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].