Download AN ATTEMPT TO PRODUCE RECOMBINANT HORSE INSULIN D

Batsaikhan.D and et al. (2016) Mongolian Journal of Agricultural Sciences №19 (03): 3-9
3
AN ATTEMPT TO PRODUCE RECOMBINANT HORSE INSULIN
D.Batsaikhan1*, S.Lkhagvasuren1, B.Munkhtogtokh2, B.Tuvshin1,
B.Bayar-Enkh1, Ya.Ganbold1
1-Institute of veterinary medicine, MULS
2-Research institute of animal husbandry, MULS
*Email: [email protected]
ABSTRACT
The present study aimed to produce horse insulin by use of recombinant DNA technology. Because the
hormones such as somatotropin, prolactin and leptin, which are practically applicable in veterinary medicine,
are also peptides, they can be obtained on the basis of this model technology. To isolate total RNA, horse
pancreas samples were collected in RNA stabilizing solution from local abattoirs, sent to the laboratory, and
stored the samples in the freezer at -800C. Molecular biological reagents and kits for total RNA extraction,
one step RT (reverse transcription)-PCR reactions, cloning, expression, and purification were used to produce
equine proinsulin. Primers were designed from NCBI gene bank data and synthesized in Macrogen Inc, Korea.
Obtained equine proinsulin was processed by enzymes trypsin and carboxypeptidase B to form mature insulin.
The purity of insulin was assessed by SDS-PAGE. Molecular weight of insulin is approximately 6.0 kDa. The
hormone was also measured by ELISA test and it was equal to 55 IU per ml of solution. Finally it can be
concluded equine insulin with 55 IU/ml activity can be produced by recombinant DNA technology.
KEY WORDS: Equine, pancreas, proinsulin, gene, primer, cloning, purification
INTRODUCTION
Insulin is a protein hormone produced by B cells of
islet of Langerhans in the pancreas, its molecular
weight is about 5,800 dalton. It is a dipeptide
consisting of 2 chains, A and B. Insulin acts on most
body tissues, except the encephalon. Insulin increases
glucose uptake in cells, the synthesis of glycogen and
fatty acids, the formation of triglycerides, and
promotes the active transport of amino acids in cells,
especially muscle cells, thereby increasing the
concentration of metabolites necessary for protein
synthesis and enhancing synthesis. In 1921, Canadian
scientists Frederick G. Banting and Charles H. Best
successfully purified insulin from a dog's pancreas
[1]. Structures of insulin in the different species are
very similar. In the study of horse and whale insulin
as compared to insulin of other mammals such as
cattle, swine and sheep, the only differences found
were in the three residues occupying positions 8-10 in
the glycil (A) chain [2]. Also, human insulin differs
from swine insulin by only amino acid residue, while
it differs from horse by 2, cattle by 3 and sheep, goat
and cat by 4 [3]. Proinsulin or prohormone of insulin
is a single-chain polypeptide of approximately 9 kDa
with three intrachain disulfide bridges, the number of
amino-acid residues varying somewhat with species
(78 in dog, 81 in cattle and sheep, 84 in cod and pig,
4
Batsaikhan.D and et al. (2016) Mongolian Journal of Agricultural Sciences №19 (03): 3-9
86 in horse, human, and rat). It is cleaved into insulin
and C (or connecting)-peptide. Synthesized in the
pancreatic B cells, it is normally present at about 5%
of the concentration of insulin, and is secreted in
small amounts into the blood [4].
Over the years scientists made continual
improvements in producing insulin. Researchers
continued to improve insulin but the basic production
method remained the same for decades. Insulin was
extracted from the pancreas of cattle and pigs and
purified. Then in the early 1980s biotechnology
revolutionized insulin production. Researchers had
already decoded the chemical structure of insulin in
the mid 1950s. They also determined the exact
location of the insulin gene at the top of human
chromosome 11 [5]. By 1977, a research team had
spliced a rat insulin gene into a bacterium that then
produced insulin. In the 1980s, researchers used
genetic engineering to manufacture a human insulin
[5,6]. As reported by Chang et al, 1998 proinsulin
which was prepared by the same procedure showed a
maximum refolding yield of 57% at the same molar
concentration at pH 10.5 [7]. In other study, 5-8 mg
of partially purified, S-sulfonated human proinsulin
was normally obtained from 1L of cultured bacteria
[8]. In 1982, the Eli Lilly Corporation produced a
human insulin that became the first approved
genetically engineered pharmaceutical product.
Without needing to depend on animals, researchers
could produce genetically engineered insulin in
unlimited supplies [1].
Despite, insulin is less applicable in veterinary
medical practices, camel (Camelus dromedarius)
recombinant insulin was produced and characterized
to use in down stream biotechnological and disease
treatment applications in Saudi Arabia, because
camels suffer from fetal diseases, which may have an
effect on the country’s economy [9].
In this study, we aimed to make an attempt of
producing recombinant equine insulin, and
characterize molecular weight and hormonal activity
of the obtained insulin preparation to use the
technology model for further studies of production of
some peptide hormones important for veterinary
medical practices by rDNA technology.
MATERIALS AND METHODS
Sample collection and RNA extraction
Horse pancreas samples were taken from 3 adult
horses, slaughtered in an abattoir in Nalaikh district,
UB city. Pancreas were removed and weighed, and
tissue samples were quickly cut into slices less than
0.5 cm in thickness and immediately submerged into
100 ml sterile glasses containing RNA Stabilization
solution within approximately 20 min after
sacrificing horses. Total RNAs were extracted from
pancreas by use of total RNA extraction kit
(Amersham Biosciences) according to the
manufacturer’s protocol. Pre-weighed (~100 mg) of
pancreatic tissues were removed from RNA
stabilization solution and extraction buffers and
immediately submerged into 1.5 ml tube containing
225 μl beta-mercaptoethanol. Samples were
homogenized thoroughly by tissue grinder. Samples
were centrifuged at 10,000 x g for 15 min at room
temperature and supernatants were dispensed
carefully by vacuum aspiration. After washing RNA
pellets the samples were centrifuged at high speed for
5 min and supernatants were aspirated without
disrupting pellets. The tubes were incubated on ice
and washed with 1.5 ml ice-cold 70% ethanol.
Samples were centrifuged at 10,000 x g and the
ethanol supernatants were discarded carefully. RNA
pellets were dissolved in 100 to 200 μl of DEPCtreated water and incubated on ice for 15 min. The
yield and purity of RNA were assessed by
spectrophotometric analysis.
Reverse transcription and cDNA amplification
First-strand cDNA of equine proinsulin was
synthesized by use of AccuPower RocketScript RTPCR Premix kit as described in the manufacturer’s
instruction (Bioneer).
Both the forward and reverse primers for
amplification of horse proinsulin cDNA were
designed on the basis of NCBI sequences of equus
caballus insulin cDNAs (Accession XM-001496805).
Equuscaballusinsulin-1-F (5`-CCA GCA GGT CAC
CGT-3`) and Equuscaballusinsulin-2-R (5`-AGT
TGC AAT AGT TCT CCA GC-3`) were synthesized
(Macrogen Inc) and utilized to amplify horse
proinsulin cDNA.
Thermocycling conditions were 60ºC for 30 min,
95ºC for 5 min followed by denaturing at 95ºC for 30
seconds and annealing at 55ºC for 30 seconds and
extension at 72ºC for a minute. The final extension
cycle was at 72ºC for 5 min. The PCR amplicons were
analyzed by 1.5% agarose gel electrophoresis.
Molecular cloning of equine proinsulin cDNA
In order to insert amplified equine proinsulin cDNA
into a linearized plasmid vector pCRT4/NT-TOPO of
TOPO-TA one-step cloning strategy (Invitrogen), the
kit was used according to manufacturer’s instructions.
Two microliter of fresh PCR product was mixed with
Batsaikhan.D and et al. (2016) Mongolian Journal of Agricultural Sciences №19 (03): 3-9
1 μl salt solution in a microcentrifuge tube, and the
total volume was brought up to 5 μl by ddH2O. One
microliter of linearized pCRT4/NT-TOPO vector was
added and the reaction mix was incubated at room
temperature for 5 min. Then the reaction mix was
incubated on ice and 1 μl was used directly to
transform competent One Shot TOP10- E. coli
DH10B (Invitrogen). The transformed cells were
plated on LB plates containing 100 mg/ml ampicillin.
The plates were incubated at 37ºC overnight to grow
colonies. Bacterial colonies were screened by
growing 10 to 20 single colonies in 6 ml of LB media
containing 100 mg/ml ampicillin for 6 to 8 hours.
Expression and purification of equine proinsulin
Recombinant equine proinsulin expression was
induced by addition of 1 mM iso-propyl-β-thiogalactopyranoside (IPTG) for 4 h at 30 0C in 55.5 mM
glucose in Luria-Bertani (LB) medium. Induction was
followed by purification of protein using AccuPrep
5
His-tagged protein Purification kit (Bioneer) as
described in the instruction of manufacturer. Protein
purity was assessed by SDS-PAGE.
Cleavage of equine proinsulin
Equine proinsulin protein was dissolved in 100 mM
Tris/H3PO4, pH 7.5, containing 0.1 % Tween 20 to
protein concentrations of 2 mg/ml. Trypsin (Sigma
Aldrich) and carboxypeptidase B (Sigma Aldrich)
were added to trypsin/ protein ratios of 2:2000 (by
mass) and carboxypeptidase B/protein ratios of
1:1000 (by mass). After 30 min, the digestion was
stopped by decreasing the pH to 3 by adding acetic
acid. Acetonitrile to 20% was added in order to
stabilize the cleavage products.
Insulin measurement
Insulin activity was measured by equine insulin
ELISA test kit (Endocrine Technologies Inc)
according to the manufacturer’s instruction.
RESULTS
Pancreas of three horses were taken after slaughtering
and a pancreas weighed 314.3±3.5 g in average
(Fig.1). Pancreatic tissue samples was excised less
than 0.5 mm in thickness from tail portion and placed
in RNA stabilization solution for total RNA isolation,
because this part contains highest number of
Langerhans islets, beta cells of which produce insulin.
Figure 1. A pancreas taken from horse carcass within 20 minutes after slaughtering
After isolation of total RNA. Both forward and
reverse primers were designed by NCBI primerBLAST software and synthesized in Macrogen Inc.
Also, equine proinsulin amino acid sequence known
from literatures was converted to the DNA nucleotide
sequence by using online In-silico Sequence
Conversion tool and attempts to design primers of
equine proinsulin were made and they were
synthesized also in Macrogen Inc. However, the
former primers were selected in the present study.
Then equine proinsulin cDNA was produced from
total RNA using a ready to use lyophilized mastermix
containing all components for first strand cDNA
synthesis and PCR reaction in one tube.
Electrophoregram shows the cDNA molecule
encoding equine proinsulin consists of approximately
260 bp nucleotides (Fig.2).
6
Batsaikhan.D and et al. (2016) Mongolian Journal of Agricultural Sciences №19 (03): 3-9
Figure 2. Equine proinsulin cDNA molecule of approximately 260 bp nucleotides
Figure 2. Equine proinsulin cDNA molecule of approximately 260 bp nucleotides
The equine proinsulin gene was cloned using contain non-recombinant vector are killed upon
non-recombinant
are killed
The
equine
proinsulin genevector
was (figure
cloned 3),using
plating. The
DNA insert wasvector
not sequenced
and upon
after
linearized
pCRT4/NT-TOPO
and contain
The
insertexpression,
was not sequenced
and after
linearized
vector (figure
3), and plating.
induction
of DNA
proinsulin
the hormone
was
expressed pCRT4/NT-TOPO
in E.coli. The vector
pCRT4-TOPO
of proinsulin
expression,
hormone
was
expressed
in selection
E.coli. ofThe
vector pCRT4-TOPO
purified by
use of AccuPrep
Histhetagged
protein
allowed direct
recombinant
via disruption induction
by kit
usewith
of Ni-NTA
AccuPrepmagnetic
His tagged
allowed
of recombinant
via disruption
purification
silica protein
resins.
of lethaldirect
E.coliselection
gene, ccdB
and blue/white
screening purified
purification
kit
with
Ni-NTA
magnetic
silica
resins.
of
lethal
E.coli
gene,
ccdB
and
blue/white
screening
is not required, because ligation of PCR product Then the proinsulin was cleaved by using Trypsin
proinsulin
cleaved by using
is
not required,
because
ligation of PCR
(SigmatheAldrich)
andwas
carboxypeptidase
B Trypsin
(Sigma
disrupts
expression
of lacZalpha-ccdB
geneproduct
fusion Then
(Sigma
Aldrich)
and
carboxypeptidase
B
(Sigma
disrupts
expression
of
lacZalpha-ccdB
gene
fusion
permitting growth of only positive recombinant Aldrich) as described in materials and methods
permitting
of onlyandpositive
section. as described in materials and methods
protein upongrowth
transformation
bacterialrecombinant
cells, which Aldrich)
protein upon transformation and bacterial cells, which section.
Figure 3. Plasmid map of pCR4 containing horse proinsulin gene
Figure(taken
3. Plasmid
map ofTA
pCR4
containing
proinsulin gene
from TOPO
cloning
manualhorse
by Invitrogen)
(taken from TOPO TA cloning manual by Invitrogen)
The molecular weight of recombinant horse insulin is approximately 6.0 kDa by SDS polyacrylamide gel
The
molecular weight
electrophoresis
(Fig.4). of recombinant horse insulin is approximately 6.0 kDa by SDS polyacrylamide gel
electrophoresis (Fig.4).
Batsaikhan.D and et al. (2016) Mongolian Journal of Agricultural Sciences №19 (03): 3-9
7
Figure 4. Electrophoregram of purified equine insulin by SDA-PAGE
Because total amount of the hormone produced in this
experiment was lower and insufficient to be used for
insulin measurement in rabbits according US
pharmacopea, equine insulin was measured by
equine insulin ELISA test and the amount was equal
to 50 IU per ml protein solution.
DISCUSSION
Our study can be the first attempt to obtain equine
insulin by use of recombinant DNA technology,
although equine insulin, including its amino acid and
nucleotide sequences, structures and physic-chemical
characteristics has been investigated and reported [1,
2, 3]. But, there is no any study toward cloning and
characterizing peptide hormones in animals by
recombinant DNA technology in our country. As a
result of the present study, a model technology for
production of recombinant equine insulin was
initially being developed in order to use this example
for further studies on production of various protein
hormones important in veterinary medical practices,
besides of its possible use for various purposes.
Human insulin is essentially important for curing
diabetes mellitus, because approximately 150 million
people have diabetes mellitus worldwide, and that
this number may well double by the year 2025 [10].
Therefore, results of human insulin studies are
extensively reported in the literatures. While
companies still sell a small amount of insulin
produced from animals-mostly porcine-from the
1980s onwards, insulin users increasingly moved to a
form of human insulin created through recombinant
DNA technology. According to the Eli Lilly
Corporation, 95% of insulin users in most parts of the
world take some form of human insulin in 2001.
Some companies have stopped producing animal
insulin completely. Companies are focusing on
synthesizing human insulin and insulin analogs, a
modification of the insulin molecule in some way [1].
Synthesizing human insulin is a multi-step
biochemical process that depends on basic
recombinant DNA techniques and an understanding
of the insulin gene. Manufacturers manipulate the
biological precursor to insulin so that it grows inside
simple bacteria. While manufacturers each have their
own variations, there are two basic methods to
manufacture human insulin. One method of
manufacturing insulin is to grow the two insulin
chains separately. This will avoid manufacturing each
of the specific enzymes needed. Manufacturers need
the two mini-genes: one that produces the A chain and
one for the B chain. Since the exact DNA sequence of
each chain is known, they synthesize each minigene's DNA in an amino acid sequencing machine,
the two chains are then mixed together and joined by
disulfide bonds through the reduction-reoxidation
reaction. An oxidizing agent (a material that causes
oxidization or the transfer of an electron) is added.
Starting in 1986, manufacturers began to use another
method to synthesize human insulin. They started
with the direct precursor to the insulin gene,
proinsulin, the connecting sequence between the A
and B chains is spliced away [11] with enzymes such
as trypsin and carboxipeptidase B [12] and the
resulting insulin is purified.
Although the amino acid sequence of insulin varies
among species, certain segments of the molecule are
highly conserved, including the positions of the three
disulphide bonds, both ends of the A chain and the C
terminal residues of the B chain [13]. In addition to
human insulin, insulin protein from different species
such as rabbit, sheep, pig, dog, horse, elephant and
camel has been studied closely. Despite the species
diversity, insulin from all investigated animals
8
Batsaikhan.D and et al. (2016) Mongolian Journal of Agricultural Sciences №19 (03): 3-9
harbors a close homology to human insulin. The
harbors a close homology to human insulin. The
amino
differences
give to
each
species'
insulin
a
harborsacid
a close
homology
human
insulin.
The
amino
acid
differences
give each
species'
insulin
a
slightly
different
structure
and
activity
because
the
amino acid
differences
giveand
each
species'
insulinthea
slightly
different
structure
activity
because
whole
protein
folds and
around
on itself
and has
slightlyinsulin
different
structure
activity
because
the
whole
insulin
protein
folds around
on itself
and has
very
where
it interacts
the
wholespecific
insulin locations
protein folds
around
on itselfwith
and has
very
specific
locations
where
it interacts
with
the
insulin
receptorlocations
on the cell.
Theitsequence
porcine
very specific
where
interactsof
the
insulin
receptor on the cell.
The sequence
ofwith
porcine
insulin
and
human
insulin
is
almost
identical,
but not
insulin
receptor
on
the
cell.
The
sequence
of
porcine
insulin and human insulin is almost identical, but not
exactly
– it differs
by
one amino
acid.
Equine but
insulin
insulin and
insulin
is almost
identical,
not
exactly
– it human
differs by
one amino
acid.
Equine insulin
is
different
by
two
amino
acids,
while
bovine
insulin
exactly
–
it
differs
by
one
amino
acid.
Equine
insulin
is different by two amino acids, while bovine insulin
by
three from
It is acids,
of interest
point out
that
is different
byhuman.
two amino
whileto
insulin
by
three from
human.
It is of interest
tobovine
point out
that
the
newly
developed
analogs
like out
Lispro,
by three
from
human. Itinsulin
is of interest
to point
that
the
newly
developed
insulin
analogs
like Lispro,
Aspart
anddeveloped
Glargine; also differ
the newly
analogsfrom
like “human
Lispro,
Aspart
and Glargine; insulin
also differ
from
“human
insulin”
in two
or more also
amino acids.from
Theoretically
Aspart and
Glargine;
“human
insulin”
in two
or more aminodiffer
acids. Theoretically
changes
in
amino
acid
could
affect
the
solubility
and
insulin”
in
two
or
more
amino
acids.
Theoretically
changes in amino acid could affect the solubility and
diffusion
properties
of
insulin
molecules
[14].
changes in properties
amino acid could
affect the
solubility[14].
and
diffusion
of insulin
molecules
Clinical
efficacy
of insulin
clearly
does
not depend
on
diffusion
properties
of
insulin
molecules
[14].
Clinical efficacy of insulin clearly does not depend on
the
species
of insulin
used. clearly
A number
ofnot
clinical
trials
Clinical
efficacy
of insulin
does
depend
on
the
species
of insulin
used. A number
of clinical
trials
have
clearly
shown
that
animal
insulins
and
human
the species
insulinthat
used.animal
A number
of clinical
trials
have
clearlyofshown
insulins
and human
insulins
are comparable
their clinical
The
have clearly
shown thatin
insulinsefficacy.
and human
insulins
are comparable
inanimal
their clinical
efficacy.
The
duration
of
action
of
human
insulin
is
slightly
shorter
insulins are
comparable
in their
clinical
efficacy.
The
duration
of action
of human
insulin
is slightly
shorter
CONCLUSION
duration
of
action
of
human
insulin
is
slightly
shorter
CONCLUSION
CONCLUSION
As a result of the present study, the following
As a result of the present study, the following
conclusions
made:
As a resultcan
of be
present study, the following
conclusions
can
bethe
made:
1.
Pancreas
of
Mongolian
weighs 314.3±3.5 g
conclusions
be made: horse
1.
Pancreascan
of Mongolian
horse weighs 314.3±3.5 g
in
average,
equine
proinsulin
cDNA 314.3±3.5
nucleotides
1. in
Pancreas
of equine
Mongolian
horse weighs
average,
proinsulin
cDNA nucleotidesg
have
approximately
260
bp
length,
and
molecular
in average,
equine proinsulin
cDNA
have
approximately
260 bp length,
andnucleotides
molecular
weight
and
hormonal
activity
of
recombinant
have approximately
260 activity
bp length,
molecular
weight
and hormonal
ofand
recombinant
equine
insulin
is
6.0
kDa
and
55.0
IU/ml
weight and
hormonal
of recombinant
equine
insulin
is 6.0 activity
kDa and
55.0 IU/ml
respectively.
equine insulin is 6.0 kDa and 55.0 IU/ml
respectively.
respectively.
ACKNOWLEDGEMENT
ACKNOWLEDGEMENT
ACKNOWLEDGEMENT
We should thank to S.Davaasuren, researcher of
We should thank to S.Davaasuren, researcher of
Molecular
laboratory, Dr.V.Batbaatar,
We should genetics
thank to S.Davaasuren,
researcher of
Molecular
genetics
laboratory, Dr.V.Batbaatar,
researcher
of
Laboratory
of microbiology
and
Molecular of
genetics
laboratory,
Dr.V.Batbaatar,
researcher
Laboratory
of microbiology
and
researcher of Laboratory of microbiology and
REFERENCES
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27Die
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than animal insulins [15]. Therefore, equine insulin,
than animal insulins [15]. Therefore, equine insulin,
different
withinsulins
only two
amino
acid residues
also
than animal
[15].
Therefore,
equinecan
insulin,
different
with only two
amino
acid residues
can
also
be
used
for
human
medicine
in
association
with
above
different
with
onlymedicine
two amino
acid residues
canabove
also
be
used for
human
in association
with
issues.
Human
synthetic
M-proinsulin
waswith
obtained
be
used
for
human
medicine
in
association
above
issues. Human synthetic M-proinsulin was obtained
in
30-35%
yield
after purification
either
anion
issues.
Human
synthetic
M-proinsulin
wasby
obtained
in
30-35%
yield
after purification
either
by
anion
exchange
chromatography
on
Q-Sepharose
or
acid
in 30-35%chromatography
yield after purification
either byoranion
exchange
on Q-Sepharose
acid
precipitation
[16].
Lee
et
al.
[17]
purified
human
exchange
chromatography
on
Q-Sepharose
or
acid
precipitation [16]. Lee et al. [17] purified human
proinuslin
from
cellular
plasma
by purified
use of DEAE
precipitation
[16].
Lee
et
al.
[17]
human
proinuslin from cellular plasma by use of DEAE
sephacell
Sephadex
columns
andoftheDEAE
yield
proinuslinand
from
cellularG-200
plasma
by use
sephacell
and
Sephadex
G-200
columns
and the yield
was
35%.
All
insulin
preparations
in
Australia
are
sephacell
G-200 columns
and the yield
was
35%.and
AllSephadex
insulin preparations
in Australia
are
standardized
to insulin
contain 100 IU/ml and
typical daily
was 35%. All
inaaAustralia
are
standardized
to contain preparations
100 IU/ml and
typical daily
dose
in human
is 0.7 IU/kg
per and
day [18].
Only
2
standardized
to contain
IU/ml
typical
daily
dose
in human
is 0.7 100
IU/kg
per day a[18].
Only
2
insulin
preparations,
concentrated
at
40
U/ml
are
dose
in
human
is
0.7
IU/kg
per
day
[18].
Only
insulin preparations, concentrated at 40 U/ml are2
registered
for veterinary
use or treatment
diabetes
insulin preparations,
concentrated
at 40of
are
registered
for veterinary
use or treatment
ofU/ml
diabetes
in
dogs
and
cats
[19].
In
our
study,
content
of
insulin
registered
for
veterinary
use
or
treatment
of
diabetes
in dogs and cats [19]. In our study, content of insulin
is
IU percats
ml of solution.
Further
andofdetailed
in 55
dogs
In our study,
content
insulin
is
55 IUand
per ml[19].
of solution.
Further
and detailed
studies
may
have
important
implications
and
would
is 55 IUmay
perhave
ml of
solution.implications
Further and
studies
important
anddetailed
would
provide
a
framework
to
disease
association
and
gene
studies may
have important
implications
provide
a framework
to disease
associationand
andwould
gene
analysis
within
diverse
populations.
provide
a
framework
to
disease
association
and
gene
analysis within diverse populations.
analysis within diverse populations.
2.
2.
2.
An attempt of recombinant DNA technology for
An attempt of recombinant DNA technology for
producing
insulin DNA
demonstrates
An attempt equine
of recombinant
technologythat
for
producing
equine
insulin demonstrates
that
recombinant
peptide
hormone
productionthat
is
producing equine
insulin
demonstrates
recombinant
peptide
hormone
production is
possible
to
be
made
under
laboratory
condition
recombinant
hormone
production
is
possible
to bepeptide
made under
laboratory
condition
and
further
studies
are
necessary
toward
down
possible
to
be
made
under
laboratory
condition
and further studies are necessary toward down
streaming
technology
for production
of down
other
and furtherthis
studies
are necessary
toward
streaming
this
technology
for production
of other
peptide
hormones,
which
are
practically
streaminghormones,
this technology
of most
other
peptide
whichfor
areproduction
practically
most
applicable
in veterinary
medicine.
peptide hormones,
which
are practically most
applicable
in veterinary
medicine.
applicable in veterinary medicine.
immunology and our colleagues in our institute for
immunology and our colleagues in our institute for
their
kind andand
invaluable
assistances
andinstitute
advices to
immunology
our colleagues
in our
their
kind and invaluable
assistances
and advicesfor
to
do
such
a
complicated
scientific
work.
their
kind
and invaluable
assistances
do
such
a complicated
scientific
work.and advices to
do such a complicated scientific work.
5.
5.
5.
6.
6.
6.
7.
7.
7.
Dictionary of Biochemistry and Molecular
Dictionary of Biochemistry and Molecular
Biology
(2ndof
ed), Oxford University
736 p
Dictionary
and press,
Molecular
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Oxford University
press,
736 p
Owerbach
D.,
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G.I.,
Rutter
W.L.,
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T.B.p
Biology
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1980.
The
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is
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on
chromosome
Owerbach
D., Bellgene
G.I.,isRutter
W.L.,
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1980.
The insulin
located
on chromosome
11
in humans,
Nature,
82-84on chromosome
1980.
The insulin
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S.J.,
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