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Protein
is one of the most important component of all organisms due to it biological
properties that have specific function and also depend on the physiological
with other molecules to regulate bodily functions. Protein perform vast
function within organism, including catalysing metabolic reaction, DNA
replication, responding to the stimuli and transporting the molecule from one
location to another. There are many type of proteins function such antibody
that bind specifically to foreign antigen like viruses and to help protect the
body from foreign material that can distrupt our normal body function (Gleba & Giritch, 2012).
Moreover, some of messenger proteins such as hormone and biomarker molecules
that transmit signal to biological processes between cells and some of them are
enzyme that assist the chemical activities that take place in the cells (Alberts et al., 2002).
Due to this properties, it is widely used molecular biology tool to study the
recombinant protein production using bacterial system.

When
we want to structure the mechanisms, pathways or other facts of the proteins,
we will find that the quantities are not sufficient from nature sources. In
that case, we need to produce high quantity and purity of proteins in short
time and cost-effective. Thus, recombinant protein is required to produce large
quantities of proteins by modifying gene sequence. By definition, recombinant
protein is encoded by recombinant DNA, which has been cloned in a system that
supports expression of the gene and translation of mRNA (Clark, Pazdernik, Clark, & Pazdernik, 2016).
Additionally, the major advantages of Escherichia coli system is simple
and well understood genetic, very easy to genetically manipulate, culturing
cost is minimal, expression is fast since the doubling time is only 20-30
minutes, well established regulatory track record, fermentation easy to scale
up, inclusion bodies easy to purify and no un-intended glycosylation and no
viral or prion contamination risks. Due to these advantages, is preferred as an
ideal system for protein expression some of the example of recombinant protein
expressed in Escherichia coli are insulin, Taq polymerase and others.

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Protein
production in Escherichia coli has been studied for over two decades.
Among of the bacterial system that available to produce heterologous protein, Escherichia
coli is the most suitable and applicable strains. It was the first
expression system introduced in biotech industry and Gram-negative bacteria is
still used widely in biopharmaceutical products. Escherichia coli offers
fast proliferation and fermentation period is few days only offering high
utilization of the degree of upstream facilities. The expression level is high
for about 1-5 g/L (Gleba & Giritch, 2012).
Strains of Escherichia coli depends on nature of works. There are
various type of strains that are specifically good for particular type of work.
So choosing the type of strains is the first thing to start the
experimentation. For example, the Escherichia coli DH5? strain is
usually for cloning routine, and Escherichia coli BL21 (DE3) is suitable
for T7 system expression as well as BL21 (DE3) pLysS usually used for
expression of T7 promoter with tight regulation (Gopal & Kumar, 2013).

Component
of the expression of gene of interest include vectors which is the DNA molecule
as the vehicle used to transfer foreign genetic material to the cells. Four
major type of vectors are plasmids, viruses, cosmids and artificial chromosomes
(Baneyx, 1999).
Moreover, vectors should have the following characteristics which are
selectable marker as well as antibiotic gene used for screening, regulatory
gene, origin of replication, T7 promoter and terminator, start and stop
codon,  to control the transcription and
translation process. Besides, expression vector have to have Shine-Delgarno
sequence that facilitate ribosome binding on the mRNA during translation and tag
or fusion protein for the stability of the protein molecule also the protease
cleavage site and multiple cloning site. Hence, this explained the basic
features of the Escherichia coli expression vectors which is needed for
cloning and the expression of the recombinant protein.

Many
theories have been proposed to explain that expression of recombinant protein
with induction of Isopropyl
?-D-1-thiogalactopyranoside (IPTG) yield
high density of the protein production. Moreover, IPTG induction is the most
efficient method to induce the expression vector. Despite of being preferable
technique to yield high density of recombinant protein, this technique has some
limitations that require culture monitoring to ensure the IPTG is added at
optimum condition. Besides, previous study conducted to investigate the
recombinant protein expression using classical method which is there is no IPTG
induction, protein also produced in large amount (Briand et al., 2016).
Thus, the study at that time had proven that, even in the absence of inducer,
the expression of target protein still can occurred.

In
contrast to a previous study, auto-induction of expression recombinant protein
were also investigated contribute large amount of protein production. As
described by Studier and collegues in 2005, auto-induction is efficient in screening
of many clones in parallel for expression and solubility of the protein. In
addition, investigation factors that affect this expression system is due to
the chemical reaction of complex media during the cultivation (Studier, 2005).
As an example, it is almost caused by the lactose, glucose and other amino acid
component that inhibit induction of T7 expression strains.  Moreover, recombinant protein also can be
expressed in un-induced expression system as previously reported by Sarabipour
and collegues in 2014. They found that expression system does not utilize IPTG
induction was still occur. Besides, the method relies on un-induced expression
in specific strains and yield high amount of protein production (Sarabipour, King, & Hristova, 2014).

However,
using bacterial expression system sometimes got their limitations such protein
solubility and the protein stability. High level of expression can also results
the insoluble material in the form of inclusion bodies and accumulate in the
cell. Some of the experiment conducted, they found that recombinant protein
expression in Escherichia coli also lack post-translational
modification as to be produced in eukaryotic system (Demain & Vaishnav, 2009).
Besides that, tag and fusion protein is used to protect from the degradation by
protease (Wang, Xiang, Wang, Wang, & Zhang, 2011).
Protein expression with no tag or fused with fusion protein may be unstable and
toxic to the bacteria system, thus make it easily to be degraded because fusion
protein increase folding, solubility and resistance to proteolysis.  Therefore, most of the tags will make our
desire protein more stable and soluble though the protein may not stay soluble
if the tag are removed.

Recombinant protein expression, Escherichia coli is the most
preferable microbial cell factory. As it is suitable host for expressing stably,
folded protein in an abundant products, but when coming across difficulties to
express protein such in eukaryotic system or in very large scale, there might
be several possible solutions to face the challenges. Nevertheless, most of
studies aiming the troubleshooting of recombinant protein expression and need
improvement from time to time as technology is growing rapidly and very
sophisticated. 

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