by
negatives
values.
increase
in
yield
or
the
modified
amarantin
accumulation,
when
compared
to
Terrific
broth
in
shake
flask,
and
almost
a
four-fold
increase
in
productivity.
According
to
previous
observations,
these
results
shown
that
the
yield
of
the
modified
amarantin
(99
mg/L)
was
higher
than
32
mg/L
obtained
using
the
nonmodified
amarantin
acidic
sub-
unit
(Luna-Suárez
et
al.,
2008),
higher
than
60
mg/L
obtained
using
the
third
region
modified
amarantin
acidic
subunit
(Luna-Suárez
et
al.,
2010)
and
higher
than
that
described
by
previous
researches
using
vectors
encoding
soybean
11S
storage
proteins
(Tai
et
al.,
1999).
The
yield
was
also
higher
than
40
mg
L−1previously
reported
on
proamarantin-His-tagged
using
pET-AMAR-6His
vector
and
the
same
E.
coli
strain
(Medina-Godoy
et
al.,
2004).
Finally,
is
higher
than
55
mg
L−1of
the
His-tag
amarantin
acidic
subunit
reported
by
our
group
(Luna-Suárez
et
al.,
2008).
3.4.
Analysis
of
soluble
and
insoluble
fractions
from
induced
cells
An
analysis
of
soluble
fraction
and
insoluble
fraction
was
car-
ried
out
in
order
to
know
in
which
fraction
is
present
our
modified
protein.
SDS–PAGE
gels
and
Western
blot
analysis
with
soluble
and
insoluble
fractions
from
induced
E.
coli
cells
after
induction
at
37 ◦C
are
presented
in
Fig.
5;
a
higher
accumulation
of
the
modified
pro-
tein
was
in
the
insoluble
fraction
(Fig.
5A
and
B,
lane
2).
This
result

C.
Castro-Martínez
et
al.
/
Journal
of
Biotechnology
158 (2012) 59–
67 65
Fig.
5.
SDS–PAGE
(A)
and
Western
blot
analyses
(B)
of
soluble
(lane
1)
and
insolu-
ble
(lane
2)
protein
fractions
of
induced
E.
coli
cells
harboring
the
pET-AC-M3-6His
expression
cassette,
experiment
at
0.3
mM
IPTG
and
37 ◦C;
M,
protein
molecular
weight
marker.
The
arrow
indicates
modified
amarantin
acidic
subunit.
suggests
that
modified
amarantin
acidic
subunit
is
accumulated
in
inclusion
bodies
in
E.
coli,
which
implies
that
an
improper
folding
of
this
subunit
is
occurring
in
the
oxidizing
environment
found
in
the
E.
coli
cytoplasm
(Fahnert
et
al.,
2004);
it
is
also
possible
that
the
folding
machinery
of
E.
coli
is
not
able
on
stabilize
the
modified
sub-
unit
through
an
appropriate
protein
structure
(Baneyx
and
Mujacic,
2004),
as
reported
by
Luna-Suárez
et
al.
(2008)
for
the
nonmodified
amarantin
acidic
subunit
and
by
Luna-Suárez
et
al.
(2010)
for
the
modified
acidic
subunit.
3.5.
Purification
of
the
modified
amarantin
acidic
subunit
For
the
first
step,
the
induced
cell
debris
of
the
insoluble
fraction
of
modified
amarantin
was
purified
using
ammonium
sulfate.
This
protein
was
detected
in
20–30%
and
30–40%
fractions;
then
these
fractions
were
electroeluted
(Fig.
6).
It
was
purified
with
a
50%
yield
to
a
∼
90%
purity
(as
judged
by
SDS–PAGE);
about
50
mg
of
pure
modified
protein
per
liter
of
Terrific
broth
culture
were
obtained,
which
is
higher
in
comparison
with
the
results
reported
for
Fig.
6.
Purification
stages
of
modified
amarantin
acidic
subunit.
Lanes:
M,
Molecular
marker;
1,
total
protein
extract;
2,
insoluble
fraction;
3,
combined
protein
precip-
itated
at
20–30%
and
30–40%
ammonium
sulfate
saturation
ranges
and
4,
electro
eluted
protein.
Luna-Suárez
et
al.
(2008)
for
the
nonmodified
amarantin
acidic
subunit
(30
mg/L).
Is
higher
than
third
region
modified
amarantin
acidic
subunit
(Luna-Suárez
et
al.,
2010).
In
addition,
our
mod-
ified
acidic
subunit
value
is
comparable
than
that
obtained
for
His-tagged
complete
proamarantin
harboring
the
plasmid
pPICZ-
AMAR6His
T7
into
Pichia
pastoris,
a
high
gene
dosage
vector
expressed
in
E.
coli
Origami
(DE3)
(Medina-Godoy
et
al.,
2004).
3.6.
2D-Electrophoresis
To
assess
the
isoelectric
point
(pI)
of
the
modified
amarantin
acidic
subunit
we
used
2D-electrophoresis.
This
protein
had
a
pI
of
5.87,
which
indicates
that
this
value
is
only
0.1
pH
units
smaller
than
the
theoretical
pI.
In
addition,
the
modified
protein
showed
a
molecular
weight
of
33.3
kDa,
assessed
by
SDS–PAGE
using
the
Quantity
One
software
(Bio-Rad,
Hercules,
CA),
this
value
is
only
0.3
smaller
than
the
theoretical
one.
3.7.
In
vitro
ACE
inhibitory
activity
In
this
paper
we
determined
in
vitro
ACE
inhibitory
activity
of
the
modified
amarantin
and
this
result
was
compared
with
previous
studies
of
potential
sources
of
ACE
inhibitory
activity.
In
this
work,
the
ACE
IC50 value
(50%
inhibitory
concentration)
of
the
digested
modified
amarantin
acidic
subunit
was
0.047
mg/mL.
In
contrast,
previous
studies
in
our
laboratory
reported
that
the
ACE
IC50 value
of
the
digested
modified
amarantin
(third
variable
region
insertion
of
4
VY
peptides)
was
0.064
mg/mL
and
the
ACE
IC50 value
of
the
digested
nonmodfied
acidic
subunit
was
0.483
mg/mL;
these
val-
ues
are
nearly
1.4-fold
and
8-fold
higher
than
the
protein
evaluated
in
this
work,
respectively.
Thus,
the
modified
protein
containing
the
antihypertensive
peptides
(four
units
of
VY
and
one
unit
of
IPP)
had
a
significant
effect
(p
<
0.05)
on
inhibition
of
the
ACE.
In
addition,
the
ACE
IC50 value
obtained
here
resulted
to
be
bet-
ter
than
reported
for
an
optimized
hydrolysis
of
Acetes
chinensis
(1.17
mg
mL−1)
proposed
as
a
potential
source
of
bioactive
peptides
(Cao
et
al.,
2011).
It
is
better
than
the
obtained
from
cowpea
Vigna
unguiculata
hydrolysate
(0.112
mg
mL−1),
the
ACE
inhibitory
activ-
ity
of
the
modified
acidic
subunit
is
better
than
the
reported
for
a
recombinant
antihypertensive
peptide
multimer
(0.1
mg
mL−1)
proposed
as
potent
antihypertensive
activity
(Rao
et
al.,
2009).
Moreover,
the
ACE
inhibitory
activity
of
the
modified
acidic
is
better
than
the
commercial
product
PeptACE
Peptides
(IC50 =
0.114
mg
mL−1)
from
Natural
Factors
Nutritional
Products
Ld.
(Coquitlam,
BC,
Canada)
reported
by
Cinq-Mars
and
Li-Chan
(2007).
Then,
we
can
consider
that
the
new
modified
amarantin
(with
vector
pET-AC-M3-6His)
has
potent
antihypertensive
activ-
ity.
Furthermore,
the
purified
modified
amarantin
may
be
used
as
food
additives
in
functional
foods
as
well
as
drugs
for
treating
and
preventing
hypertension.
4.
Concluding
remarks
In
this
study,
the
amarantin
acidic
subunit
was
genetically
mod-
ified;
we
inserted
two
antihypertensive
peptides.
The
modified
protein
was
expressed
in
E.
coli,
and
identified
by
SDS–PAGE
and
Western
blot
analyses;
it
was
less
stable
than
the
nonmodified
one.
Moreover,
using
a
simplified
factorial
design
approach,
we
set
up
an
efficient
expression
protocol
that
allowed
the
increase
of
the
expression
on
modified
amarantin
in
E.
coli
(up
to
99
mg
L−1),
which
is
significantly
higher
than
shake
flasks
conditions.
The
cul-
ture
media,
the
temperature,
the
speed
to
agitation
and
the
air
flow
were
important
parameters
that
determine
the
yields
and
produc-
tivity
of
the
antihypertensive
protein
from
amaranth
seeds.
The
air
flow
is
the
condition
that
most
significantly
influences
modified
amarantin
expression,
and
is
adversely
affected
only
by
speed
of

66 C.
Castro-Martínez
et
al.
/
Journal
of
Biotechnology
158 (2012) 59–
67
agitation
above
300
rpm.
The
hydrolyzed
protein
had
a
high
ACE
inhibitory
activity,
nearly
10
times
more
active
than
the
nonmodi-
fied
sample.
The
successfully
expression
of
this
protein,
together
with
the
two-step
purification
procedure
should
facilitate
its
further
inves-
tigation.
Thus,
the
modified
amarantin
with
ACE
inhibitory
peptides
could
be
used
as
food
additive
in
functional
foods
as
well
as
drug
for
treating
and
preventing
hypertension.
Finally,
this
study
pro-
vides
us
with
some
insight
into
the
possible
large-scale
production
protocols
for
other
promising
biopeptides
into
proteins.
Acknowledgment
C.C.M.
thanks
CONACYT-México
for
a
scholarship
to
carry
out
this
study.
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