4 Discussion
Inclusion bodies are mainly produced by over expression of recombinant
proteins, which intricate and costly denaturation and refolding
procedures are needed to recover biologically active proteins. Besides,
the final soluble refolded protein yields are typically very low because
of protein aggregation forming by interactions between the proteins
hydrophobic areas. In this study, the amino acid sequence of FPOX was
evaluated by protparam server as a bioinformatics tool. The evaluation
of FPOX amino acid sequence showed that it contains 437 amino acids with
about 52% of hydrophobic amino acids that is prone to form inclusion
body. It has been reported that a protein with a more hydrophobic part,
it will probably produces more aggregates [21]. The aggregation
behavior of a protein could be slowly changed by alteration of the
non-polar composition by only a few percent [22]. Under different
conditions of expression including the use of various temperatures
(16-37 °C) and IPTG concentrations (0.1-1 mM), FPOX was predominantly
expressed as inclusion body aggregates. The major section of FPOX
inclusion bodies are considered as large particles. It has been shown
that inclusion bodies can be formed intracellularly with a distinctive
size range of 0.2-1.5 μm as refractile particles [23]. Inclusion
bodies can intensify to more than 1 μm in diameter [24], a big part
of a single bacterial cell, and therefore can obviously be visible under
a microscope. Inclusion bodies are mainly composed of recombinant
protein (up to 99%), but contain also both chaperones and membrane
parts that join to the inclusion bodies during formation [24, 25]
and is in agreement with our study that the major part of inclusion body
is composed of recombinant FPOX based on SDS-PAGE analysis. Typically,
high concentrations of chaotropes and denaturants such asGdnHCl and urea
are used to solubilize inclusion bodies. While, the use of high
concentrations of these compounds leads to disrupt completely the
structure of protein that sometimes, causes protein aggregation during
refolding process [26]. In the present work, FPOX inclusion bodies
were solubilized by high concentration of urea (8 M), however, the
soluble FPOX showed no activity. Therefore, for depletion of urea and
refolding process FPOX was dialyzed against lower concentrations of
urea. However, the whole of protein aggregated during the elimination of
urea that can be attributed to the structure disruption of protein in
the presence of high level of urea. In this case, by traditional
urea-denatured procedure, FPOX was only soluble in urea 8 M, while,
recently, Qi et al. proposed a freeze-thawing method that can be used to
directly solubilize a considerable of inclusion body in low
concentration of urea [19]. In this context, by traditional
urea-denatured procedure, inclusion bodies of enhanced green fluorescent
preotein (EGFP) and catalytic domain of human macrophage metalloelastase
(MMP-12-CAT) could be solubilized in urea 5-8 M and are not soluble in
lower concentrations of urea (0-3 M). While, by freeze-thawing method,
both proteins inclusion bodies could highly solubilize in different
concentrations of urea (1-8 M), and maximum solubilization of them was
observed in urea 2 M that the solubility of these proteins in urea 2 M
by freeze-thawing method is comparable with their solubility in urea 8 M
by traditional urea-denatured procedure [19]. In this study, by
freeze-thawing method, FPOX was soluble by about 30 % in low
concentrations of urea (1-2 M) while was not soluble in urea 0.5 M. The
alkaline pH away from protein isoelectric point plays an important role
in aggregation destabilizing of inclusion body, therefore contributes to
the solubility of protein in the presence of low concentration of urea
[19]. It has been reported that the important agent of inclusion
body solubilization by freeze-thawing procedure is cold temperature
stress and the formation of ice crystals during freezing process
compared to other factors [19]. However, after dialyzing the soluble
FPOX in urea 1 and 2 M, it showed a weak activity. Totally, refolding
efficiency is low, leading to decreased yield of the final
product[27]. In both strains of E. coli,fpox gene was
expressed as inclusion body; however, the presence of FPOX in the
soluble phase was more in the shuffle strain. E. coli BL21 (DE3)
which no contains pivotal proteases such as OmpT is the greatest broadly
used prokaryotic expression host thatapplied as an important standard
among other expression hosts [10]. The reteplase was produced inE. coli BL21strain and it was found that all of the recombinant
protein is expressed as insoluble form [28] that is in agreement
with ourresult. In addition, by resolving codon bias problem in
Rosetta-gami B and SHuffle strains of E. coli , these strains are
also used to increase the solubility of heterologous proteins which have
been manipulated to create correct folding of proteins containing
disulfide bonds in E. coli [10, 29]. However, by expressing
reteplase with 9 disulfide bonds in these strains, no expression was
observed in the soluble form [28]. In case of reteplase, it was
formed as inclusion body in BL21, Rosetta-gami B and SHuffle strains ofE. coli which the greatest level of inclusion bodies was produced
in E. coli BL21 [28]. In another study, the greatest level of
FGF-1was produced in Shuffle strain with a maximum of soluble/insoluble
ratio than the two other strains [30] that is in accordance with the
solubility of FPOX in Shuffle strain compared to BL21 in our study.
However, the amount of FPOX in cytoplasmic fraction as soluble phase was
yet low; therefore, we applied other methods for increasing the
solubility of FPOX. It has been showed that the solubility of
recombinant proteins can be increased by adding compatible solutes
during protein expression [27]. In this study, we added sorbitol and
arginine to the culture medium during the protein expression. In the
presence of sorbitol, the solubility of FPOX had a negligible increase
compared to arginine and control. In agreement with our study, the
solubility of green fluorescent protein (GFP) was increased in the
presence of sorbitol in culture medium [27]. Sorbitol is applied
frequently as proteins stabilizer in vitro [31, 32]. The
inhibition of the native conformations unfolding to the
misfolded/unfolded states can be took place by sorbitol using a
mechanism similar to that of other polyhydric alcohols [33, 34].
Finally, sorbitol by conversion to fructose-6-phosphate is entered into
the glycolysis and helps to the ATP production [35, 36]. It has been
indicated that solutes which could potentially help to ATP production by
the cell and interact favorably with the side chains of protein and
stabilize them against inactivation, are effective solubilizers
[27]. In the presence of arginine in the culture medium, the growth
of bacteria and thus the level of FPOX expression were decreased. In
this context, Prasad et al. showed that the growth of bacteria in the
presence of arginine was decreased.TheGFP activity in the soluble phase
enhanced till 0.2 M arginine and then decreased [27]. In another
study, The solubilization of active GFP from insoluble phase was
obtained in higher concentration of arginine at 1 and 2 M. So, in our
study the used level of arginine may be low and should be optimized
according to the expression conditions [37]. In the present work, we
used pET22b as expression vector for the expression of fpox gene
which contains pelB signal peptide for the secretion of expressed
protein into periplasmic space in E.coli . Transfer of protein
from the inner membrane to periplasm is mediated by PelB signal peptide
[38]. In the periplasm, the signal peptide is cut by signal
peptidase and the mature protein is folded and transported across the
outer membrane [39]. In different studies, PelB has successfully
been used to secrete various enzymes into periplasmic space in E.
coli . In a study, active nattokinase was successfully secreted into
periplasmic space in E. coli using PelB and the native signal
peptide of nattokinase [40]. The level of inclusion bodies formation
of lipase from Pseudomonas fuorescens BJ-10 was 20.8 % of that
formed by non-tag expression during the use of PelB signal peptide
[12]. It seems that periplasmic space has much less protease
activity compared to the cytoplasm. Additionally, because of less
contaminating proteins in the periplasmic space, the purification of
recombinant proteins is easier than cytoplasmic fraction. Besides, since
periplasmic space has a more oxidative environment compared to the
cytoplasm, thus, the correct formation of disulfide bonds can be
facilitated [41]. According to our results, among proteases,
alkaline protease efficiently produced Fru-ValHis from HbA1c. In this
context, Hirokawa et al. selected an Aspergillus protease as the
most effective enzyme for releasing Fru-ValHis as substrate for FPOX
based on screening among different proteases [5]. In another study
by Hirokawa et al. different proteases with various sources of plant,
bacterial, fungal and yeast were investigated that they concluded among
different proteases, neutral protease with bacterial origin
(Bacillus polymyxa ) could efficiently liberate Fru-ValHis from
HbA1c [4]. It seems that proteases from bacterial source
(Bacillus genus) are a suitable enzyme for producing the
substrate of FPOX. In our study, FPOX showed activity for using in HbA1c
enzymatic method. In traditional HbA1c kits, 10-(Carboxymethyl amino
carbonyl)-3,7-bis (dimethylamino) phenothiadine sodium is mainly used as
coloring agent. However, we used TMB as coloring agent in HbA1c
enzymatic method for the first time. TMB has been used as a more
accurate, sensitive, cheap and non mutagenic coloring agent as peroxisae
substrate [42]. Thus, the use of TMB in HbA1c enzymatic method can
be economically useful and can be used to detect low concentrations of
H2O2. The activity of our FPOX enzyme
was compared with FPOX enzyme from HbA1c commercial Kit. For blood
samples from diabetic patients, similar results were obtained but our
results were gained after 20min instead of 5min obtained by the
commercial kit. The higher time can be attributed to the lower
concentration of our FPOX compared to the kit FPOX. Because of,more
enzymes are available to bind to the substrate is caused to increase the
reaction speed [43].