Evaluating the Five Escherichia coli Derivative

Strains as Platform for Arginine Deiminase

Overproduction
Sara Abdollahi
Shiraz University of Medical Sciences
Mohammad Hossein Morowvat (  mhmorowvat@sums.ac.ir )
Shiraz University of Medical Sciences https://orcid.org/0000-0003-4466-3175
Amir Savardashtaki
Shiraz University of Medical Sciences
Cambyz Irajie
Shiraz University of Medical Sciences
Sohrab Najafipour
Fasa University of Medical Science
Younes Ghasemi
Shiraz University of Medical Sciences

Original article

Keywords: Arginine deiminase, Enzyme activity, Escherichia coli, Host cell physiology, Recombinant
protein production

Posted Date: November 23rd, 2020

DOI: https://doi.org/10.21203/rs.3.rs-112137/v1

License:   This work is licensed under a Creative Commons Attribution 4.0 International License.
Read Full License

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Abstract
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 modified expression strains. This study attempted to evaluate the
five host strains including BL21 (DE3), Rosetta (DE3), DH5α, XL1-BLUE and SHuffle 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 five strains exhibited a significant 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 efficacy evaluation of the five studied strains for the
production of similar pharmaceutical enzymes. The strains also could be analyzed in terms of
proteomics.

Key Points
All the five strains exhibited a significant band at 46 kDa which indicates successful production of
arginine deiminase (ADI).
E. coli BL21 (DE3) was recognized as the best expression platform for ADI production.
The obtained ADI from BL21 (DE3) and Rosetta (DE3) had the most activity.

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;
Karyolaimos et al. 2020). Using E. coli as the host microorganism has many advantages (Rosano and
Ceccarelli 2014) including fast growth kinetics, fast high-density cultivation (Sezonov et al. 2007;
Shiloach and Fass 2005), ease of genetic manipulation, relatively inexpensive cultivation, easy and fast
plasmid transformation with heterologous DNA (Pope and Kent 1996), rapid expression, recombinant
protein production over a period of one day, known metabolic pathways and the development of

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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), flagellar 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 SHuffle T7 which can be both
SHuffle-K12 and SHuffle-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 deficient in genes encoding OmpT
and Lon proteases. It has a good result for the production of recombinant proteins because OmpT can
degrade proteins during purification (Grodberg and Dunn 1988).

Rosetta (DE3) host strains are BL21 derivatives engineered which harbor the pRARE plasmid to enhance
the expression of eukaryotic proteins at high frequencies that contain rare codons used in E. coli. Rosetta
(DE3) strain carries genes for Arg (AGG, AGA and CGG), Ile (AUA), Leu (CUA), proline (CCC) and glycine
(GGA).This strain is deficient in Lon and OmpT proteases (Jia and Jeon 2016; Loyevsky et al. 2003; Terpe
2006).

The other most frequently used E. coli K-12 laboratory strain is DH5α which provides a critical platform
for routine cloning. Transformation with high efficiency and the lack of nonspecific endonuclease I
(endA1) has led to high-quality plasmid DNA (Anton and Raleigh 2016; Song et al. 2015; Taylor et al.
1993).

SHuffle T7 Competent E. coli are designed to catalyze the formation of disulfide bonds within the
cytoplasm of E. coli for their folding and activity (Lobstein et al. 2012). This is achieved by the genetic

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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 five host strains including BL21 (DE3), Rosetta (DE3), DH5α, XL1-
BLUE and SHuffle 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 five E. coli strains were used for arginine deiminase production: BL21 (DE3), Rosetta (DE3),
DH5α, XL1-BLUE and SHuffle T7.

In order to investigate the bacterial growth rate of these five 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 CaCl2 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

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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 final 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 final 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 H3PO4, 6M H2SO4,
and 2 mM NH4Fe (SO4)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 defined 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
significance 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 five 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

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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 five E. coli hosts: BL21 (DE3), Rosetta
(DE3), DH5α, XL1-BLUE and SHuffle 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 significant 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 five E. coli strains.

For quantification 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 significantly 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
Fig. 3.

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
significance of different bacterial host strain for increasing the ADI enzymatic activity.

In the present study we investigated five E. coli strains for to improve the arginine deiminase production,
including BL21 (DE3), Rosetta (DE3), DH5α, XL1-BLUE and SHuffle 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 SHuffle T7.

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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 disulfide 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 SHuffle cells is a proper candidate for expressing the proteins that need disulfide bonds for
their folding and activity. There are two versions of engineered SHuffle strains with two distinctive E. coli
strain backgrounds, SHuffle K-12 and SHuffle B (Ren et al. 2016). In this research we used SHuffle 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 five 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 SHuffle 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 find 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 significant difference compared with other ADI enzymes from DH5α, SHuffle 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 efficient expression system with respect to
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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 find 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, financial 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. Najafipour contributed to
conception, design, acquisition, analysis, and interpretation; All authors contributed to design, acquisition,

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and analysis. All authors critically revised the manuscript, gave final approval, and agreed to be
accountable for all aspects of the work.

Acknowledgements

This study was a part of PhD dissertation of Sara Abdollahi, proposed and approved in Department of
Medical Biotechnology, School of Advanced Medical Sciences and Technologies, Shiraz University of
Medical Sciences, Shiraz, Iran.

ORCID IDs

Sara Abdollahi https://orcid.org/0000-0001-5730-9285

Mohammad Hossein Morowvat https://orcid.org/0000-0003-4466-3175

Amir Savardashtaki https://orcid.org/0000-0003-1700-9426

Cambyz Irajie https://orcid.org/0000-0001-8438-2146

Sohrab Najafipour https://orcid.org/0000-0001-5224-5876

Younes Ghasemi https://orcid.org/0000-0003-4172-0672

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Figures

Figure 1

Microbial growth curves of five E. coli strains at 37 °C, 120 rpm for 10 h using optical density
measurement method at 600 nm

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Figure 2

A) SDS-PAGE analysis of ADI expression induced with IPTG in five E. coli hosts: BL21 (DE3), SHuffle T7
and Rosetta (DE3). Lane M: protein molecular weight marker (KD), Lane 1: (negative control) total E. coli
BL21 (DE3) lysate without induction, Lane 2: (negative control) total E. coli BL21 (DE3) lysate after
induction, Lane 3: E. coli BL21 (DE3) harboring plasmid pET3a-ADI without induction Lane 4: E. coli BL21
(DE3) harboring plasmid pET3a-ADI after induction, Lane 5: SHuffle T7 harboring plasmid pET3a-ADI
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without induction Lane 6: SHuffle harboring plasmid pET3a-ADI after induction, Lane 7: Rosetta (DE3)
harboring plasmid pET3a-ADI without induction Lane 8: Rosetta (DE3) harboring plasmid pET3a-ADI after
induction, B) SDS-PAGE analysis of ADI expression induced with IPTG in DH5α, XL1-BLUE. Lane M:
protein molecular weight marker (KD), Lane 1: DH5α harboring plasmid pET3a-ADI without induction
Lane 2: DH5α harboring plasmid pET3a-ADI after induction, Lane 3: XL1-BLUE harboring plasmid pET3a-
ADI without induction Lane 4: XL1-BLUE harboring plasmid pET3a-ADI after induction

Figure 3

Quantitative gel densitometry measurements of gel lanes (in the range of 46 kDa) as determined using
ImageJ software. 1: (negative control) BL21 (DE3) (not-transformed), 2: BL21 (DE3), 3. SHuffle T7, 4:
Rosetta (DE3), 5: DH5α, 6: XL1-BLUE

Figure 4

The specific supernatant enzyme activity for the obtained ADI from each studied E. coli strain

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