Protein inexpensive nutritional component such as Luria Bertani broth

Protein expression strongly related to the core of the molecular
mechanisms, which is the central dogma. It is the basic familiarity in the
molecular biology that deal with the base-pairing nature of nucleic acids and
structural nature of DNA, RNA and protein. The central dogma is the process of
how information is transferred between the DNA, RNA, and protein. Moreover, it
is basically expression of the idea of base sequence information in protein
that never converted back into DNA or RNA. Moreover, the basic process that
responsible for gene expression are transcription, RNA processing and
translation. Transcription is chemically and enzymatically where the DNA is
transcribed into mRNA while translation is the process conversion of mRNA that
decoded from transcription process to the proteins with the help of ribosomes
and other component in translation process.

Production of recombinant proteins generally contains two major
steps: molecule cloning and protein expression. Prior to transform into host
system, the foreign DNA or the gene of interest within the recombinant DNA
construct may or may not be expressed in the host machinery. The choice of the
host cell will initiate the step for the whole process. All of the strength and
the weakness of the host cell’s system may be affect the protein production.
The advantages using Escherichia coli as the host will generate high
density of the cultures. This is because Escherichia coli has the fast
growth kinetics which growth cycle approximately every 20 minutes (Sørensen & Mortensen, 2005). Moreover, being the most versatile organisms in the expression of
prokaryotic cells, there are large number of complex media that can be made
from readily available and inexpensive nutritional component such as Luria
Bertani broth that consist of tryptone, sodium chloride and yeast extract to
support the growth of the bacteria in the medium.

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Within
the domain of Escherichia coli expression, most popular approach for
producing protein is the coding sequence of the T7 system (Kevin, Vo, Michaelis, & Paddon, 1997).
T7 system or T7lac promoter
is an ideal system and very useful for producing recombinant protein. Subsequently,
to achieve expression of the recombinant protein, researchers have removed this
region and replace with gene of interest in sort of trick to produce the
desired protein. The key component of Escherichia coli expression
system is the lac operon that allow the vector to express the desired
protein. Using this system, the recombinant DNA such in pET vectors will be
introduced in to a T7 expression host such as Escherichia coli BL21
(DE3) and in the presence of inducer, the protein will be expressed.

Generally, T7 RNA polymerase is encoded by transcriptional control
of lacUV5 promoter in the bacterial genome.  Lactose repressor acts both at lacUV5
promoter to transcribe the gene of interest (Clark et al., 2016) and at the T7lac promoter in the vector (pET system) to
block the binding of T7 RNA polymerase to transcribe the target gene.  Induction using IPTG will expressed any
protein in bacterial is controlled by the lac operon. An operon is the
collection of genes and just downstream of T7 promoter. For instance, lactose
will cause the induction but, in the presence of readily metabolize carbon
source (glucose), induction will be difficult to occur. It is because in the
presence of both sugar, glucose will be utilized first and then the T7lac
promoter will be induced by the lactose.

Another essential point, lower level of glucose will cause the
cyclic adenosine monophosphate (cAMP) to produce and activate the lac operon (Rosano & Ceccarelli, 2014). This will promote the production of enzymes such ?
-galactosidase, transacetylase and permease. Then, lactose will be supressed by
the presence of glucose in the cell and hence, inactivate the basal expression
of theT7lac promoter. T7 expression system (Escherichia coli) can
be induced by lactose or its non-hydrolyzed analog which is isopropyl
?-D-1-thiogalactopyranoside (IPTG). Typically bacteria do not used lactose as a
food source, but when it is given enough lactose, lactose will bind to
repressor protein and will cause the reduction of two enzyme which are
?-galactosidase and permease (Christina et al., 2004). Besides, ?-galactosidase will hydrolyse disaccharide lactose to
monosaccharide glucose and lactose and hence, bacteria can continue to grow
with the presence of food source. Therefore, protein expression will be
regulated. However, there is a problem using the inducible T7 expression
system, because even in small amount of T7 RNA polymerase, it can lead abundant
expression of target protein.According to Studier, F.W (1990), even in the absence of IPTG,
there is some expression of T7 RNA polymerase from lacUV5 promoter in
the bacterial genome and basal expression of the target protein as illustrated
in the figure above. All of the recombinant protein expressed in Escherichia
coli may in the normal functioning of the cell and may be toxic to the
bacteria. Therefore, the strategy to encounter un-intend induction is by
culturing the bacterial cell in certain complex media. Complex media can
support growth of a wide range of Escherichia coli strain with different
nutritional requirement because it containing important elements such yeast
extract and casein (Studier, 2005). As described by Grossman et al., composition of the complex media
can induce substantial amounts of target protein upon saturation in the absence
of inducer. This is because, induction during saturation would stress the cell
and lactose initially as the inducer was not responsible for un-intended
induction but that cyclic AMP is required (Grossman et al., 1998). In addition, they found that using mutant cell, cyclic AMP does
not function appropriately although within the induction, thus it is affect the
stability and amount protein production. Thus, this modification help to
regulate protein expression upon saturation without adding inducing.

Another opinion, recombinant protein expression in Escherichia
coli had been proved to produce high yield and does not require IPTG
induction. According to a study by Sarabipour and collegues, they discovered,
without IPTG induction via un-induced expression for a period (12 to 18 hours)
does not require optimization in BL21 (DE3). 
Besides, glucose prevent induction by lactose and the amino acid prevent
the induction by lactose during log phase together with high rates of aeration
inhibit the induction. By doing experimental analysis, the proposed this
phenomenon was called auto-induction. Auto-induction is caused by the small
amounts of lactose in media that can be inhibited by the presence of glucose (Sarabipour et al., 2014). Thus, they suggested glucose did not significantly suppress the
production of recombinant protein in the absence of inducer, IPTG. As illustrated
in the figure below, the uptakes of lactose from media will inhibit theRecently, a group of researchers discovered a self-inducible
heterologous protein expression system in Escherichia coli. They found
that, this is a suitable strategy to overcome some problems that had been
observed in regulating protein expression using IPTG such as toxicity
production, cost and culture monitoring for larger scale. Therefore, they reported
Self-Inducible Expression (SILEX) system based on new strain that allow intrinsic
efficient auto-induction without any changes of culture medium (Briand et al., 2016). Moreover, SILEX system consist of genetically engineered BL21
(DE3) strain and SILEX plasmid that encoding human heat shock protein 70 (Hhsp70)
and second plasmid is encoding for the target gene and hence this is make the
SILEX is the simplest inducible expression system can be used. From their experimental
analysis, they have mentioned the correlation between hHsp70 and auto-induction
of its expression. hHsp70 is a stress protein that presents as anti-aggregation
function so, it will interact with the expressed protein with folding properties
(Mosser et al., 1997). It will prevent the leaky expression and degradation of protein production
in the bacterial cell. In addition, as proposed by Studier et al., an
auto-inducing medium did not require IPTG induction and this technique lead the
substantial expression of recombinant protein. Hence, above all the strong evidence,
it support SILEX system has improved recombinant protein expression by the
interaction between two proteins (hHsp70 and expressed target protein).

As conclusion, there are many techniques in recombinant protein
expression have been studied as un-intended induction, auto-induction and SILEX
system have proved to be generally useful producing protein without IPTG
induction. Despite of these techniques which more convenient than IPTG
induction, protein expression also can be done in high throughput and more
efficient on commercial scale.