Evaluating the Five Escherichia coli Derivative Strains as Platform for Arginine Deiminase Overproduction

Escherichia coli is one of the most preferred host microorganisms for the production of recombinant proteins due to its well-characterized genome, availability of various expression vectors and host strains. Choosing a proper host strain for the overproduction of a desired recombinant protein is very important because of the diversity of genetically modied expression strains. This study attempted to evaluate the ve host strains including BL21 (DE3), Rosetta (DE3), DH5α, XL1-BLUE and SHue in terms of arginine deiminase (ADI) production and enzyme activity. Arginine deiminase (ADI) was chosen a bacterial enzyme which degrades L-arginine. It is effective in treatment of some types of human cancers like melanoma and hepatocellular carcinoma (HCC) which are arginine-auxotrophic. Five mentioned E. coli strains were cultivated. The pET-3a was used as the expression vector. The competent E. coli cells were obtained through CaCl 2 method. It was then transformed with the construct of pET3a-ADI using heat shock strategy. The ADI production levels were examined by 10% SDS-PAGE analysis. The ability of host strains for expression of the requested recombinant protein was compared. The enzymatic activity of the obtained recombinant ADI from each studied strain was assessed by a colorimetric 96-well microtiter plate assay. All the ve strains exhibited a signicant band at 46 kDa. BL21 (DE3) produced the highest amount of ADI protein followed by Rosetta (DE3). The following activity assay showed that ADI from BL21 (DE3) and Rosetta (DE3) had the most activity. There are some genetic and metabolic differences among the various E. coli strains, leading to differences in the amount of recombinant protein production. The results of this study can be used for the ecacy evaluation of the ve studied strains for the production of similar pharmaceutical enzymes. The strains also could be analyzed in terms of proteomics.


Introduction
One of the host microorganisms of choice for the production of recombinant proteins is Escherichia coli which has been extensively used for various biotechnology purposes (Ferrer-Miralles et al. 2009; fermentation methods and conditions of its cultivation (Kang and Seong 2020;Sahdev et al. 2008;Sandomenico et al. 2020;Sørensen and Mortensen 2005). E. coli B and K-12 strains are among the most generally used bacterial hosts for recombinant proteins production on an industrial scale (Marisch et al. 2013). The source of these two strains are supposedly both from normal commensals of the human gut, and their derivatives have existed in the laboratory since 1922 and before 1918, respectively (Daegelen et al. 2009). E. coli B and K-12 MG1655 strains have been compared to each other in terms of genetic and biochemical properties. It was found that their genomes had about 99% sequence identity in size and structure in 92% of their genomes (Jeong et al. 2009). They also differ noticeably in distribution of insertions sequences and other changes because of horizontal gene transfer (Jeong et al. 2009). Differences between B and K12 strains include the absence of ompT proteases in BL21 (DE3) (Jeong et al. 2015), agellar component genes and the dcm, which encodes the DNA cytosine methylase (Jeong et al. 2009).
Most frequently used E. coli B and K-12 host strains which are widely available for protein expression including B [Rosetta (DE3) and BL21 (DE3)], K-12 (DH5α, XL1-Blue) and SHu e T7 which can be both SHu e-K12 and SHu e-B.
The expression host strain has a crucial role in recombinant protein expression. These host strains do not produce some harmful natural proteases in order to keep the expression plasmid stable. There are many helpful host strains for a variety of individual applications. One of the most common E. coli host strains for protein expression which are widely available laboratory strains including BL21 (DE3). BL21 is able to grow quickly in minimal media (Chart et al. 2000;Sørensen and Mortensen 2005). BL21 (DE3) is a strain which extensively used for recombinant proteins production under the control of T7 RNA polymerase (Studier 1990;Studier and Moffatt 1986). BL21 is de cient in genes encoding OmpT and Lon proteases. It has a good result for the production of recombinant proteins because OmpT can degrade proteins during puri cation (Grodberg and Dunn 1988).
The other most frequently used E. coli K-12 laboratory strain is DH5α which provides a critical platform for routine cloning. Transformation with high e ciency and the lack of nonspeci c endonuclease I (endA1) has led to high-quality plasmid DNA (Anton and Raleigh 2016;Song et al. 2015;Taylor et al. 1993).
SHu e T7 Competent E. coli are designed to catalyze the formation of disul de bonds within the cytoplasm of E. coli for their folding and activity (Lobstein et al. 2012). This is achieved by the genetic deletion of the chromosomal copies of gor and trxB which expresses active cytoplasmic DsbC. (Gon et al. 2006;Ren et al. 2016).
XL-1 Blue are K strains which are used in cloning and protein expression are great hosts for routine cloning applications using plasmid or lambda vectors, And are the most popular strain for blue-white screening (Bullock 1987).
The aim of this study is to compare the ve host strains including BL21 (DE3), Rosetta (DE3), DH5α, XL1-BLUE and SHu e T7, in terms of growth rate, ADI expression levels and protein activity. It has been widely exploited for different cancer therapy purposes such as melanoma and hepatocellular carcinoma (HCC). Choosing the best host organism could increase the ADI production concnetration, activity and yield. As far as we know, there is no similar report on this topic.

Materials And Methods
Bacterial strains, culture media and growth A total of ve E. coli strains were used for arginine deiminase production: BL21 (DE3), Rosetta (DE3), DH5α, XL1-BLUE and SHu e T7.
In order to investigate the bacterial growth rate of these ve strains, optical density of these cultures was measured at 600 nm using a spectrophotometer every one hour during ten hours.
Luria Bertani broth (LB) media (Sigma-Aldrich, St. Louis, MO, USA) containing yeast extract, 5.0 g/L; tryptone, 10.0 g/L; and NaCl, 10.0 g/L was exploited for cell growth and preservation. The optical density measurement at 600 nm was exploited hourly during 10 h to determine the growth pattern for each studied strain. LB culture medium was used as the blank for this purpose. Plasmid construction, vector and molecular cloning pET-3a vector was used as the expression vector. The pET3a-ADI construct was designed and synthetized by CinnaGen company (Tehran, Iran). The competent host cells were produced by routine CaCl 2 method and transformation was performed through heat shock method with the construct of pET3a-ADI (Morowvat et al. 2014).

Protein Expression
One-milliliter of overnight cultures in 50 mL of LB broth containing 50 µg/mL ampicillin (Sigma-Aldrich, St. Louis, MO, USA) at 37 ºC was inoculated into 50 mL of the mentioned culture medium. It was then incubated at 37 ºC. For protein expression, induction was carried out with 1 mM IPTG during the exponential growth phase when the cultures reached about 0.6 in OD 600 . Then, the cultures were incubated for a further 4 h at 37 ºC.

SDS-page Analysis
For analysis of arginine deiminase production, the prokaryotic host cells were centrifuged at 7000 rpm for 10 min. According to the Laemmli method, the total cell protein was assessed using 10% SDS-PAGE analysis (Morowvat et al. 2014). Gel densitometry was performed using the publicly available ImageJ software 1.52a (Girish and Vijayalakshmi 2004).

ADI Activity Assay
The ADI activity was measured by using the E. coli pellets from 1 mL of the nal culture medium. It was suspended in 1 mL lysis buffer containing 1 mM EDTA, 150 mM NaCl, 100 mM phosphate buffer, 1 mM PMSF and 2 mg/mL lysozyme. The suspension was then incubated for 30 min at 30 ºC. After this step, the soluble protein-containing fractions were separated using centrifugation at 13,000 rpm at 4 ºC for 10 min. 20 µL of clear supernatant was exploited to perform the ADI activity assay. The mentioned assay was performed by measuring the concentration of citrulline as the nal product of the enzymatic reaction. It was performed as a colorimetric 96-well microtiter plate assay by as described by Knipp and Vasak (Knipp and Vašák 2000). The arginine solution (40 µL) was added to the ADI soluble fraction (20 µL) in a 96-well microtiter plate. After this step, the enzyme reaction was commenced. The mixture was then incubated at 37 ºC using a water bath for 30 min. Subsequently, the color developing reagent (COLDER) was added (200 µL) to the enzymatic reaction. The COLDER solution was composed of 1 Vol. of 80 mM diacetyl monoxime (DAMO), 2.0 mM thiosemicarbazide, three Vol. of 3M H 3 PO 4 , 6M H 2 SO 4 , and 2 mM NH 4 Fe (SO 4 ) 2 . Finally, the microtiter plate was heated for 15 min at 95 ºC for color development. The absorbance was measured at 530 nm after cooling to the room temperature. To measure the concentration of the produced citrulline, a calibration curve was generated (0-400 µM citrulline). One unit (U) of activity was de ned as the amount of enzyme to produce one µmol citrulline per minute at 37 °C under the assay conditions (Noh et al. 2004).

Bradford Assay
For measuring the concentration of total protein in each sample we used Bradford assay. It is a rapid and sensitive method for measuring the protein values in the microgram range. In this method, the Coomassie Brilliant Blue was attached to the protein and consequently, it changed the wavelength from 465 nm to 595 nm. Therefore, at a wavelength of 595 nm, the protein-color complex measurements were performed.

Statistical analysis
Analysis of variance (ANOVA) with the statistical difference at 5% was selected to determine the signi cance of the observed results. The GraphPad prism version 8.02 (GraphPad Software, La Jolla California, USA) was employed for the statistical analysis.

Results
With evaluating the ve strains growth curves over 10 h, we found that all of them follow an almost identical reproduction pattern (Fig. 1). The arginine deiminase (ADI) gene was optimized and cloned computationally in NdeI and BamHI recognition sites of pET-3a (+) expression vector, and this gene was located under the IPTG-inducible control T7 promoter.
Transformation And Expression Of ADI pET3a-ADI constructs were transformed successfully in all the ve E. coli hosts: BL21 (DE3), Rosetta (DE3), DH5α, XL1-BLUE and SHu e T7. When the cell density reached to OD 600 of 0.6 for each host strains, the induction was performed with 1 mM IPTG at 37 ºC for four hours. The SDS-PAGE analysis of ADI expression showed a single signi cant band at 46 kDa as expected due to molecular weight of the Mycoplasma ADI for each sample. Figure 2 shows the comparison of ADI expression in insoluble fraction of the ve E. coli strains.
For quanti cation of protein expression, the ImageJ gel densitometry analysis was employed. It measures the protein bands. It was revealed that the intensities of protein yield for BL21 (DE3) and Rosetta (DE3) were signi cantly higher than those of other host strains tested. The relative band intensities of the ADI protein in each strain in the range of 46 kDa were derived and results were shown in

Enzyme Activity
As shown in Fig. 4, the arginine deiminases which have been expressed in BL21 (DE3) and Rosetta (DE3) had higher enzyme activity compared to those were expressed in other strains. These results implied the signi cance of different bacterial host strain for increasing the ADI enzymatic activity.
In the present study we investigated ve E. coli strains for to improve the arginine deiminase production, including BL21 (DE3), Rosetta (DE3), DH5α, XL1-BLUE and SHu e T7. We compared their growth rate, ability for production yield, enzyme activity and concentration of total protein of each strain.

Discussion
Arginine deiminase (ADI) is an important therapeutic enzyme which metabolize the L-arginine (Liu et al. 1995). The hepatocellular carcinoma (HCC) and melanoma which are recognized auxotrophic situation for arginine are the major types of human cancer which might be treated with the recombinant ADI (Ensor et al. 2002). To decrease its immunogenicity, the PEGylated form of arginine deiminase (ADI-PEG20) is practiced as an anticancer therapeutic (Han et al. 2016;Yang et al. 2010). After its application, the local reservoir L-arginine depletes. Eventually, it inhibits the growth of arginine-auxotrophic tumor cells (Ni et al. 2008). It has been granted as an orphan drug by the food and drug administration (FDA) and European medicines agency (EMA) (Shen and Shen 2006;Zhu et al. 2010).
After analyzing the SDS PAGE and ImageJ software results, it was found that E. coli BL21 (DE3) and Rosetta (DE3) produced the highest amount of ADI followed by DH5α, XL1-BLUE and SHu e T7.
Enzyme activity can be changed by a variety of factors, such as temperature, pH, and concentration. So, it makes sense that the ADI enzymes which are produced in different bacterial hosts have different enzyme activity.
Choosing a proper host is very important in biotechnology. Due to its diverse molecular networks, each species exhibits different behaviors when expressing recombinant proteins (de Moura et al. 2020;Li and Huang 2018;Rai et al. 2020). The most frequent prokaryotic strain which has been very widely used to express recombinant proteins is E. coli BL21 (DE3) (Makino et al. 2011;Rosano and Ceccarelli 2014). The Rosetta (DE3) strain is engineered to boost the expression of genes containing rare codons. Due to the mutations of trxB and gor genes it can increase disul de bond formation in the cytosolic fraction. We have to consider that the use of these strains, usually enhances the levels of protein production but sometimes can lead to a decrease in protein solubility (Fathi-Roudsari et al. 2016;Rosano and Ceccarelli 2014). Both of BL21 (DE3) and Rosetta (DE3) are E. coli B strains. Characteristics such as the absence of two main proteases OmpT and Lon protease enhanced permeability make E. coli B a desirable host for the production of genetically engineered proteins.
Engineered SHu e cells is a proper candidate for expressing the proteins that need disul de bonds for their folding and activity. There are two versions of engineered SHu e strains with two distinctive E. coli strain backgrounds, SHu e K-12 and SHu e B (Ren et al. 2016). In this research we used SHu e K-12 for ADI overexpression.
In a study by Tegel et al. the impact of two E. coli expression strains on the recombinant human protein production was assessed. Results showed an improved expression yield with using the Rosetta (DE3). They suggested this strain can be an appropriate choice for high-throughput protein production (Tegel et al. 2010). Wang et al evaluated GsiA-GFP fusion-protein expression in ve E. coli expression strains. The most productive strain for expressing GsiA-GFP fusion-protein was E. coli BL21 (DE3) (Wang et al. 2011). Fathi-Roudsari et al. compared three E. coli strains in recombinant production of reteplase. The results showed that BL21 (DE3) has the highest level of expression in inclusion bodies followed by Rosetta (DE3) and SHu e T7 (Fathi-Roudsari et al. 2016).
As we have described previously, the ADI is an enzyme which degrades arginine in HCC and melanoma tumor cells which are sensitive to arginine depletion. In this project we intended to analyze these E. coli B and K-12 host in expressing ADI protein and to evaluate their activity.
Previous studies have cloned various ADI genes and expressed in E. coli strains to nd their functions in arginine metabolism, cell growth, and biological activities including anti-tumor activity (Ni et al. 2008). Among all the ADI enzymes, those which were produced in BL21 (DE3) and Rosetta (DE3) had the most activity and signi cant difference compared with other ADI enzymes from DH5α, SHu e T7 and XL1-BLUE strains.
Our results revealed some differences among the two K-12 and B strains for an unstudied recombinant protein for human cancer therapy. The B strains had the most e cient expression system with respect to their higher expression level, ADI enzymes amount and also the higher enzyme activity. Each recombinant system has a lot of differences in many factors such as growth behavior, yields of product, and other characteristics. The most appropriate host strain should be chooses based on the characteristics of the requested protein. All of the examined strains have been successful in expressing the ADI protein, but some strains seem to be more successful. A part of this success is due to the physicochemical properties of this protein. Therefore, to nd out more, the exact molecular mechanism of recombinant protein overexpression needs to be further investigated.
The results of this study could be useful in choosing the best host strain and optimizing the production of ADI enzyme. For notable production of soluble proteins in E. coli, some strategies need to be considered such as engineering the hosts or analyzing environmental parameters and manipulating the strength of promoter.

Declarations
Ethics approval and consent to participate Not applicable.

Consent for publication
Not applicable.

Availability of data and material
All generated or analyzed data, the exploited softwares and materials were included in this published article. The generated results during the current study are available from the corresponding author on reasonable request.

Competing of interests
The authors declare that they have no competing interests, nancial or otherwise in this study. The manuscript is approved by all the authors.

Funding
This work was supported by Research and Technology Deputy of Shiraz University of Medical Sciences, Shiraz. Iran (Grant no. 98-01-36-19817).
Authors contribution S. Abdollahi, M. H. Morowvat and Y. Ghasemi contributed to conception, design, acquisition, analysis, interpretation, and drafted the manuscript; A. Savardashtaki, C. Irajie, and S. Naja pour contributed to conception, design, acquisition, analysis, and interpretation; All authors contributed to design, acquisition, and analysis. All authors critically revised the manuscript, gave nal approval, and agreed to be accountable for all aspects of the work.