Advances in Environmental Biology Saccharomyces PYNIU-AT)

Advances in Environmental Biology, 7(13) November 2013, Pages: 4519-4528
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Advances in Environmental Biology
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Evaluating Alfa-1 Antitrypsin Properties in Saccharomyces Yeast (Practical study on
PYNIU-AT)
1
Sahar Majdi, 2Abbas Sahebghadam lotfi, 3Maryam Ghodrati siahmazgi, 4Reyhaneh Chelongar, 4Raheleh Behrooznam, 4Samira Vaziri,
Mehdi Manbachi, 6Masoumeh Rajabi Bazl, 7Masoud Negahdary
5
1
Young Researchers and Elite Club, Tehran Medical Branch, Islamic Azad University, Tehran, Iran.
Department of Clinical Biochemistry, TarbiatModares University, Tehran, Iran.
3
Department of Biochemistry, Science & Research Branch, Islamic Azad University, Tehran, Iran.
4
Department of Biology, Payame Noor University, I.R. of IRAN.
5
Department of Horticulture, Science and Research Branch, Tehran, Islamic Azad University, Tehran, Iran.
6
Department of Clinical Biochemistry, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
7
Young Researchers and Elites Club, Marvdasht Branch, Islamic Azad University, Marvdasht, Iran.
2
ARTICLE INFO
Article history:
Received 12 September 2013
Received in revised form 25
November 2013
Accepted 29 November 2013
Available online 16 January 2014
Key words:
Serpins, PYINU-AT, Saccharomyces,
Glycosylatedness, Alpha-1 Antitrypsin
ABSTRACT
Serpins according to their structure are proteins and those studies about fourth structural
forming or folding are interesting because of their structural changes which are
extremely interesting and including unstable conditions. Serpins percentage in high
eukaryotes, plants and viruses are still not known in born homology against animaly
and plants in prokaryotes and mycology. Al serpins are not working like a protein’s
inhabit or rather some of them are known as other types proteinase like cross-class. For
example crmA, virus serpins, with changing its activity restrain (1) interlokin enzymes
and SccAAI or antigen-cubic cells, cysteine restrain proteinase in papain groups. Noninhabitoryserpins show different activities which are including chaperon figures
(thermal shock proteins with 47KD molecular weight) or HSP47 and proteins which are
transmitted hormones like binding globulin to (CBG). Serpins vocalize around 20%
from discovered proteins in blood plasma. The current study is going to investigate the
purity separation of determination of the properties and optimization of the alpha-1
Antitrypsin expression increase in Saccharomyces cerevisiae. In order to separate and
purify this protein, some properties such as molecular mass, the amount and capacity of
inhibiting are also measured. It is worth mentioning that the materials and performing
technique in the present study have been determined in the following order: strains,
plasmids, cultivation environments, transformation, protein property determination, and
expression increase.
© 2013 AENSI Publisher All rights reserved.
To Cite This Article: Sahar Majdi, Abbas Sahebghadam lotfi, Maryam Ghodrati siahmazgi, Reyhaneh Chelongar, Raheleh Behrooznam,
Samira Vaziri, Mehdi Manbachi, Masoumeh Rajabi Bazl, Masoud Negahdary, Evaluating Alfa-1 Antitrypsin Properties in Saccharomyces
Yeast (Practical study on PYNIU-AT). Adv. Environ. Biol., 7(13), 4519-4528, 2013
INTRODUCTION
Serpins are the main parts of the protein with the different duties and turnovers which is including halter
and also unhalter activities such as vocalization of lobe [15]. Serpins of clinically interesting because of the
mutation which is available in their structure cause many diseases such as coagulation defects and a number of
diseases also caused by structural changes and polymerized protein molecule [20, 11]. Restricting motion of
human plasma proteases was determined by Fermi also Pernossi in 1984, in addition the central inhibitor
clarifying for anti-proteolytic was at the beginning decomposed by Shultz in 1955, and it was named Alpha-1
Antitrypsin. Human alpha 1-antitrypsin (AAT), a serum glycoprotein, is one of the best-known models of serine
protease inhibitors (serpins) superfamily [1, 17]. Alpha 1-proteinase is the main neutral serine proteinase in
serum, especially Elastase, which enjoys three different additional Oligo-Saccharides, and it is 12% of its
weight. Iso-electric pH of this protein varies between 4.4 and 7.4. An average amount of about 34g of this
protein is synthesized by liver and secretes into serum [3, 5, 26]. In addition, AAT exists in the other types of
cells, including plackets, pancreas megakaryocytes, langerhans islands [8, 14]. Its density is between 1.5 up to
3.5 gr/lit, and its plasmid half-life is between 4 to 8 days [22]. AAT forms a strong complex with proteases
which is an irreversible reaction [4, 16, 23]. Ultimately, reduction of secretion results in this inhibitor shortage,
and then leads to some health problems such as pulmonary emphysema [19, 28]. Giving pure AAT to these
Corresponding Author: Masoud Negahdary, Young Researchers and Elites Club, Marvdasht Branch, Islamic Azad
University, Marvdasht, Iran
E-mail: [email protected]
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Masoud Negahdary et al, 2013
Advances in Environmental Biology, 7(13) November 2013, Pages: 4519-4528
patients decreases the disease side effects, and due to its broad treatment application, protein exploitation
process could be designed from human blood [24]. Hence, its recombinant production through various sources
such as Eukaryotic and prokaryotic micro-organisms is important for genetic manipulation facility and mass
production [29]. In this regard, since AAT is the result of mass produced Glycosylated, consistent yeast,
Eukaryotic micro-organisms is preferred. After AAT expression in Saccharomyces cerevisiae yeast, expression
increased through fermentor, and the properties of this protein, including molecular mass, and inhibition are
measured.
Materials and Methods
The intracellular serpins:
Serpins in high Eukaryotes are devided into zgroups:
The intra cellular serpins or ovoserpins making a complete clear group and are the ancestor of extracellular
serpins. The adjustment of cell behavior is a completely understandable subject under the obscurities of serpins.
Megsin is a member of serpines which is sustaining the megacaryocyte of marrow. Plasminogen has an ability
to restrain tumor necrosis factor which is known as an element of apoptosis and MENT is along with
agglomerating cromatine. Some of the interacellarserpins or (ovoserpins) are performing the restraining roll. For
example PI-6 restrains captain G but the activities of interacelluharserpins still unknownable. Any way by the
exception of the ovoalbomyne which is a non-inhibitory serpins, all the interacelluralserpins including parts of
knee is for hibitory activities. OVO albumen which is the main part of the egg call it as an albumen , first of all
use as a sauing proteins elements. Recent studies show that ovo albumen from its structural arrangement during
embryonic development the extracellular serpins. The extracellular serpins; extracellular serpins are devided in
8 group called as a/b/c/d/e/f/g/h. Among them the biggest group is a, which is contouningserpins like alfa-1antitrypsine the members of this group are in relationship with wide range of processess which the common one
is inspiring the protease. For example R A S P (calistatin) alfa -1-antitrypsine, antikimotrypsine are available in
this group. Some of the non-inhibitory serpins such as CBG which are transmitting the hormones and those
globolines which are adjoining to (TBG-tirocsine) and peptide hormones (the carrier of angiotansionogen,
vterineserpines like UTMP(uterine milk protein) and UFAP (utera ferine associated protein) are including in
extracellular serpins in b/c/e groups. Uterine serpines are extremely branching and receptacle non-inhibitory
knee area which their activity still mysterious. Joining ovine UTMP to the growth factor which has surveyed
indicate that this serpine is completely effective to seprate the active from pregnant uterus. In group F, PEDF
(pigmenteEthelium Derived Factor) serpines is available. Which is imagine that is a not o filli factor and α2 (anti
plasmine) which is known as a serpine in moray have common ancestry with mammels in group F. group d is
including cofactor F (heparin). Group (g) including C1 restrainer and in h group hsp 47 serpines are available
which never have restrainer activity, and exactly they act like active pap ones in folding procelagen. Micro
organisms which are used in researching for doing this research they use unidirectional bacteria and zymogenic
which is indicating bellow:
1. DH5α lineage (achrichiakli) it’s a bacteria lineage which is produced by stratagene company .it is used
for reproduction and keeping pelasmids. Its genetic profile as follows: (F –gyr A 96 Nlae, rec AL thi-1 hsd R 17
r-k mtk)
2. Zymogenic lineage it is used for AAT recombinant protein, as long as it has shown in genetic plans,
(ura3) tubidged has been activated in this zymogenic lineage. Genetic profile (MATa pep4: his 3 prb - ∆1.6R
could 1, his 3-20, ura 3-52)
3. Plasmid PYINU-at, vector indicates α1-AT human zymogenic one vector in dicate 2μ based.this image
indicate that how vector Manu fractured?
Chemical materials and biologic usages in researching: subculture of bacteria LB (Luria Bertani subculture
of hiquid Bacteria (LB)
Bactopepton(Dif Co ) 10 g
Zymogenic extract (Dif Co) 5g
Salt (NaCl) (Merck) 10g
Distilled water to 1litre volume
Subculture of bacteria solid LB
Bacto peptone (Dif Co) 10 g
zymogenic extract (Dif Co) 5g
Salt (Merck) (NaCl) 10g
Aga, powder (Mir Midia) 15g
Distilled water (to 1 liter volume)
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Masoud Negahdary et al, 2013
Advances in Environmental Biology, 7(13) November 2013, Pages: 4519-4528
Put these materials into the clean flasks then add distilled water up to 1liter volume. After that all these
materials dissolve in water, strill the culture environment by auto clave.
Results and Discussion
According for table 1, SDS-PAGE gel is related to saccharomyces cerevisiae, a zymogeni fermentation
which is containing AAT gen with inspiration and cycle engine is depending on oxygen from left to right zero
indicate a time before on inspiration and 1 to 5 hours regularly indicate after inspiration.
Table1: zymogene fermentation of saccharomyces cerevisiae including AAT Gen along with starting of inspiration at 2 after insemination
with IPTG0.4m M and cycle enginge which is depending on oxygen
explanation
Time
The time after OD600
Speed on Temp.
Weather
PH
according to inspiration
minute
pressure
hours
according to
vvm
100μg/ml
1
0
0.07
150
30
1
7
ampicilin
2
1
0.1
150
30
1
7
mM0.4IPTG
3
2
0.309
150
30
1
7
100μg/ml
4
3
0.62
200
30
1
7
ampicilin
5
4
0.71
210
30
1
7
6
5
0.9
215
30
1
7
7
6
1.1
220
30
1
7
1.2
1
0.8
0.6
0.4
Mean: 0.54
SD: 0.39
0.2
0
1
2
3
4
5
6
7
The time after inspiration (according to hours)
Diagram. 1: Fermentation of Saccharomyces cerevisiae yeast containing AAT gene accompanied with
induction start at time 2 after insemination and oxygen-dependent revolution
According to these cases at the continuances of zymogene fermentation of saccharomyces cerevisiae with
AAT Gen and the starting of inspiration during 3/4/5/6/7 hours after insemination with IPTG 0.4MM with cycle
engine which is related to oxygen had done.
Tables 2 to 6 with graphs indicate those explanations.
Table 2: zymogene fermentation of saccharomyces cerevisiae including AAT Gen along with starting of inspiration at 3 after insemination
with IPTG0.4m M and cycle enginge which is depending on oxygen
explanation
Time according The time after OD
Speed on
Weather
pressure
Temp.
PH
to hours
inspiration
600
minute
according to vvm
100μg/ml ampicilin
1
0
0.1
150
30
1
7
2
1
0.3
150
30
1
7
3
2
0.45
179
30
1
7
mM0.4IPTG100μg/ml 4
3
0.69
185
30
1
7
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Masoud Negahdary et al, 2013
Advances in Environmental Biology, 7(13) November 2013, Pages: 4519-4528
ampicilin
5
6
7
8
4
5
6
7
0.80
1.11
1.3
1.5
203
236
254
273
30
30
30
30
1
1
1
1
7
7
7
7
1.6
1.4
1.2
1
0.8
0.6
Mean: 1.13
0.4
SD: 0.81
0.2
0
1
2
3
4
5
6
7
8
The time after inspiration (according to hours)
Diagram. 2: Fermentation of Saccharomyces cerevisiae yeast containing AAT gene accompanied with
induction start at time 3 after insemination and oxygen-dependent revolution
Table 3: zymogene fermentation of saccharomyces cerevisiae including AAT Gen along with starting of inspiration at 4 after insemination
with IPTG0.4m M and cycle enginge which is depending on oxygen
explanation
Weather
pressure
Time according The time after
Speed on
PH
OD 600
Temp.
to hours
inspiration
minute
according to
vvm
100μg/ml
1
0
0.1
150
30
1
7
ampicilin
2
1
0.3
150
30
1
7
3
2
0.7
180
30
1
7
4
3
0.85
195
30
1
7
mM0.4IPTG 100μg/ml 5
4
0.82
220
30
1
7
ampicilin
6
5
1.2
260
30
1
7
7
6
1.56
270
30
1
7
8
7
1.61
280
30
1
7
9
8
1.8
320
30
1
7
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Masoud Negahdary et al, 2013
Advances in Environmental Biology, 7(13) November 2013, Pages: 4519-4528
1.8
1.6
1.4
1.2
1
0.8
Mean: 0.95
0.6
SD: 0.59
0.4
0.2
0
1
2
3
4
5
6
7
8
9
The time after inspiration (according to hours)
Diagram. 3: Fermentation of Saccharomyces cerevisiae yeast containing AAT gene accompanied with
induction start at time 4 after insemination and oxygen-dependent revolution
Table 4: zymogene fermentation of saccharomyces cerevisiae including AAT Gen along with starting of inspiration at 5 after insemination
with IPTG0.4m M and cycle enginge which is depending on oxygen
explanation
Weather
Time according The time after
Speed on
pressure
PH
OD 600
Temp.
to hours
inspiration
minute
according to
vvm
100μg/ml ampicilin
1
0
0.2
150
30
1
7
2
1
0.4
180
30
1
7
3
2
0.7
205
30
1
7
4
3
0.9
215
30
1
7
5
4
1.3
255
30
1
7
mM0.4IPTG 100μg/ml 6
5
1.77
290
30
1
7
ampicilin
7
6
1.80
310
30
1
7
8
7
1.90
335
30
1
7
9
8
2.2
355
30
1
7
10
9
2.3
390
30
1
7
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Masoud Negahdary et al, 2013
Advances in Environmental Biology, 7(13) November 2013, Pages: 4519-4528
2.5
2
1.5
Mean: 1.29
1
SD: 0.77
0.5
0
1
2
3
4
5
6
7
8
9
10
The time after inspiration (according to hours)
Diagram. 4: Fermentation of Saccharomyces cerevisiae yeast containing AAT gene accompanied with
induction start at time 5 after insemination and oxygen-dependent revolution
Table 5: zymogene fermentation of saccharomyces cerevisiae including AAT Gen along with starting of inspiration at 6 after insemination
with IPTG0.4m M and cycle enginge which is depending on oxygen
explanation
Weather
Time according The time after
Speed on
pressure
PH
OD 600
Temp.
to hours
inspiration
according to
minute
vvm
100μg/ml ampicilin
1
0
0.12
150
30
1
7
2
1
0.4
170
30
1
7
3
2
0.65
190
30
1
7
4
3
1.107
200
30
1
7
5
4
1.6
266
30
1
7
6
5
1.8
280
30
1
7
mM0.4IPTG
7
6
1.9
290
30
1
7
100μg/ml ampicilin
8
7
1.95
300
30
1
7
9
8
2.3
325
30
1
7
10
9
2.4
300
30
1
7
11
10
2.41
390
30
1
7
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Masoud Negahdary et al, 2013
Advances in Environmental Biology, 7(13) November 2013, Pages: 4519-4528
3
2.5
2
1.5
Mean: 1.49
1
SD: 0.81
0.5
0
1
2
3
4
5
6
7
8
9
10
11
The time after inspiration (according to hours)
Diagram. 5: Fermentation of Saccharomyces cerevisiae yeast containing AAT gene accompanied with
induction start at time 6 after insemination and oxygen-dependent revolution
Table 6: zymogene fermentation of saccharomyces cerevisiae including AAT Gen along with starting of inspiration at 7 after insemination
with IPTG0.4m M and cycle enginge which is depending on oxygen
explanation
Weather
Time according The time after
Speed on
pressure
PH
OD 600
Temp.
to hours
inspiration
according to
minute
vvm
100μg/ml ampicilin
1
0
0.12
150
30
1
7
2
1
0.40
170
30
1
7
3
2
0.65
190
30
1
7
4
3
1.180
200
30
1
7
5
4
1.60
270
30
1
7
6
5
1.80
280
30
1
7
7
6
1.85
290
30
1
7
mM0.4IPTG
8
7
2.15
300
30
1
7
100μg/ml ampicilin
9
8
2.30
325
30
1
7
10
9
2.34
350
30
1
7
11
10
2.5
389
30
1
7
12
11
2.85
390
30
1
7
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Masoud Negahdary et al, 2013
Advances in Environmental Biology, 7(13) November 2013, Pages: 4519-4528
3
2.5
2
1.5
Mean: 1.6
1
SD: 0.87
0.5
0
1
2
3
4
5
6
7
8
9
10
11
12
The time after inspiration (according to hours)
Diagram 6: Fermentation of Saccharomyces cerevisiae yeast containing AAT gene accompanied with induction
start at time 7 after insemination and oxygen-dependent revolution
Conclusion:
Serpin A1, also known as Alpha-1 antitrypsin (AAT), is a prototype member of the serpin super family of
the serine protease inhibitors [12]. This serine protease inhibitor blocks the protease, neutrophil elastase. Alpha1 antitrypsin is mainly produced in the liver and acts as an antiprotease [18, 13]. Its principal function is to
inactivate neutrophil elastics, preventing tissue damage. Alpha1-antitrypsin, an acute phase protein and the
classical neutrophil elastase inhibitor, is localized within lipid rafts in primary human monocytes in vitro [27]. It
association with monocytes is inhibited by cholesterol depleting/efflux-stimulating agents (nystatin, filipin,
MbetaCD (methyl-beta-cyclodextrin) and oxidized low-density lipoprotein (oxLDL) and conversely, enhanced
by free cholesterol. Furthermore, SerpinA1/monocyte association per se depletes lipid raft cholesterol as
characterized by the activation of extracellular signal-regulated kinase 2, formation of cytosolic lipid droplets,
and a complete inhibition of oxLDL uptake by monocytes [7, 25]. Previous population studies have suggested
that heterozygote status for the AAT gene (SerpinA1) is a risk factor for chronic rhinosinusitis with nasal
polyposis (CRSwNP). Alpha-1 antitrypsin deficiency is a recently identified genetic disease that occurs almost
as frequently as cystic fibrosis. It is caused by various mutations in the SerpinA1 gene, and has numerous
clinical implications [2]. Alpha-1 antitrypsin deficiency is an inherited disease affecting the lung and liver. In
the liver, alpha-1 antitrypsin deficiency may manifest as benign neonatal hepatitis syndrome; a small percentage
of adults develop liver fibrosis, with progression to cirrhosis and hepatocellular carcinoma [6, 10]. It’s most
important physiologic functions are the protection of pulmonary tissue from aggressive proteolytic enzymes and
regulation of pulmonary immune processes [21]. It is apparent that Zymogen IA is similar to hog pepsinogen in
several respects. It is known that hog pepsinogen is stable in a mild alkaline medium in which hog pepsin is
unstable. The observation that gastricsin and human pepsin are produced from the activation of the Zymogen IA
fraction suggests two possibilities. (a) Zymogen IA is a common precursor for gastricsin and human pepsin, and
(b) Zymogen IA consists of two zymogens, pepsinogen and a zymogen for gastric sin. The evidences in favor of
the “common precursor” are (i) Zymogen IA appeared as a single boundary in ultracentrifugation studies and as
a single band in starch gel electrophoresis; (ii) the ratio of pepsin to gastricsin varied as a function of the
activating pH whereas the total amount of enzyme produced from Zymogene. Gastricsin and human pepsin were
both found to be present as a zymogen in human gastric mucosa. In the attempts to purify the zymogen, a
fraction (IA) has been obtained from the alkaline extract of human gastric mucosa after ammonium sulfate
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Masoud Negahdary et al, 2013
Advances in Environmental Biology, 7(13) November 2013, Pages: 4519-4528
precipitation and diethylaminoethyl cellulose chromatography. Zymogen IA behaved as a single component in
ultracentrifugation and starch gel electrophoresis. It was stable in a solution of pH 8.5. However, once acidified
to pH 5 or below, it was no longer stable at pH 8.5. When acid-activated Zymogen IA was fractionated in an
Amberlite IRC-50 column or by starch gel electrophoresis, gastric sin and human pepsin were obtained. The
physicochemical and enzymatic properties of the enzymes obtained from the activation of Zymogen IA were
identical to gastric sin and human pepsin isolated from human gastric juice. It appeared that both enzymes might
be derived from this same zymogen. Single-chain pro-urokinase (pro-uPA) is present both in the medium and
lysate of the A431 epidermoid carcinoma cell line. Most of the cell-associated pro-uPA is on the cell surface, as
shown by indirect immunofluorescence and by surface lodination. Pro-uPA is not an integral membrane protein
but is bound to a specific surface receptor that is completely saturated [9]. A mild acid treatment uncovers the
surface receptors by dissociating pro-uPA. Resaturation of uncovered receptors has been studied by reincubating
cells in normal medium; within 40 min, 50% of the free sites are reoccupied. Excess uPA-specific antibodies
prevent rebinding of ligand to the receptors. Thus, A431 cells first secrete uPA, which then binds to the surface
receptor. We propose that the synthesis of uPA and uPA receptor by the same cell may provide a pathway for
the activation of the metastatic potential of malignant cells.
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