Vol. 72 | No.

12 | Dec 2016 International Journal of Sciences and Research

REFOLDING OF PROTEIN, NIES39_A07830 FROM Arthrospira platensis

AND ITS L-ASPARAGINASE ACTIVITY

Asep A. Prihanto (Corresponding Author)

(Dept. Fish Processing Technology, Faculty of Fisheries and Marine Science, Brawijaya University,
Indonesia/ asep_awa@ub.ac.id)
(Center for Coastal and Marine Studies, Brawijaya University, Indonesia/ asep_awa@ub.ac.id)

Happy Nursyam
(Dept. Fish Processing Technology, Faculty of Fisheries and Marine Science, Brawijaya University,
Indonesia/happy_nursyam@yahoo.com)

Abstract
L-Asparaginase (E.C. 3.5.1.1) is an amidohydrolase enzyme which catalyze L-asparagine to L-
aspartate and ammonia. This enzyme has an important applications on medicine and food
processing aid. In this research, L-asparaginase from food grade microorganism, Arthrospira
platensis was cloned and expressed in Escherichia coli system. The cloned gene expressed in
the inclusion body form. It was found that deduced protein was located and accumulated in the
pellet fraction, as inclusion bodies. Hence, to achieve an active form of the enzyme, it was
needed to be correctly folded with steps as follow solubilization and refolding. Here, we report
the recovering activity of the enzyme throughout our refolding process. Several denaturant
agents were used to completely denature a protein from inclusion body. Urea, triton-X 100,
and sarkosyl are able to denature the protein. The refolding process was conducted by dilution
and dialysis methods. After experienced refolding process, L-asparaginase activity was
detected. The highest activity was achieved from dialysis methods (64 U). We, therefore,
reported the activity in the refolded enzyme even though the process needs to be optimized.

Keywords: Refolding, inclusion body, L-asparaginase, food grade, Arthrospira platensis,

Introduction
L-Asparaginase (E.C. 3.5.1.1) is an amidohydrolase enzyme which catalyze L-asparagine to L-
aspartate and generate ammonia. This enzyme has an important application on medicine and
food processing aid. For medicine, this enzyme has long been known for its role in the
therapeutic of cancer, mainly for acute lymphoblastic leukemia. This enzyme able to reduce an
existence of acrylamide, a carcinogenic compound in food products which their raw materials
are L-asparagine rich materials.
Bacteria is a favored source for L-Asparaginase. It is mainly due to the easiness and cost-
effectiveness in culture and production. Escherichia coli and Erwinia carotovora are the two
famous L-asparaginase source for therapeutic aid. Unfortunately, several side effects such
hypersensitivity, anaphylaxis reaction, and short half-life and have been reported. For
commercially available food processing aid, yeast derived L-asparaginase is may be the only
one. Thus, the new source of L-asparaginase is needed to be explored. Marine organism is still
underexplored source of L-asparaginase (Prihanto and Wakayama, 2016). Marine

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cyanobacteria as a source of L-asparaginase is relatively new. Furthermore, only few papers

have been reported in recent decade.
One of L-asparaginase gene from marine cyanobacterial, A. platensis is NIES39_A07830. The
gene were cloned successfully cloned in pET 22 plasmid under E. coli RGB DE3 expression
host (unpublished data). The expression, unfortunately form inclusion body. The protein
aggregate, inclusion body of NIES39_A07830 protein is nonfunctional protein which absent
from L-asparaginase activity. Therefore, here we reported an effort to functionalize the protein
by using refolding technique.

Literature Review
Heterologous recombinant expressions can be conducted by using several number of options.
Escherichia coli expression system is perhaps the most convenient and frequently use by
researchers. This expression systems offers easiness of culture, short life cycle, and easiness in
genome manipulation (Gopal and Kumar, 2013; Rosano and Ceccarelli, 2014). Even though, E.
coli expression offering the easiness in many ways, one drawbacks such Inclusion body
expression may appears.
Inclusion body or intracellular protein aggregates indicates misfolded of expressed protein.
Expressed Protein accumulate in the inappropriate cells compartment due overloading or
sometimes malfunction of protein control pathways in the cell (Kopito, 2000). Different
strategies can be used to overcome E coli system expression problem, (ie, changing vector,
expression host, culture environment, co-expression of other genes and gene manipulation).
Other approach to achieve active fraction of expressed protein are refolding strategy. This
approach contains two main steps namely, solubilization and refolding (Singh and panda, 2005).
Inclusion bodies cannot be solubilized using mechanical treatment such as French press,
sonication, and bead beating. They will solubilize in chemical agents, chaotropes agents such
urea, guanidine hydrochloride or detergents like triton X and Sodium Dedocyl Sulphate (SDS).
After the protein is soluble, the next step is refolding process. It is a trial and error process, even
rational design and recipes may be provided (Basu et al., 2011). Hence, overall obtaining
functional active of inclusion body is still nuisance tasks.

Methodology
Cloning gene.
The gene of protein NIES_A07830 from A. platensis was extracted. The gene were then
applified with a primers ( FNIES-A07 5-
GTAAATTGTACACATATGGGTGTTAATGTG-3 and
RNIES-A07 5-AGGGTAAGTGGCTCGAGTTTTGGCTAAC -3) contained NdeI and XhoI.
Amplified gene were purified and ligated to plasmid (pET 22b) with already restricted by using
same restriction enzyme. Transformations were performed with E. coli JM 109 for plasmid
amplification and E. coli RGB (DE3) for expression.
The cells were pre-cultured in 5 ml LB medium containing 1% glucose and appropriate amount
of 50 g ampicillin/ml, 30 g kanamycin/ml, and 34 g chloramphenicol/ml for overnight at
25-37 C. 1% pre-cultured celld were inoculated to 200 ml LB medium with the some
antibiotics as in pre-culture. After the cell reached OD600= 0.6-0.8, an isopropyl -D-
thiogalactoside (IPTG) in a final concentration of 0 1 mM was used for induction. The
incubation was continued for 16-h.

Refolding

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Prior to refolding process, solubilization of inclusion body was conducted. The pellet was
washed twice with 10 mM Tris-HCl buffer (pH 7.5) and added same buffer containing different
concentration of urea, Triton X-100, and sarkosyl for solubilization. Two different refolding
procedures given by Kuhelj et al., (1995); Batas et al., (1999); Joo et al. (2007) were followed
with modification. One-step dilution methods 40-fold into eight different refolding buffers
containing (Tris-HCl, PMFS, arginine, EDTA, Urea) at room temperature for 1224 h. The
protein was refolded via the gradual removal of urea upon dialysis into the buffer containing
0.1 M KPB (pH 7) with different concentration of urea at 4C. The details of the refolding
methods is shown in Tab. 1.

Table 1. Refolding methods

Dilution Method
No Buffer pH PMFS Arginine EDTA Urea
1 Tris-HCl 7 0.1 M 0.25 M 5 mM 10 mM
2 Tris-HCl 7 0.1 M 0.25 M 10 mM 10 mM
3 Tris-HCl 7 0.1 M 0.5 M 5 mM 10 mM
4 Tris-HCl 7 0.1 M 0.5 M 10 mM 10 mM
5 Tris-HCl 8 0.1 M 0.25 M 5 mM 10 mM
6 Tris-HCl 8 0.1 M 0.25 M 10 mM 10 mM
7 Tris-HCl 8 0.1 M 0.5 M 5 mM 10 mM
8 Tris-HCl 8 0.1 M 0.5 M 10 mM 10 mM
Dialysis Method
9 Buffer pH Urea
10 KPB 7 6M
11 KPB 7 4M
12 KPB 7 3M
13 KPB 7 2M
14 KPB 7 1M
15 KPB 7 0.5 M
16 KPB 7 0M

Enzyme assay
L-asparaginase activity was assayed following Sheng et al., (1993) method with slight
modification. The reaction aliquot, comprising of 15 mM L-asparagine, and samples, 400 l
pH 7 incubated on 37 C for 15 minutes. To stop the reaction, reaction aliquot was heated on
100 C for 2 minutes. Ninhidrin 0.01% was added and then incubated on 37 C for 3 hour.
After centrifugation with 6000 rpm for 5 minutes, the supernatant was read with absorbance of
340 nm.

SDS-PAGE analysis
Protein bands analysis followed Laemli, (1970) method with slight modification. SDS-PAGE
with 10% separating gel and 5% stacking gel was developed. The samples (10 l sample +
loading dye buffer) were loaded in each sample well. The gels were run at a constant voltage
for about 60- 120 min. followed by staining in Coomassie Brilliant Blue (CBB). De-staining
was achieved by allowing the gel for overnight under room temperature.

Results

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The gene encoding NIES39_A07830 was successfully cloned in pET-22b plasmid. The gene
was expressed in E. coli Rosetta Gami B DE3. The expression of NIES39_A07830 was found
in the pellet (Fig. 1). The activity of the enzyme was not found in neither supernatant nor pellet.

Fig 1. The expression of L-asparaginase in pET system. Protein molecular weight of RGB grown in 37C, IPTG
(0, 0.1, 0.5 and 1 mM) 1-4 soluble, 5-8 insoluble fraction. Black arrow indicated deduced expression of
NIES39_A07830

Solubilization
Pellet expression is also called inclusion body (IB). The inclusion body expression should be
refolded for gaining active form of enzyme. In this effort, the IB was solubilized by using
several chaothropic and detergent agents. The solubilization of IB using Urea, showed high
result of solubilization (Fig. 2). The concentration of urea from 2 mol to 8 mol, gave similar
result where IB expression of NIES39_A07830 found on solubilized fraction (S).

Fig. 2 Urea Solubilization (2M, 4M, 6M, 8M), S is soluble and I is insoluble fractions.

The solubilization also conducted by using Triton-X 100 dan sarkosyl. Triton-X 100
solubilization by using concentration of 0.25 %, 0.5 %, 0.75 %, 1 % were failed to solubilized the IB
( Fig. 3). In this treatment, Triton-X 100 was not suitable for solubilization agent. The IB was still

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found in insoluble fractions. It was indicated that for solubilizing the protein, Triton-X 100
failed to solubilize protein even after 1% of concentration.

Fig. 3 Triton X-100 Solubilization (0.25 %, 0.5 %, 0.75 %, 1 %)

Sarkosyl solubilization resulted in rather smear band of protein. Solubilization of inclusion

body on 0.5% sarkosyl indicate the least soluble protein. This result also similar in 2% sarkosyl.
Triss buffer containing 2% sarkosyl generate soluble fraction of inclusion body, even though
the protein of inclusion body was still found in insoluble fraction. Hence, the sarkosyl is may
be not suitable for solubilizing agent.

Fig. 3 Sarkosyl Solubilization (0.5 %, 1 %, 1.5 %, 2 %)

Refolding and L-Asparaginase Activity

L-asparaginase activity assay of protein NIES39_A07830 revealed that not all method resulted
in the same activity. The Highest activity was obtained from dialysis procedure. It was found

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that the activity of L-asparaginase was 64 U. It was almost two times higher than that of diluted
NIES39_A07830 (38 U).

Fig. 4 The best refolding result of after subjecting 10 mM Tris-HCl buffer (pH 7.5) containing 6 M Urea (M=
marker, 1= dilution method, 2= dialysis method)
Discussion
Protein in the form of inclusion body is absent from its functional activity. It is because
the folded form of the protein is not in normal condition. Fortunately, it can be returned to
functionally active protein. Two steps, namely of solubilization and refolding of the protein are
the two important for it. Proteins should be prepared in solubilization buffer solutions that
contains chaotropic, detergents, and reducing agents. Next step is refolding in the buffer which
usually containing the same agent in lesser concentration.
Successful solubilization can be investigated by knowing the protein whether in soluble
of insoluble fraction after centrifuged. If protein is still reside in insoluble fraction, it assumed
that the solubilization process was not succeed. In this investigation, three solubilizing agents
(urea, triton-X and sarkosyl) were used for solubilizing NIES_A0378 protein. With 4 M of urea,
the IB was successfully solubilized. In contrast, sarkosyl gave a smear result on protein band.
In addition, triton-X with the concentration of 2% even failed to solubilize NIES_A0378 protein.
Urea is considered as a powerful protein denaturant. The effectiveness of urea was probably
modulated by pH and ionic strength (Palmer and Wingfield, 2004; Hummon et al., 2007).
Among those three of solubilizing agents, urea was accounted as the best solubilizing
agent. Urea, demonstrated milder solubilization (only 4 M urea) conditions. It will give an
advantage such as higher final refolding yields (Puri et al., 1992). Other advantage is the low
price of urea. The only small drawback is probably the fact that solubilization of aggregate
protein by urea is pH dependent. Hence, pH controlling is needed during solubilizing process
(Khurana et al., 1995).
Refolding of NIES_A0378 is to change protein conformation from solubilized, unfolded
to folded state which is a native functional protein. We investigated two different procedures,
namely dilution and dialysis to refold the protein. In dilution without the presence of low
concentration of denaturant, unfolded protein will collapse into a rigid structure, hence, we
included the 10 mM Urea. The most applicable concentration is protein dependent, for example
the stability of the protein (Tsumoto et al., 2003). Step wise dialysis of unfolded protein resulted
in also active protein. In this method, unfolded protein sample was brought to equilibrium with
high denaturant concentration to low denaturant. In this steps, the equilibrium form of protein
is important to achieve correct and appropriate form of fold of protein.

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As inclusion bodies contain relatively pure and intact proteins, protein refolding is an
important process to obtain active recombinant proteins from inclusion bodies. However,
conventional refolding methods, such as dialysis and dilution, are time consuming and, often,
recovered yields of active proteins are low, and a trial-and-error process is required to achieve
success. Every protein is different and unique, hence there is no the best condition for refolding
and obtaining active protein (Albert et al., 2002). The agents in the protein refolding buffer is
sometimes interfering the bioactivity. Hence, it will affected L-asparaginase activity of the
refolded protein.

Conclusions
In this study, we reported protein refolding protocol for NIES39_A07830, which provided
success protocol to achieve an active recombinant protein of L-asparaginase enzyme. Urea is
the best denaturant to solubilize the protein. The dialysis method resulted in the less protein
bands in the refolding process. In addition, we were able to achieved active recombinant protein
of L-asparaginase enzyme.

Acknowledgements
We thanks to Institute of Research and Community Service, Faculty of Fisheries and Marine
Science, Brawijaya University for providing grant and support for this study.

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