GST Fusion Assisted Overexpression and Purification of Recombinant Parasite Lactate

Dehydrogenase Enzyme in Escherichia coli

Ramdhani Haryanti1, Sulaiman N. Depemade2, Muhamad Ali2*
1

Magister Management of Animal Husbandry Resources Program University of Mataram, Jl.

Majapahit No. 62, Mataram, Indonesia, 83125; 2Laboratory of Microbiology and Biotechnology
Faculty of Animal Husbandry Universy of Mataram,
Jl. Majapahit No. 62 Mataram, Indonesia, 83125.

*Corresponding author: ali.molbiotech@gmail.com

Abstract
Recently, Escherichia coli is one of the well-established and most popular organisms
for the production of recombinant proteins. However, expression levels and solubility may be
issue, since some proteins are generated in low amount and aggregate in inclusion body.
Fusion proteins have become essential for the overexpression and solubility improvement of
recombinant proteins in E. coli. In this study, parasite Lactate dehydrogenase-encoding gene
was fused in the C-terminal of glutathione-s-transferase gene and subsequently expressed in
E. coli BL21. Expression levels and purification results of the fused protein were determined
by SDS-PAGE. The SDS-PAGE result shows that the 58 kDa band corresponding to the GSTpLDH protein was successfully overexpressed and purified using GSTrap column. Our
results are not only useful for robust production of parasite Lactate dehydrogenase, but also
helpful for the enzyme purification.
Keywords: parasite Lactate Dehydrogenase (pLDH), Glutathione-s-Transferase (GST),
Fusion Protein, Escherichia coli, GSTrap column

Introduction
Parasite lactate dehydrogenase (pLDH) is an enzyme produced by the anaerobic
glycolysis malaria-causing parasite, Plasmodium, which infects red cells to produce ATP.
pLDH have been found in four species of malaria and for each species there are different
isomers (Kusuma et al., 2014). The enzyme has led Plasmodium able to live in the red blood
cells to produce NAD from NADH to be used in the process of glycolysis (Berwal et al.,
2008). This enzyme works to break down the lactic acid to pyruvic acid is used as an energy
source for Plasmodium.
pLDH can be used to diagnose malaria, evaluate the results of treatment, as well as
for screening anti-malarial drugs (Alfiah et al., 2009; Nwazue, 2014). The existence of this
enzyme in the blood of a person can be a clue that in the blood is contained Plasmodium
blood so the owners are known to suffer malaria. Berwal et al. (2008) and Ali et al. (2013)
mentions another benefit pLDH is as a target antimalarial compounds, which could be used to
find new drugs for malaria. PLDH crystal structure indicates amino acids near the "active
site" that allows the binding of the enzyme with the inhibitor. The formation of the bond
between pLDH with such inhibitors would cause pLDH inactive so that Plasmodium can not
survive.
pLDH to produce in large quantities, the production of recombinant enzyme in the
bacteria E. coli is an option that is widely used today (Ali, 2015). Ali et al (2005) adds that,

the use of E. coli bacteria is very popular lately because the technology is well established,
has been supported by the tools and materials are available commercially and can produce
proteins in a relatively short time (12 hours). The use of E. coli BL21 is advantageous in
addition to more efficiently produce proteins and to produce acetic acid and prevent a drop in
pH can affect the growth media. However, Rosano and Cecarelli (2014) states that the
production of recombinant proteins in E. coli often produces lower amounts of protein quality
is not adequate, especially for difficult proteins are expressed (difficult-to-express proteins).
Berwal et al. (2008) have expressed pLDH recombinant E. coli also produce lower amounts
of protein.
Rosano and Cecarelli (2014) states that one of the strategies to increase the expression
of recombinant proteins in E. coli is to use fusion technology (merge with other proteins),
including by glutath S-transferase (GST). Glutathione S-transferase (GST) is a cytosolic
enzyme multifunctional group plays an important role in detoxifying electrophilic compounds
by conjugation with glutathione (GSH). Glutathione S-transferase is a multifunctional
enzyme complex that plays a role in detoxification of electrophilic xenobiotic compounds
(Griscelli et al., 2004). On the premise above, then this study will be reviewed enzyme
expression pLDH fused to GST enzyme in the bacteria E. coli BL21. Through the fusion is
expected pLDH be expressed (produced) a sufficient number of expected quality.

Methodology
This study will be conducted at the Laboratory of Microbiology and Biotechnology
Faculty of Animal Husbandry Universitas Mataram implemented for 3 months. First step to
preparation of Lisogeny Broth (LB). Isolates starting from the stage inoculation of bacteria in
the form of glycerol stock (at a temperature of -20C) on a solid LB medium containing 50 ug
/ ml ampicillin (concentration of working solution) then incubated in the incubator at 37 C
for 12 hours. After that, a single colony was inoculated to manufacture starter using 100 ml of
liquid LB medium containing 50 ug / ml ampicillin. Then the culture in shaker at 37 C with
a velocity of 120 rpm for 12 hours. Recombinant Protein Expression of GST-pLDH starter
bacteria were incubated 24 hours at a temperature of 37C inoculated back in liquid LB
medium containing 50 ug / ml ampicillin. Incubation was carried out at a temperature of 37C
with a shaker speed of 120 rpm and input IPTG was then added different concentrations and
testing results by SDS PAGE preparation. The variables were observed in this study is the
thickness of the protein band to express recombinant proteins (factors affecting such
expression IPTG concentration and temperature optimum).

Result and Discussion

The results showed that the expression of protein msg looks pretty good. This is likely
because the concentration of IPTG (1 mM) used was appropriate, the expression of the
protein bands pLDH looks nice. In the use of IPTG to note is more or less a given because it
can give toxic effects or will not be able terekspresikannya pLDH recombinant protein.
Snustad and Simmons (2003) states that the use of IPTG begins with IPTG bound at the
binding site for the repressor protein inducers. The addition of inducer (IPTG) repressor
resulted in changes in the structure of the binding site for the repressor protein with a binding
site for the repressor to the operator. coli can produce recombinant proteins in large numbers
very quickly and cheaply. But one of the problems encountered in the production of
recombinant proteins in E. coli is the formation of inclusion body recombinant proteins
beragregat namely in the form insoluble in the cytoplasm.

M
kDa
130
100
70

GST-pLDH

55
35
25
15
10

Figure 1. The test results of SDS-PAGE pGEX-pLDH

The presence of15
excessive expression process can be burdensome and inhibit the
secretion of recombinant proteins by the cell. This causes a lot of protein can not be secreted
into the site of bond formation disulfide, folding of proteins into proteins active and mature
(prisplasmik) that will form a collection or aggregates of the protein insoluble (inclusion
body) (Sorenson and Mortensen, 2004). So from that conducted this solubility test
(Solubilization) to change the protein insoluble (insoluble) or the so-called inclusion body
into a soluble protein (soluble) in the sample used (recombinant protein GST-pLDH). This
was followed by phases of characterization using SDS-PAGE to see the profile of
recombinant proteins

Conclusion
Based on the research, it was concluded that the GST-pLDH has a molecular weight
of about 58 kDa.

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