Safety Aspect of Recombinant Protein Produced by *Escherichia coli*: Toxin Evaluation with Strain and Genomic Approach

*Iman Permana Maksum, Ahmad Nabiel, Safri Ishmayana and Ukun M.S. Soedjanaatmadja*

#### **Abstract**

*Escherichia coli* is a Gram-negative bacteria which is well known for its pathogenic properties that can cause serious food poisoning, mostly indicated by diarrhea or other severe symptoms. Despite of its well-known properties due to its ability to produce toxin, most of *E. coli* strains are harmless and even beneficial especially in recombinant protein production. This bacterium is suitable for protein recombinant host since it has rapid growth, high expression rate, and well-known genome. Various proteins have been produced using *E. coli* expression systems, with therapeutic protein for medical application being the most notably produced. Apart from that, our group succeeded in producing beta galactosidase from a wild type *E. coli* strain B130. Furthermore, recombinant human serum albumin was successfully produced using *E. coli* strains BL21 (DE3). However, studies on *E. coli* toxin contamination in recombinant protein productions, strains, and genomic comprehension are indispensable, particularly in therapeutic protein. Therefore, this chapter will discuss the safety aspects of recombinant therapeutic proteins in terms of toxin contamination by strain and genomic approaches.

**Keywords:** *E. coli*, genomic maps, recombinant therapeutic proteins, toxin

#### **1. Introduction**

*Escherichia coli* is a member of *Enterobacteria* family which can be found in gastrointestinal tracts [1–3]. In general, it is well known to cause broad diseases, including gastrointestinal problems. Aside the fact that *E. coli* was normal to be found in colon, a number of its strains were discovered with the ability to produce toxins. Shiga toxin *E. coli* (STEC) and enterotoxin *E. coli* (ETEC) were groups of *E. coli* strains that have the ability to produce toxin that may cause several diseases, such as diarrhea [1, 4, 5].

Although *E. coli* may cause numerous gastrointestinal diseases; in fact, strains that are responsible for pathogenic properties were relatively minor in numbers. Furthermore, *E. coli* was considered harmless and even useful as a host for producing recombinant proteins. Even this bacteria becomes favorite host chosen in industrial and medical applications since it has rapid growth, well-characterized

gene, and its ability to grow under aerobic and anaerobic system, and facilitates to form high cell density culture (HCDC) [6–8].

The discussion about advantage in producing recombinant proteins and worries of toxins of *E. coli* is like talking about two opposite sides of a coin. This will certainly raise a question "Is it safe to produce recombinant protein in *E. coli*? Will it be toxin-free contamination?" Therefore, this chapter will discuss the safety aspects of recombinant protein produced by *E. coli* against toxins using genomic and strains approach.

#### **2. Toxin produced by** *Escherichia coli*

Several pathogenic *E. coli* strains are known to be responsible for broad diseases, from mild to complicated cases. It is varying from mild diarrhea, hemorrhagic colitis, to hemolytic uremic syndrome. Among the pathogenic strains, STEC is an example of common strains which occupy high number in *E. coli* serotypes that produce toxin called Shiga toxins (Stx) [1, 2, 5, 9–11]. While STEC is a common pathogenic example, it belongs to a larger group named enterohemorrhagic *E. coli* (EHEC); also, there still exist numerous pathogenic *E. coli* and cause different diseases and complications. Pathogenic *E. coli* were classified in **Table 1** along with its diseases they caused and virulence factors [1].

Considering the number of pathogenic *E. coli*, it is useful to classify the toxins' properties and structure. It will be convenience to determine whether the toxins belong to organic compound or peptide-based structure; therefore, we could analyze contamination probabilities in terms of producing recombinant protein. Most of the virulence factors stated in **Table 1** were protein attached in bacterial membrane with the role of adhesion or recognition to host cell [12]. Meanwhile, shiga toxin, heat-stable and heat-labile toxin, and other cytotoxins were protein released by pathogenic *E. coli*. These toxins have specific receptors to induce invagination to the host cell, while their virulence mechanism also differs depending on the nature of each toxin and their molecular target [4].

STEC serotypes vary and differ in number of incidences, although the O157:H7 is a serotype considered to be responsible of numerous outbreaks. Shiga toxin occupies AB5 structure (see **Figure 1**), the catalytic subunit A (StxA) and homopentamer of subunit B (Stx B) as recognition site to globotetraosylceramide (Gb3/Gb4), which are present in the host cell surface, which leads to invagination of the toxin. STEC can produce either Stx1 (Stx1 and Stx1c), Stx2 variant (Stx2, Stx2c, Stx2d, Stx2e, and Stx2f) or range combination of both variants [4, 12]. Once invagination succeeds, catalytic subunit A would disrupt cell metabolism by inhibiting elongation factordependent aminoacyl tRNA binding (see detailed mechanism in [4]). The highly specific RNA *N-*glycosidase activity cleaves adenine base in eukaryotic ribosomal RNA, precisely at 28S subunit on the α-sarcin loop located in position 4324 [4].

Meanwhile, heat-labile (LT) and heat-stable (ST) toxin belong to ETEC groups. Nevertheless, LT enterotoxin shares similar structures to Stx which occupy AB5 conformation. Subunit A acts as a toxin by binding to its receptor, guanylyl cyclase C (GC-C). The interaction will activate guanine nucleotide protein Gsα by ADP-ribosylation, which trigger stimulation of secretion by cAMP-dependent mechanism. Elevated numbers of cAMP cause CTFR channel to secrete water and ions, thus generating diarrhea [3]. By contrast, ST structure is relatively simple. The STa class was made up with 18–19 cysteine-rich amino acids, while STb has 48 amino acids. ST virulence acts by triggering secretion of water and ions by triggering signaling cascade through guanylyl cyclase C (GC-C) in intestine [13, 14]. The structure of both ST and LT is shown in **Figure 2**.

**45**

**Figure 1.**

*StxB [3].*

*Safety Aspect of Recombinant Protein Produced by* Escherichia coli*: Toxin Evaluation…*

**Pathogenic Diseases Virulence factors Reference**

Bfp, intimin, LEE

Shiga toxins, intimin, Bfp

Heat-labile and heat-stable

toxins, CFA

Daa, AIDA

invasion

hemolysin

capsule

*AAF, aggregative adhere fimbria; AIDA, adhesion involved in diffuse adherence; Bfp, bundle-forming pili; CFA, colonization factor antigen; Daa, diffuse adhesin; Ipa, invasion plasmid antigen; LEE, locus for enterocyte* 

Shigellosis-like Shiga toxin, hemolysin,

AAFs, cytotoxins

cellular invasion, Ipa

Type 1 fimbriae, cellular

Type 1 and P fimbriae, AAFs,

S fimbriae, K1 capsule

Type 1 and P fimbriae, K1

Diarrhea in children

Hemorrhagic colitis, hemolytic uremic syndrome

Traveler's diarrhea

Diarrhea in children

Acute diarrhea in children

Crohn's diseaseassociated

Lower urinary tract infection and systemic infection

Neonatal meningitis

Food-borne source disease [1]

*Crystal structure of shiga-like toxin in* E. coli *(PDB: 1R4P). (A) Transversal view of Stx subunits, StxA represented as teal-colored chain, while StxB presented in rainbow color; (B) axial view of homopentamer* 

*DOI: http://dx.doi.org/10.5772/intechopen.92031*

**Enteric** *E. coli*

(EPEC)

(ETEC)

(EAEC)

(EIEC)

(DAEC)

(AIEC)

(UPEC)

(APEC)

*effacement.*

**Table 1.**

*coli* (EHEC)

Enteropathogenic *E. coli*

Enterohaemorrhagic *E.* 

Enterotoxigenic *E. coli*

Enteroaggregative *E. coli*

Diffusely adherent *E. coli*

Adherent invasive *E. coli*

**Extraintestinal** *E. coli* Uropathogenic *E. coli*

Neonatal meningitis *E. coli* (NMEC)

Avian pathogenic *E. coli*

*Classification of pathogenic* E. coli*.*

Enteroinvasive *E. coli*

*Safety Aspect of Recombinant Protein Produced by* Escherichia coli*: Toxin Evaluation… DOI: http://dx.doi.org/10.5772/intechopen.92031*


*AAF, aggregative adhere fimbria; AIDA, adhesion involved in diffuse adherence; Bfp, bundle-forming pili; CFA, colonization factor antigen; Daa, diffuse adhesin; Ipa, invasion plasmid antigen; LEE, locus for enterocyte effacement.*

#### **Table 1.**

E. Coli *Infections - Importance of Early Diagnosis and Efficient Treatment*

form high cell density culture (HCDC) [6–8].

**2. Toxin produced by** *Escherichia coli*

its diseases they caused and virulence factors [1].

of each toxin and their molecular target [4].

structure of both ST and LT is shown in **Figure 2**.

and strains approach.

gene, and its ability to grow under aerobic and anaerobic system, and facilitates to

The discussion about advantage in producing recombinant proteins and worries of toxins of *E. coli* is like talking about two opposite sides of a coin. This will certainly raise a question "Is it safe to produce recombinant protein in *E. coli*? Will it be toxin-free contamination?" Therefore, this chapter will discuss the safety aspects of recombinant protein produced by *E. coli* against toxins using genomic

Several pathogenic *E. coli* strains are known to be responsible for broad diseases,

Considering the number of pathogenic *E. coli*, it is useful to classify the toxins' properties and structure. It will be convenience to determine whether the toxins belong to organic compound or peptide-based structure; therefore, we could analyze contamination probabilities in terms of producing recombinant protein. Most of the virulence factors stated in **Table 1** were protein attached in bacterial membrane with the role of adhesion or recognition to host cell [12]. Meanwhile, shiga toxin, heat-stable and heat-labile toxin, and other cytotoxins were protein released by pathogenic *E. coli*. These toxins have specific receptors to induce invagination to the host cell, while their virulence mechanism also differs depending on the nature

STEC serotypes vary and differ in number of incidences, although the O157:H7 is a serotype considered to be responsible of numerous outbreaks. Shiga toxin occupies AB5 structure (see **Figure 1**), the catalytic subunit A (StxA) and homopentamer of subunit B (Stx B) as recognition site to globotetraosylceramide (Gb3/Gb4), which are present in the host cell surface, which leads to invagination of the toxin. STEC can produce either Stx1 (Stx1 and Stx1c), Stx2 variant (Stx2, Stx2c, Stx2d, Stx2e, and Stx2f) or range combination of both variants [4, 12]. Once invagination succeeds, catalytic subunit A would disrupt cell metabolism by inhibiting elongation factordependent aminoacyl tRNA binding (see detailed mechanism in [4]). The highly specific RNA *N-*glycosidase activity cleaves adenine base in eukaryotic ribosomal RNA, precisely at 28S subunit on the α-sarcin loop located in position 4324 [4]. Meanwhile, heat-labile (LT) and heat-stable (ST) toxin belong to ETEC groups. Nevertheless, LT enterotoxin shares similar structures to Stx which occupy AB5 conformation. Subunit A acts as a toxin by binding to its receptor, guanylyl cyclase C (GC-C). The interaction will activate guanine nucleotide protein Gsα by ADP-ribosylation, which trigger stimulation of secretion by cAMP-dependent mechanism. Elevated numbers of cAMP cause CTFR channel to secrete water and ions, thus generating diarrhea [3]. By contrast, ST structure is relatively simple. The STa class was made up with 18–19 cysteine-rich amino acids, while STb has 48 amino acids. ST virulence acts by triggering secretion of water and ions by triggering signaling cascade through guanylyl cyclase C (GC-C) in intestine [13, 14]. The

from mild to complicated cases. It is varying from mild diarrhea, hemorrhagic colitis, to hemolytic uremic syndrome. Among the pathogenic strains, STEC is an example of common strains which occupy high number in *E. coli* serotypes that produce toxin called Shiga toxins (Stx) [1, 2, 5, 9–11]. While STEC is a common pathogenic example, it belongs to a larger group named enterohemorrhagic *E. coli* (EHEC); also, there still exist numerous pathogenic *E. coli* and cause different diseases and complications. Pathogenic *E. coli* were classified in **Table 1** along with

**44**

*Classification of pathogenic* E. coli*.*

**Figure 1.**

*Crystal structure of shiga-like toxin in* E. coli *(PDB: 1R4P). (A) Transversal view of Stx subunits, StxA represented as teal-colored chain, while StxB presented in rainbow color; (B) axial view of homopentamer StxB [3].*

**Figure 2.**

*Enterotoxin secreted by ETEC. (A) Heat-labile enterotoxin (PDB: 1tii) [16]; (B) heat-stable enterotoxin, STa class (1etn) [17]; (C) heat-stable enterotoxin, STb class (1ehs) [18].*

The fact both STEC and ETEC toxins (Stx and LT, and ST, respectively) are peptide based elucidates its origin that were genetically listed in their DNA. These toxins were made under central dogma of protein synthesis. Therefore, analysis through genomic approach on recombinant *E. coli* host is possible to be conducted.

#### **3.** *E. coli* **as host for recombinant protein expression**

The production of recombinant proteins in microbial systems was started in 1970 and continued to boom in 1980 with the production of insulin. There is no doubt that this method has revolutionized and widened the field of biochemistry [19]. The ability to express large quantity of protein with less effort, relative to manual synthesis, allows industrial processes to produce in commercial scale. However, several considerations should be discussed before executing the production such as, appropriate vector, location of the protein of interest (whether as soluble fraction or inclusion bodies), optimum condition (pH, medium, temperature, aerobic/ anaerobic system), genetic design for convenience of purification, and at the top of it, microbial selection [7, 8, 20].

*E. coli* become preferred microbes in terms of recombinant protein host among researchers and industrial use. The simplicity of its expression system, compared to other higher level organism, and large quantity of well-characterized genomic database offer advantages in constructing the vector to be used [20]. A plenty number of research regarding *E. coli* also become an advantage to give amount of consideration of various expression conditions. Nevertheless, *E. coli* expression system has limited post-translational modification, which means that some proteins that require modification, such as alkylation or glycosylation, may not be perfectly expressed in *E. coli*. However, several strains of *E. coli* have the ability to perform specific post-translational modification [19, 21]. Therefore, we provide a simple summary on recombinant proteins produced by *E. coli* along with strains and expression strategies in **Table 2**.

Among recombinant proteins mentioned in **Table 2**, hEGF and hPT-2 are examples of therapeutic protein. Regarding its use in medical interests, therapeutic proteins produced in *E. coli* have to be safe for administration into human bodies; therefore, purification steps and any contaminants present become a huge concern in producing recombinant protein. Idetifying location of protein target is a prominent fundamental to determine source of contamination and to predict any possible contamination. Understanding the protein location also helps with the purification strategies needed to separate contaminants, specifically toxins, with the result that highly pure proteins were recovered. Choi et al*.* [29] through **Figure 3** classify locations of protein expressed in *E. coli* and its general purification steps needed.

**47**

steps [21, 33].

which is used for recombinant protein expression.

*Safety Aspect of Recombinant Protein Produced by* Escherichia coli*: Toxin Evaluation…*

P64k *E. coli* K12 GC366 Soluble intracellular, using

Human prethrombin-2 (hPT-2) *E. coli* TOP10F' Fusion protein with intein,

ArcticExpress

Human serum albumin *E. coli* BL21 (DE3) Extracellular, using TorA

Hepcidin *E. coli* BL21 (DE3) Inclusion bodies, fused with

pLys

(DE3)

*Summary of recombinant protein using* E. coli *expression system and its expression strategy.*

**Recombinant proteins Strain Expression strategies Ref.**

BL21 (DE3) Extracellular, using OmpA

vector

*E. coli* RR1 LacZ Soluble intracellular as fusion protein

pTWIN1

*E. coli* BL21 (DE3) Extracellular using TorA signal

vector

*E. coli* DH5α Soluble protein, designed with

His-tag

His-tag

*E. coli* BL21 (DE3) Extracellular, using OmpA

pM-152 vector

CBD-*Ssp* DnaB-hPT-2

Soluble intracellular using

peptide constructed on pD881

constructed in pD881 vector

constructed on pD881 vector

co-expression of prefoldin, chaperonin HSP60, and sHSP

Intracellular expression using

Soluble intracellular, using Strep-tag and combination of

His- and Trx-tag

constructed on pET-20b(+)

[22]

[23]

[24]

[25]

[26]

[6]

[27]

[28]

[29]

[30]

[31]

[32]

The distinction of protein location is affected by either the nature of the expres-

Based on protein location, toxin contamination can be investigated. Both Stx and LT-ST toxins are secreted by *E. coli*, increasing the risk of contamination when the protein of interest is produced extracellularly. Even so, since extracellular protein exists in soluble state, purification might not be impossible. Whereas intracellular expression may put more concern at contamination risk since toxins might be clumped together in the form of inclusion bodies. This case may put more consideration in solubilization and purification process. However, these allegations are only an assessment of risk factors with the assumption that toxins are produced in *E. coli*,

sion system or the protein construction design. Both extracellular and intracellular strategies on expressing protein give its own advantages and disadvantages. Extracellular expression offers simple purification, improved folding, and soluble products. This strategy can be achieved using signal peptide, co-expression with phospholipase, or co-expression with chaperon [33, 34]. In contrast, intracellular expression prefers inclusion bodies formation. While inclusion bodies give easy separation and prevent protease degradation, it has complex purification steps and refolding process is compulsory. Fusion partners, such as intein, often added in gene construction in intracellular works to provide efficient strategy in purification

*DOI: http://dx.doi.org/10.5772/intechopen.92031*

Human prethrombine-2 (hPT-2) *E. coli* BL21 (DE3)

Keratinase *E. coli* AD494(DE3)

Pig liver esterase (γ-PLE) *E. coli* origami

α-Cyclodextrin glycosyltransferase

(hEGF)

(hEGF)

(PFA)

**Table 2.**

SpA-βgal (*Staphylococcus* protein A-*E. coli β*-galactosidase)

Human epidermal growth factor

Human epidermal growth factor

*Pyrococcus furiosus* α-amylase


*Safety Aspect of Recombinant Protein Produced by* Escherichia coli*: Toxin Evaluation… DOI: http://dx.doi.org/10.5772/intechopen.92031*

#### **Table 2.**

E. Coli *Infections - Importance of Early Diagnosis and Efficient Treatment*

**3.** *E. coli* **as host for recombinant protein expression**

*class (1etn) [17]; (C) heat-stable enterotoxin, STb class (1ehs) [18].*

it, microbial selection [7, 8, 20].

**Figure 2.**

expression strategies in **Table 2**.

The fact both STEC and ETEC toxins (Stx and LT, and ST, respectively) are peptide based elucidates its origin that were genetically listed in their DNA. These toxins were made under central dogma of protein synthesis. Therefore, analysis through genomic approach on recombinant *E. coli* host is possible to be conducted.

*Enterotoxin secreted by ETEC. (A) Heat-labile enterotoxin (PDB: 1tii) [16]; (B) heat-stable enterotoxin, STa* 

The production of recombinant proteins in microbial systems was started in 1970 and continued to boom in 1980 with the production of insulin. There is no doubt that this method has revolutionized and widened the field of biochemistry [19]. The ability to express large quantity of protein with less effort, relative to manual synthesis, allows industrial processes to produce in commercial scale. However, several considerations should be discussed before executing the production such as, appropriate vector, location of the protein of interest (whether as soluble fraction or inclusion bodies), optimum condition (pH, medium, temperature, aerobic/ anaerobic system), genetic design for convenience of purification, and at the top of

*E. coli* become preferred microbes in terms of recombinant protein host among researchers and industrial use. The simplicity of its expression system, compared to other higher level organism, and large quantity of well-characterized genomic database offer advantages in constructing the vector to be used [20]. A plenty number of research regarding *E. coli* also become an advantage to give amount of consideration of various expression conditions. Nevertheless, *E. coli* expression system has limited post-translational modification, which means that some proteins that require modification, such as alkylation or glycosylation, may not be perfectly expressed in *E. coli*. However, several strains of *E. coli* have the ability to perform specific post-translational modification [19, 21]. Therefore, we provide a simple summary on recombinant proteins produced by *E. coli* along with strains and

Among recombinant proteins mentioned in **Table 2**, hEGF and hPT-2 are examples

of therapeutic protein. Regarding its use in medical interests, therapeutic proteins produced in *E. coli* have to be safe for administration into human bodies; therefore, purification steps and any contaminants present become a huge concern in producing recombinant protein. Idetifying location of protein target is a prominent fundamental to determine source of contamination and to predict any possible contamination. Understanding the protein location also helps with the purification strategies needed to separate contaminants, specifically toxins, with the result that highly pure proteins were recovered. Choi et al*.* [29] through **Figure 3** classify locations of protein

expressed in *E. coli* and its general purification steps needed.

**46**

*Summary of recombinant protein using* E. coli *expression system and its expression strategy.*

The distinction of protein location is affected by either the nature of the expression system or the protein construction design. Both extracellular and intracellular strategies on expressing protein give its own advantages and disadvantages. Extracellular expression offers simple purification, improved folding, and soluble products. This strategy can be achieved using signal peptide, co-expression with phospholipase, or co-expression with chaperon [33, 34]. In contrast, intracellular expression prefers inclusion bodies formation. While inclusion bodies give easy separation and prevent protease degradation, it has complex purification steps and refolding process is compulsory. Fusion partners, such as intein, often added in gene construction in intracellular works to provide efficient strategy in purification steps [21, 33].

Based on protein location, toxin contamination can be investigated. Both Stx and LT-ST toxins are secreted by *E. coli*, increasing the risk of contamination when the protein of interest is produced extracellularly. Even so, since extracellular protein exists in soluble state, purification might not be impossible. Whereas intracellular expression may put more concern at contamination risk since toxins might be clumped together in the form of inclusion bodies. This case may put more consideration in solubilization and purification process. However, these allegations are only an assessment of risk factors with the assumption that toxins are produced in *E. coli*, which is used for recombinant protein expression.

**Figure 3.** *Scheme of protein expressed in* E. coli *and its following purification steps [33].*

#### **4. Safety aspects of recombinant protein production against toxin**

Using comprehensive understanding of toxin origin, specifically Shiga toxin and enterotoxin, it is clear that these toxins were peptide based and generated by certain gene in STEC and ETEC. The gene *stx* was responsible for producing the Stx toxin using central dogma of *E. coli*, reciprocally to ST and LT encoding gene. Moreover, *E. coli* strains that are commonly used for recombinant protein work are also known. Therefore, it is possible to examine the safety aspect of recombinant protein against toxin through genetic alignment between common *E. coli* strains in recombinant work and toxin genes. Here, *E. coli* BL21 (DE3) (ACC: NC\_012892) and K-12 MG1655 (ACC: U00096.3) were used as representative. While toxin genes used are Stx (ACC: AY143336.1), LT (ACC: JQ031712), and ST (ACC: P22542.1).

In term of the existence of *stx* gene, common recombinant host strains are absence of the *stx* gene. Therefore, since the strains were clearly different, it is considerably safe to use *E. coli* as recombinant host without neglecting other contaminants.

#### **5. Expression and characterization of** *HSA* **gene in** *E. coli* **BL21 (DE3)**

This step started with growing *E. coli* BL21 (DE3) [pD881-torA-HSA] transformant as starter culture at 200 rpm, 37°C for 16–18 hours. Then starter culture was moved as much as 1% into 25 mL Luria-Bertani medium containing kanamycin as selection marker. *E. coli* BL21 (DE3) cell culture was grown until OD600nm reached 0.8 for induction. Before induction was performed, 1 mL sample from culture was separated as protein fraction before induction (t0). Induction was initiated by adding L-rhamnose into the expression medium to bring the final concentration to 4 mM. To obtain protein fraction in cytoplasm, sonication method was used. Lysate from six *E. coli* BL21 (DE3) [pD881-torA-HSA] transformant colonies showed that HSA was expressed in cytoplasm, it was characterized with the presence of ±67.0 kDa and in the SDS-PAGE electrophoresis [28]. The result of expression is presented in **Figure 4**.

**49**

**Figure 5.**

*20 hours of induction [35].*

*Safety Aspect of Recombinant Protein Produced by* Escherichia coli*: Toxin Evaluation…*

**6. Recombinant hEGF expression in** *E. coli* **BL21 (DE3)**

*induction; (Ki) colony transformant 1–6 after induction L-rhamnose induction [28].*

centrifuged at 3000 g, 4°C for 20 minutes [35].

*E. coli* BL21 (DE3) [pD881-PelB] that has been characterized was grown in 5 mL

LB medium containing kanamycin for about 16–18 hours at 37°C with 200 rpm shaking. Then, 1000 μL *E. coli* BL21 (DE3) [pD881-PelB] culture was transferred into 100 mL LB medium containing kanamycin in shaken flask. Native *E. coli* BL21 (DE3) was also grown and received the same treatment as the transformant. The culture was incubated until OD600nm reached 0.7. *E. coli* BL21 (DE3) [pD881-PelB] culture was taken as a sample (before L-Rhamnose induction) and transferred into a microtube and centrifuged at 3000 g, 4°C for 20 minutes. L-rhamnose was added into *E. coli* BL21 (DE3) [pD881-PelB] and native *E. coli* BL21 (DE3) culture to give final concentration of 2 mM. Incubation was continued at 37°C for 20 hours with 200 rpm shaking. Culture of the *E. coli* BL21 (DE3) [pD881-PelB] was taken as a sample (after L-rhamnose induction) and transferred into 2 microtubes then was

*Electropherogram of proteins expressed by* E. coli *BL21 (DE3) [pD881-PelB] with varying L-rhamnose concentration. Lane M is the protein markers. Lane (a-e) are soluble fractions of the medium with 40 μM, 1 mM, 2 mM, 4 mM, and 6 mM L-rhamnose as inducer, respectively. While lane (f-i) are inclusion bodies obtained from cell with 1 mM, 2 mM, 4 mM and 6 mM L-rhamnose as inducer. Samples were collected after* 

*SDS-PAGE electropherogram of HSA soluble fraction in cytoplasm after induction using 4 mM L-rhamnose from* E. coli *BL21 (DE3) [pD881-torA-HSA] colonies. (M) Protein marker; (t0) fraction before L-rhamnose* 

*DOI: http://dx.doi.org/10.5772/intechopen.92031*

**Figure 4.**

*Safety Aspect of Recombinant Protein Produced by* Escherichia coli*: Toxin Evaluation… DOI: http://dx.doi.org/10.5772/intechopen.92031*

#### **Figure 4.**

E. Coli *Infections - Importance of Early Diagnosis and Efficient Treatment*

**4. Safety aspects of recombinant protein production against toxin**

*Scheme of protein expressed in* E. coli *and its following purification steps [33].*

Using comprehensive understanding of toxin origin, specifically Shiga toxin and enterotoxin, it is clear that these toxins were peptide based and generated by certain gene in STEC and ETEC. The gene *stx* was responsible for producing the Stx toxin using central dogma of *E. coli*, reciprocally to ST and LT encoding gene. Moreover, *E. coli* strains that are commonly used for recombinant protein work are also known. Therefore, it is possible to examine the safety aspect of recombinant protein against toxin through genetic alignment between common *E. coli* strains in recombinant work and toxin genes. Here, *E. coli* BL21 (DE3) (ACC: NC\_012892) and K-12 MG1655 (ACC: U00096.3) were used as representative. While toxin genes used are Stx (ACC: AY143336.1), LT (ACC: JQ031712), and ST (ACC: P22542.1).

In term of the existence of *stx* gene, common recombinant host strains are absence of the *stx* gene. Therefore, since the strains were clearly different, it is considerably safe to use *E. coli* as recombinant host without neglecting other contaminants.

**5. Expression and characterization of** *HSA* **gene in** *E. coli* **BL21 (DE3)**

This step started with growing *E. coli* BL21 (DE3) [pD881-torA-HSA] transformant as starter culture at 200 rpm, 37°C for 16–18 hours. Then starter culture was moved as much as 1% into 25 mL Luria-Bertani medium containing kanamycin as selection marker. *E. coli* BL21 (DE3) cell culture was grown until OD600nm reached 0.8 for induction. Before induction was performed, 1 mL sample from culture was separated as protein fraction before induction (t0). Induction was initiated by adding L-rhamnose into the expression medium to bring the final concentration to 4 mM. To obtain protein fraction in cytoplasm, sonication method was used. Lysate from six *E. coli* BL21 (DE3) [pD881-torA-HSA] transformant colonies showed that HSA was expressed in cytoplasm, it was characterized with the presence of ±67.0 kDa and in the SDS-PAGE electrophoresis [28]. The result of expression is

**48**

**Figure 3.**

presented in **Figure 4**.

*SDS-PAGE electropherogram of HSA soluble fraction in cytoplasm after induction using 4 mM L-rhamnose from* E. coli *BL21 (DE3) [pD881-torA-HSA] colonies. (M) Protein marker; (t0) fraction before L-rhamnose induction; (Ki) colony transformant 1–6 after induction L-rhamnose induction [28].*

#### **6. Recombinant hEGF expression in** *E. coli* **BL21 (DE3)**

*E. coli* BL21 (DE3) [pD881-PelB] that has been characterized was grown in 5 mL LB medium containing kanamycin for about 16–18 hours at 37°C with 200 rpm shaking. Then, 1000 μL *E. coli* BL21 (DE3) [pD881-PelB] culture was transferred into 100 mL LB medium containing kanamycin in shaken flask. Native *E. coli* BL21 (DE3) was also grown and received the same treatment as the transformant. The culture was incubated until OD600nm reached 0.7. *E. coli* BL21 (DE3) [pD881-PelB] culture was taken as a sample (before L-Rhamnose induction) and transferred into a microtube and centrifuged at 3000 g, 4°C for 20 minutes. L-rhamnose was added into *E. coli* BL21 (DE3) [pD881-PelB] and native *E. coli* BL21 (DE3) culture to give final concentration of 2 mM. Incubation was continued at 37°C for 20 hours with 200 rpm shaking. Culture of the *E. coli* BL21 (DE3) [pD881-PelB] was taken as a sample (after L-rhamnose induction) and transferred into 2 microtubes then was centrifuged at 3000 g, 4°C for 20 minutes [35].

#### **Figure 5.**

*Electropherogram of proteins expressed by* E. coli *BL21 (DE3) [pD881-PelB] with varying L-rhamnose concentration. Lane M is the protein markers. Lane (a-e) are soluble fractions of the medium with 40 μM, 1 mM, 2 mM, 4 mM, and 6 mM L-rhamnose as inducer, respectively. While lane (f-i) are inclusion bodies obtained from cell with 1 mM, 2 mM, 4 mM and 6 mM L-rhamnose as inducer. Samples were collected after 20 hours of induction [35].*

Based on SDS-PAGE electrophoresis of the protein produced by the *E. coli* cell at varying concentration of L-rhamnose as inducer (**Figure 5**), it can be concluded that the best concentration of L-rhamnose that induces the production of the protein of interest was 4 mM because it produces more target protein, either in the insoluble fraction of the medium or in the form of inclusion bodies at t(20). The results also indicate that not all rhEGF translocated into the periplasm were secreted to medium. The hEGF was expressed in *E. coli* BL21 (DE3) with molecular weight of 6.2 kDa. The result of expression is presented in **Figure 5** [35].

Apart from that, our group succeeded in producing beta-galactosidase from a wild type *E. coli* strain B130, with high purity. Kinetical parameter (Km and Vmax) of the enzyme were 2.417 × 10<sup>−</sup><sup>4</sup> mol and 4.664 × 10<sup>−</sup><sup>4</sup> mol.minute<sup>−</sup><sup>1</sup> , respectively [36].

#### **7. Conclusions**

*E. coli* is renowned by its pathogenic properties, specifically in causing gastrointestinal disease. While in contrast, the same species also being helpful in expressing recombinant protein. Thus, contrary properties leave questions in terms of safety in expressing recombinant protein. Pathogenic *E. coli* strains were identified and classified in accordance with the disease caused. While most of pathogenic group gain its virulence by their membrane protein, some of it secretes toxins, like Stx from STEC or LT and ST from ETEC group. This toxin-secreting *E. coli* were important to understand contamination risk in recombinant protein. All three toxins were considered as peptide-based structure, in which production relies on respective genes. Alignment of toxin genes to commonly used *E. coli* in recombinant work makes a way to investigate toxin presence in recombinant-host *E. coli*. The BL21 (DE3) and K-12 MG1655 strains used as representative in alignment process, which generate non-overlapping alignment. This clears up the risk of toxin contamination on recombinant protein since the absence of toxin gene in these strains. Therefore, expressing recombinant protein, especially therapeutic protein, in *E. coli* was considered to be safe against toxin.

#### **Acknowledgements**

The authors would like to acknowledge the Executive Board of Universitas Padjadjaran, Dean of the Faculty of Mathematics and Natural Sciences, Head of the Department of Chemistry, Head of the Laboratory, and the entire staff of the Laboratory of Biochemistry.

#### **Funding sources**

Academic Leadership Grant from the Directorate of Research, Public Services, and Innovations, Universitas Padjadjaran.

**51**

**Author details**

Ukun M.S. Soedjanaatmadja\*

provided the original work is properly cited.

Iman Permana Maksum, Ahmad Nabiel, Safri Ishmayana and

Natural Sciences, Universitas Padjadjaran, Sumedang, Indonesia

\*Address all correspondence to: ukun\_28@yahoo.com; ukun@unpad.ac.id

Laboratory of Biochemistry, Department of Chemistry, Faculty of Mathematics and

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

*Safety Aspect of Recombinant Protein Produced by* Escherichia coli*: Toxin Evaluation…*

*DOI: http://dx.doi.org/10.5772/intechopen.92031*

*Safety Aspect of Recombinant Protein Produced by* Escherichia coli*: Toxin Evaluation… DOI: http://dx.doi.org/10.5772/intechopen.92031*

## **Author details**

E. Coli *Infections - Importance of Early Diagnosis and Efficient Treatment*

6.2 kDa. The result of expression is presented in **Figure 5** [35].

the enzyme were 2.417 × 10<sup>−</sup><sup>4</sup>

considered to be safe against toxin.

**Acknowledgements**

Laboratory of Biochemistry.

and Innovations, Universitas Padjadjaran.

**Funding sources**

**7. Conclusions**

Based on SDS-PAGE electrophoresis of the protein produced by the *E. coli* cell at varying concentration of L-rhamnose as inducer (**Figure 5**), it can be concluded that the best concentration of L-rhamnose that induces the production of the protein of interest was 4 mM because it produces more target protein, either in the insoluble fraction of the medium or in the form of inclusion bodies at t(20). The results also indicate that not all rhEGF translocated into the periplasm were secreted to medium. The hEGF was expressed in *E. coli* BL21 (DE3) with molecular weight of

Apart from that, our group succeeded in producing beta-galactosidase from a wild type *E. coli* strain B130, with high purity. Kinetical parameter (Km and Vmax) of

*E. coli* is renowned by its pathogenic properties, specifically in causing gastrointestinal disease. While in contrast, the same species also being helpful in expressing recombinant protein. Thus, contrary properties leave questions in terms of safety in expressing recombinant protein. Pathogenic *E. coli* strains were identified and classified in accordance with the disease caused. While most of pathogenic group gain its virulence by their membrane protein, some of it secretes toxins, like Stx from STEC or LT and ST from ETEC group. This toxin-secreting *E. coli* were important to understand contamination risk in recombinant protein. All three toxins were considered as peptide-based structure, in which production relies on respective genes. Alignment of toxin genes to commonly used *E. coli* in recombinant work makes a way to investigate toxin presence in recombinant-host *E. coli*. The BL21 (DE3) and K-12 MG1655 strains used as representative in alignment process, which generate non-overlapping alignment. This clears up the risk of toxin contamination on recombinant protein since the absence of toxin gene in these strains. Therefore, expressing recombinant protein, especially therapeutic protein, in *E. coli* was

The authors would like to acknowledge the Executive Board of Universitas Padjadjaran, Dean of the Faculty of Mathematics and Natural Sciences, Head of the Department of Chemistry, Head of the Laboratory, and the entire staff of the

Academic Leadership Grant from the Directorate of Research, Public Services,

mol.minute<sup>−</sup><sup>1</sup>

, respectively [36].

mol and 4.664 × 10<sup>−</sup><sup>4</sup>

**50**

Iman Permana Maksum, Ahmad Nabiel, Safri Ishmayana and Ukun M.S. Soedjanaatmadja\* Laboratory of Biochemistry, Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran, Sumedang, Indonesia

\*Address all correspondence to: ukun\_28@yahoo.com; ukun@unpad.ac.id

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### **References**

[1] Allocati N, Masulli M, Alexeyev MF, Di Ilio C. *Escherichia coli* in Europe: An overview. International Journal of Environmental Research and Public Health. 2013;**10**(12):6235-6254

[2] Beutin L, Geier D, Zimmermann S, Aleksic S, Gillespie HA, Whittam TS. Epidemiological relatedness and clonal types of natural populations of *Escherichia coli* strains producing Shiga toxins in separate populations of cattle and sheep. Applied and Environmental Microbiology. 1997;**63**(6):2175-2180

[3] Fraser ME, Fujinaga M, Cherney MM, Melton-Celsa AR, Twiddy EM, O'Brien AD, et al. Structure of Shiga toxin type 2 (Stx2) from *Escherichia coli* O157:H7. The Journal of Biological Chemistry. 2004;**279**(26):27511-27517. [cited 01 March 2020]. Available from: https:// www.rcsb.org/structure/1R4P

[4] Johannes L, Römer W. Shiga toxins from cell biology to biomedical applications. Nature Reviews Microbiology [Internet]. 2010;**8**(2): 105-116. DOI: 10.1038/nrmicro2279

[5] Hunt JM. Shiga toxin-producing *Escherichia coli* (STEC). The Journal of Clinical and Laboratory Medicine. 2010;**30**(1):21-45. DOI: 10.1016/j. cll.2009.11.001

[6] Maksum IP, Utama E, Sriwidodo ST. Extracellular secretion of recombinant human epidermal growth factor by using trimethylamine N-oxide reductase a (TORA) signal peptide in *Escherichia coli* BL21 (DE3). Journal of Pharmaceutical Sciences and Research. 2017;**9**(6):1007-1016

[7] Chae YK, Kim SH, Markley JL. Relationship between recombinant protein expression and host metabolome as determined by two-dimensional

NMR spectroscopy. PLoS One. 2017;**12**(5):e0177233

[8] Rosano GL, Ceccarelli EA. Recombinant protein expression in *Escherichia coli*: Advances and challenges. Frontiers in Microbiology. 2014;**5**(APR):1-17

[9] Yang X, Bai X, Zhang J, Sun H, Fu S, Fan R, et al. *Escherichia coli* strains producing a novel Shiga toxin 2 subtype circulate in China. International Journal of Medical Microbiology [Internet]. 2020;**310**(1):151377. DOI: 10.1016/j. ijmm.2019.151377

[10] Melton-celsa AR. Shiga toxin (Stx) classification, structure, and function. 2014:1-13

[11] Reis MHL, Matos DP, Pestana De Castro AF, Toledo MR, Trabulsi LR. Relationship among enterotoxigenic phenotypes, serotypes, and sources of strains in enterotoxigenic *Escherichia coli*. Infection and Immunity. 1980;**28**(1):24-27

[12] Farrokh C, Jordan K, Auvray F, Glass K, Oppegaard H, Raynaud S, et al. Review of Shiga-toxin-producing *Escherichia coli* (STEC) and their significance in dairy production. International Journal of Food Microbiology [Internet]. 2013;**162**(2): 190-212. DOI: 10.1016/j.ijfoodmicro. 2012.08.008

[13] Weiglmeier PR, Rösch P, Berkner H. Cure and curse: *E. coli* heat-stable enterotoxin and its receptor guanylyl cyclase C. 2010;**2**(9): 2213-2229

[14] Taxt A, Aasland R, Sommerfelt H, Nataro J, Puntervoll P. Heat-stable enterotoxin of enterotoxigenic *Escherichia coli* as a vaccine target. Infection and Immunity. 2010;**78**(5):1824-1831

**53**

*Safety Aspect of Recombinant Protein Produced by* Escherichia coli*: Toxin Evaluation…*

[22] Cheng J, Wu D, Chen S, Chen J, Wu J. High-level extracellular production of α-cyclodextrin

glycosyltransferase with recombinant *Escherichia coli* BL21 (DE3). Journal of Agricultural and Food Chemistry.

[23] Pérez ER, Suárez GJ, Diaz NE, Rodríguez SR, Menéndez CE, Balaguer DH, et al. Scaling-up fermentation of *Escherichia coli* for production of recombinant P64k protein from *Neisseria meningitidis*. Electronic Journal of Biotechnology [Internet]. 2018;**33**:29-35. DOI: 10.1016/j.ejbt.2018.03.004

[24] Strandberg L, Veide A, Enfors SO. Production of the hybrid protein staphylococcal protein A/*Escherichia coli* β-galactosidase with *E. coli*. Journal of Biotechnology. 1987;**6**(3):225-238

[25] Silaban S, Gaffar S, Simorangkir M, Maksum IP, Subroto T. Construction and optimization of prethrombin-2 human genes in *E. coli* for the production of active thrombin. Journal of Physics Conference Series. 2019;**1374**(1):6

[26] Silaban S, Gaffar S, Simorangkir M, Maksum IP, Subroto T. Effect of IPTG Concentration on Recombinant Human Prethrombin-2 Expression in *Escherichia coli* BL21(DE3). Arctic Express. 2019

Sriwidodo S, Maksum IP. Optimization extracellular secretion of recombinant human epidermal growth factor (hEGF) in *Escherichia coli* BL21 (DE3) pD881- OmpA-hEGF by using response surface method (RSM). International Journal of Research in Pharmaceutical Sciences.

[28] Maksum IP, Lestari A, Fauzia RP, Rachman SD, Soedjanaatmadja UMS. *Escherichia coli* BL21(DE3) expression system using TorA signal peptide for recombinant human albumin (rHA) secretion. International Journal of

[27] Indriyani A, Anggraeni NI,

2019;**10**(3):1824-1831

2011;**59**(8):3797-3802

*DOI: http://dx.doi.org/10.5772/intechopen.92031*

[15] Ozaki H, Sato T, Kubota H, Hata Y, Katsube Y, Shimonishi Y. Molecular structure of the toxic domain of heat-stable enterotoxin produced by a pathogenic strain of *Escherichia coli*. The Journal of Biological Chemistry.

1991;**266**:5934-5941

1996;**266**(9):5934-5941

structure/1ehs

[18] Sukumar M, Rizo J, Wall M, Dreyfus LA, Kupersztoch YM, Gierasch LM. The structure of

[19] Demain AL, Vaishnav P.

biotechadv.2009.01.008

2017;**45**(9):5285-5293

Production of recombinant proteins by microbes and higher organisms. Biotechnology Advances [Internet]. 2009;**27**(3):297-306. DOI: 10.1016/j.

[20] Kim S, Jeong H, Kim EY, Kim JF, Lee SY, Yoon SH. Genomic and

transcriptomic landscape of *Escherichia coli* BL21(DE3). Nucleic Acids Research.

[21] Maksum IP. Sriwidodo Y, Ekspresi Protein Rekombinan secara Intraselular pada Inang *Escherichia coli*. Sumedang: Alqaprint Jatinangor; 2019. p. 92

*Escherichia coli* heat-stable enterotoxin b by nuclear magnetic resonance and circular dichroism. Protein Science. 1995;**4**:1718-1729 [cited 01 March 2020] Available from: https://www.rcsb.org/

[16] van den Akker F, Sarfaty S, Twiddy EM, Connell TD, Holmes R, Hol WG. Crystal structure of a new heat-labile enterotoxin, LT-IIb. Structure; 1996;**4**(6):665-678

[17] Ozaki H, Sato T, Kubota H, Hata Y, Katsube Y, Shimonishi Y. Molecular structure of the toxin domain of heat-stable enterotoxin produced by a pathogenic strain of *Escherichia coli*. A putative binding site for a binding protein on rat intestinal epithelial cell membranes. The Journal of Biological Chemistry.

*Safety Aspect of Recombinant Protein Produced by* Escherichia coli*: Toxin Evaluation… DOI: http://dx.doi.org/10.5772/intechopen.92031*

[15] Ozaki H, Sato T, Kubota H, Hata Y, Katsube Y, Shimonishi Y. Molecular structure of the toxic domain of heat-stable enterotoxin produced by a pathogenic strain of *Escherichia coli*. The Journal of Biological Chemistry. 1991;**266**:5934-5941

[16] van den Akker F, Sarfaty S, Twiddy EM, Connell TD, Holmes R, Hol WG. Crystal structure of a new heat-labile enterotoxin, LT-IIb. Structure; 1996;**4**(6):665-678

[17] Ozaki H, Sato T, Kubota H, Hata Y, Katsube Y, Shimonishi Y. Molecular structure of the toxin domain of heat-stable enterotoxin produced by a pathogenic strain of *Escherichia coli*. A putative binding site for a binding protein on rat intestinal epithelial cell membranes. The Journal of Biological Chemistry. 1996;**266**(9):5934-5941

[18] Sukumar M, Rizo J, Wall M, Dreyfus LA, Kupersztoch YM, Gierasch LM. The structure of *Escherichia coli* heat-stable enterotoxin b by nuclear magnetic resonance and circular dichroism. Protein Science. 1995;**4**:1718-1729 [cited 01 March 2020] Available from: https://www.rcsb.org/ structure/1ehs

[19] Demain AL, Vaishnav P. Production of recombinant proteins by microbes and higher organisms. Biotechnology Advances [Internet]. 2009;**27**(3):297-306. DOI: 10.1016/j. biotechadv.2009.01.008

[20] Kim S, Jeong H, Kim EY, Kim JF, Lee SY, Yoon SH. Genomic and transcriptomic landscape of *Escherichia coli* BL21(DE3). Nucleic Acids Research. 2017;**45**(9):5285-5293

[21] Maksum IP. Sriwidodo Y, Ekspresi Protein Rekombinan secara Intraselular pada Inang *Escherichia coli*. Sumedang: Alqaprint Jatinangor; 2019. p. 92

[22] Cheng J, Wu D, Chen S, Chen J, Wu J. High-level extracellular production of α-cyclodextrin glycosyltransferase with recombinant *Escherichia coli* BL21 (DE3). Journal of Agricultural and Food Chemistry. 2011;**59**(8):3797-3802

[23] Pérez ER, Suárez GJ, Diaz NE, Rodríguez SR, Menéndez CE, Balaguer DH, et al. Scaling-up fermentation of *Escherichia coli* for production of recombinant P64k protein from *Neisseria meningitidis*. Electronic Journal of Biotechnology [Internet]. 2018;**33**:29-35. DOI: 10.1016/j.ejbt.2018.03.004

[24] Strandberg L, Veide A, Enfors SO. Production of the hybrid protein staphylococcal protein A/*Escherichia coli* β-galactosidase with *E. coli*. Journal of Biotechnology. 1987;**6**(3):225-238

[25] Silaban S, Gaffar S, Simorangkir M, Maksum IP, Subroto T. Construction and optimization of prethrombin-2 human genes in *E. coli* for the production of active thrombin. Journal of Physics Conference Series. 2019;**1374**(1):6

[26] Silaban S, Gaffar S, Simorangkir M, Maksum IP, Subroto T. Effect of IPTG Concentration on Recombinant Human Prethrombin-2 Expression in *Escherichia coli* BL21(DE3). Arctic Express. 2019

[27] Indriyani A, Anggraeni NI, Sriwidodo S, Maksum IP. Optimization extracellular secretion of recombinant human epidermal growth factor (hEGF) in *Escherichia coli* BL21 (DE3) pD881- OmpA-hEGF by using response surface method (RSM). International Journal of Research in Pharmaceutical Sciences. 2019;**10**(3):1824-1831

[28] Maksum IP, Lestari A, Fauzia RP, Rachman SD, Soedjanaatmadja UMS. *Escherichia coli* BL21(DE3) expression system using TorA signal peptide for recombinant human albumin (rHA) secretion. International Journal of

**52**

E. Coli *Infections - Importance of Early Diagnosis and Efficient Treatment*

NMR spectroscopy. PLoS One.

[8] Rosano GL, Ceccarelli EA. Recombinant protein expression in *Escherichia coli*: Advances and challenges. Frontiers in Microbiology.

[9] Yang X, Bai X, Zhang J, Sun H, Fu S, Fan R, et al. *Escherichia coli* strains producing a novel Shiga toxin 2 subtype circulate in China. International Journal of Medical Microbiology [Internet]. 2020;**310**(1):151377. DOI: 10.1016/j.

[10] Melton-celsa AR. Shiga toxin (Stx) classification, structure, and function.

[11] Reis MHL, Matos DP, Pestana De Castro AF, Toledo MR, Trabulsi LR. Relationship among enterotoxigenic phenotypes, serotypes, and sources of strains in enterotoxigenic *Escherichia* 

*coli*. Infection and Immunity.

[12] Farrokh C, Jordan K, Auvray F, Glass K, Oppegaard H, Raynaud S, et al. Review of Shiga-toxin-producing *Escherichia coli* (STEC) and their significance in dairy production. International Journal of Food

Microbiology [Internet]. 2013;**162**(2): 190-212. DOI: 10.1016/j.ijfoodmicro.

[14] Taxt A, Aasland R, Sommerfelt H, Nataro J, Puntervoll P. Heat-stable enterotoxin of enterotoxigenic *Escherichia coli* as a vaccine target. Infection and Immunity.

[13] Weiglmeier PR, Rösch P, Berkner H. Cure and curse: *E. coli* heat-stable enterotoxin and its receptor

guanylyl cyclase C. 2010;**2**(9):

2010;**78**(5):1824-1831

1980;**28**(1):24-27

2012.08.008

2213-2229

2017;**12**(5):e0177233

2014;**5**(APR):1-17

ijmm.2019.151377

2014:1-13

[1] Allocati N, Masulli M, Alexeyev MF, Di Ilio C. *Escherichia coli* in Europe: An overview. International Journal of Environmental Research and Public Health. 2013;**10**(12):6235-6254

[2] Beutin L, Geier D, Zimmermann S, Aleksic S, Gillespie HA, Whittam TS. Epidemiological relatedness and clonal types of natural populations of *Escherichia coli* strains producing Shiga toxins in separate populations of cattle and sheep. Applied and Environmental Microbiology.

1997;**63**(6):2175-2180

**References**

[3] Fraser ME, Fujinaga M, Cherney MM, Melton-Celsa AR, Twiddy EM, O'Brien AD, et al. Structure of Shiga toxin type 2 (Stx2) from *Escherichia coli* O157:H7. The Journal of Biological Chemistry. 2004;**279**(26):27511-27517. [cited 01 March 2020]. Available from: https://

www.rcsb.org/structure/1R4P

[4] Johannes L, Römer W. Shiga toxins from cell biology to biomedical

applications. Nature Reviews Microbiology [Internet]. 2010;**8**(2): 105-116. DOI: 10.1038/nrmicro2279

cll.2009.11.001

2017;**9**(6):1007-1016

[5] Hunt JM. Shiga toxin-producing *Escherichia coli* (STEC). The Journal of Clinical and Laboratory Medicine. 2010;**30**(1):21-45. DOI: 10.1016/j.

[6] Maksum IP, Utama E, Sriwidodo ST. Extracellular secretion of recombinant human epidermal growth factor by using trimethylamine N-oxide reductase a (TORA) signal peptide in *Escherichia coli* BL21 (DE3). Journal of Pharmaceutical Sciences and Research.

[7] Chae YK, Kim SH, Markley JL. Relationship between recombinant protein expression and host metabolome as determined by two-dimensional

Research in Pharmaceutical Sciences. 2019;**10**(4):3319-3324

[29] Peng S, Chu Z, Lu J, Li D, Wang Y, Yang S, et al. Co-expression of chaperones from *P. furiosus* enhanced the soluble expression of the recombinant hyperthermophilic α -amylase in *E. coli*. Cell Stress and Chaperones. 2016;**21**(3):477-484

[30] Zhang H, Yuan Q, Zhu Y, Ma R. Expression and preparation of recombinant hepcidin in *Escherichia coli*. Protein Expression and Purification. 2005;**41**:409-416

[31] Lin H-H, Yin L-J, Jiang S-T. Cloning, expression, and purification of *Pseudomonas aeruginosa* keratinase in *Escherichia coli* AD494 (DE3) pLysS expression system. Journal of Agricultural and Food Chemistry. 2009;**494**:3506-3511

[32] Brüsehaber E, Schwiebs A, Schmidt M, Bottcher D, Bornscheuer UT. Production of pig liver esterase in batch fermentation of *E. coli*. Origami. Applied Microbiology and Biotechnology. 2010;**86**(5): 1337-1344

[33] Choi JH, Keum KC, Lee SY. Production of recombinant proteins by high cell density culture of *Escherichia coli*. Chemical Engineering Science. 2006;**61**(3):876-885

[34] Maksum IP. Sriwidodo, Indriyani A. Sistem Ekspresi Protein Rekombinan dalam *Eschercia coli* secara Ekstraselular. Sumedang: Alqaprint Jatinangor; 2018. p. 207

[35] Sriwidodo MIP, Riswanto N, Rostinawati T, Subroto T. Extracellular secretion recombinant of human epidermal growth factor (hEGF) using pectate lyase B (PelB) signal peptide in *Escherichia coli* BL21(DE3).

International Journal of Research in Pharmaceutical Sciences. 2016;**8**(1):1-8

[36] Ningsih E, Padmadisastra T, Soedjanaatmadja UMS. Isolasi Pemurnian dan Karakterisasi Enzim beta-galaktosidase dari *Escherichia coli* B-130. Bandung: Laporan Tugas Akhir Penelitian. Universitas Padjadjaran; 1994

**55**

Section 2

Diagnosis and Treatment

Section 2
