**2. Expression of recombinant proteins in the cytoplasm of** *E. coli*

The breakthrough achievements in the construction of recombinant DNA molecules [1] and the transformation of chimeric DNA constructs into *E. coli* [2] in the early 1970s have paved the way for rapid advances in the development of recombinant DNA approaches to the expression of a wide collection of useful/valuable proteins of various origins. Due to the aforementioned fine properties of *E. coli*, this Gram-negative bacterium has been extensively studied and exploited for use as a host to facilitate expression of heterologous proteins (**Table 1**).

#### **2.1 Fusion protein approach**

A common strategy in the expression of heterologous proteins is to fuse the target protein with a fusion partner, of which a familiar example is the enzyme β-galactosidase (β-Gal) expressed intracellularly in *E. coli*. Being well-characterized in terms of its structure and regulation of expression [27, 28], in the early days, β-Gal was one of the few *E. coli* products to be employed as a reporter protein, for which convenient detection assays [29] were available. Fusing the short mammalian somatostatin (Som) comprising only 14 amino acids (aa) to β-Gal, in 1977, Itakura et al. demonstrated (**Figure 2**), for the first time, successful expression of bioactive recombinant somatostatin in *E. coli* [9]. In the work, Som was fused to β-Gal through the application of oligonucleotide assembly. Thus, expression of the two proteins, which was under the regulatory controls of the Lac operon, would result initially in a β-Gal-Som precursor. The β-Gal component played two important roles in the fusion: first, it offered a facile screening assay for the selection of potentially positive clones expressing β-Gal-Som; second, it served as a guardian protecting Som from being attacked by proteolytic degradation from the N-terminus.

Since Som is a short polypeptide consisting of only 14 aa residues [30], which does not consist of Met as a member, in engineering the aforementioned β-Gal-Som fusion, a Met residue was intentionally inserted precisely between β-Gal and Som, thus resulting in a β-Gal-Met-Som precursor in the work [9]. In vitro cleavage with


**Table 1.**

*Various approaches for heterologous proteins expression in* E. coli.

the chemical, CNBr, which specifically attacks Met, resulted in the separation of the fusion to yield authentic Som comprising 14 aa as the final product, which was subsequently shown to be bioactive (**Figure 2**) [9].

A similar approach was also applied to express bioactive human insulin in *E. coli* in 1979 (**Figure 2**), once again, taking advantage of β-Gal as the fusion partner and the absence of a Met residue in the polypeptide [10].

However, the above two examples appear to be the exception rather than the rule. Despite the application of β-Gal to serving as a fusion partner in many other cases of recombinant protein expression, due to the presence of one or more Met residues in the target proteins, the intriguing tactic of employing CNBr to cleave the fusion precursors to free the desired final protein to be impractical for routine use.

#### **2.2 Direct expression**

If a heterologous protein is insusceptible to proteolytic degradation by *E. coli* proteases, perhaps a simple method to achieve expression of the protein in *E. coli* is to clone its gene determinant downstream from a regulatory region comprising both the promoter and RBS sequences carried on a suitable expression vector. Examples

**31**

**Figure 2.**

**2.3 Applications of affinity tags**

*Escherichia coli: A Versatile Platform for Recombinant Protein Expression*

including human growth hormone [11], human hemoglobin [31], interleukin [32],

*Schematic diagram depicting the major components included in various expression constructs for recombinant protein expression in E. coli. Regulation region ( ) includes the promoter ( ), operator, and ribosome binding site. Other components including the ATG start codon ( ), and coding sequences for fusion tag ( ), e.g., β-Gal in Approach 1(a), GST, 6xHis-tag, or MBP in Approach 1(b); signal peptide ( ) in Approach 4; chitin-binding domain ( ); target proteins ( ); intein ( ) are also shown. Cleavage sites on fusion* 

The alignment between the target gene and its expression regulatory elements could be conveniently achieved using site-directed mutagenesis. However, the translation initiator, N-formyl-methionine (fMet), which is present in proteins formed in bacteria, may cause adverse effects on the bioactivity and stability of the target protein [33]. The efficiency of removal of fMet in the cell is incomplete and is

highly dependent on the adjacent two residues next to the initiator [34, 35]. Various strategies have been described to remove fMet from heterologous proteins including the use of both in vivo and in vitro approaches [34, 36, 37]. However, none of the available protocols is able to result in a homogeneous product that is free of fMet [34, 36]. The target protein, being contaminated by the presence of the undesirable fMet-bearing variant, may exhibit increased immunogenicity [33] and reduced levels of stability and bioactivity [34], which might have a correlation with fMet which has been speculated to serve as a degradation signal [38].

A major goal in recombinant DNA expression is to achieve efficient production of a target protein on a large scale. Common strategies including the use of: (1) plasmids with increased copy numbers such as the ones employing runaway replicons [39, 40]; (2) strong transcriptional control signals including PL, Tac, and T7 promoters [41, 42]; (3) efficient ribosome binding sites such as the Shine-Dalgarno sequence [43]; (4) inducible promoters which may be activated by heat shocking [44], light induction [45] or chemicals, e.g., isopropyl

etc., have been expressed in *E. coli* using this approach (**Figure 2**).

*proteins (Approach 1a and 1b) and intein-target protein precursors are indicated ( ).*

*DOI: http://dx.doi.org/10.5772/intechopen.82276*

*Escherichia coli: A Versatile Platform for Recombinant Protein Expression DOI: http://dx.doi.org/10.5772/intechopen.82276*

#### **Figure 2.**

*The Universe of Escherichia coli*

the chemical, CNBr, which specifically attacks Met, resulted in the separation of the fusion to yield authentic Som comprising 14 aa as the final product, which was

**Approach Examples Section** 

Tagged protein expression Human insulin-like growth factor I

Secretion/excretion Human epidermal growth factor

Fusion expression Hormone somatostatin 2.1 [9, 10] Human insulin Direct expression Human growth hormone 2.2 [11–15] Human fibroblast interferon Human leukocyte interferon (LeIF A) Human leukocyte interferon (LeIF B) Mouse granulocyte-macrophage colony stimulating factor (mGM-CSF)

(IGF-1)

Human parathyroid hormone (hPTH) Human fibroblast growth factor 21 (hFGF21) Intein-mediated Cre recombinase 3 [5, 6, 19,

> Human basic fibroblast growth factor (bFGF)

> > (Secretion)

Hirudin Human growth hormone (hGH) Human granulocyte-macrophage colony stimulating factor (Hgm-CSF) Human epidermal growth factor (Excretion)

**concerned**

20] Cecropin

**References**

2.3 [16–18]

4 [21–26]

A similar approach was also applied to express bioactive human insulin in *E. coli* in 1979 (**Figure 2**), once again, taking advantage of β-Gal as the fusion partner and

However, the above two examples appear to be the exception rather than the rule. Despite the application of β-Gal to serving as a fusion partner in many other cases of recombinant protein expression, due to the presence of one or more Met residues in the target proteins, the intriguing tactic of employing CNBr to cleave the fusion precursors to free the desired final protein to be impractical for routine use.

If a heterologous protein is insusceptible to proteolytic degradation by *E. coli* proteases, perhaps a simple method to achieve expression of the protein in *E. coli* is to clone its gene determinant downstream from a regulatory region comprising both the promoter and RBS sequences carried on a suitable expression vector. Examples

subsequently shown to be bioactive (**Figure 2**) [9].

*Various approaches for heterologous proteins expression in* E. coli.

the absence of a Met residue in the polypeptide [10].

**30**

**Table 1.**

**2.2 Direct expression**

*Schematic diagram depicting the major components included in various expression constructs for recombinant protein expression in E. coli. Regulation region ( ) includes the promoter ( ), operator, and ribosome binding site. Other components including the ATG start codon ( ), and coding sequences for fusion tag ( ), e.g., β-Gal in Approach 1(a), GST, 6xHis-tag, or MBP in Approach 1(b); signal peptide ( ) in Approach 4; chitin-binding domain ( ); target proteins ( ); intein ( ) are also shown. Cleavage sites on fusion proteins (Approach 1a and 1b) and intein-target protein precursors are indicated ( ).*

including human growth hormone [11], human hemoglobin [31], interleukin [32], etc., have been expressed in *E. coli* using this approach (**Figure 2**).

The alignment between the target gene and its expression regulatory elements could be conveniently achieved using site-directed mutagenesis. However, the translation initiator, N-formyl-methionine (fMet), which is present in proteins formed in bacteria, may cause adverse effects on the bioactivity and stability of the target protein [33]. The efficiency of removal of fMet in the cell is incomplete and is highly dependent on the adjacent two residues next to the initiator [34, 35].

Various strategies have been described to remove fMet from heterologous proteins including the use of both in vivo and in vitro approaches [34, 36, 37]. However, none of the available protocols is able to result in a homogeneous product that is free of fMet [34, 36]. The target protein, being contaminated by the presence of the undesirable fMet-bearing variant, may exhibit increased immunogenicity [33] and reduced levels of stability and bioactivity [34], which might have a correlation with fMet which has been speculated to serve as a degradation signal [38].

#### **2.3 Applications of affinity tags**

A major goal in recombinant DNA expression is to achieve efficient production of a target protein on a large scale. Common strategies including the use of: (1) plasmids with increased copy numbers such as the ones employing runaway replicons [39, 40]; (2) strong transcriptional control signals including PL, Tac, and T7 promoters [41, 42]; (3) efficient ribosome binding sites such as the Shine-Dalgarno sequence [43]; (4) inducible promoters which may be activated by heat shocking [44], light induction [45] or chemicals, e.g., isopropyl β-D-1-thiogalactopyranoside (IPTG); (5) a codon-optimized gene sequence [46, 47]; and (6) an efficient plasmid maintenance system. These various methods have been commonly applied, either individually or in conjunction with a fusion approach, to achieving efficient expression of target proteins in *E. coli*.

Although high yields of products may result from the application of above mentioned expression approaches, oftentimes, the products present themselves as insoluble inclusion bodies or aggregates. Unfortunately, these inclusion bodies are composed of denatured and misfolded proteins, which are functionally inactive [48, 49]. Due to the rearrangements of disulfide bridges in the aggregates, despite going through the processes of denaturation and renaturation, the target proteins are unlikely able to regain their functional activities [48].

Fusion of a target protein to an affinity tag presents a viable approach to not only the purification of the final product, but also the preservation of the product as a soluble protein. It has been shown that protein tags such as maltose-binding protein [50], glutathione S-transferase [51], small ubiquitin modifying protein [52, 53], and thioredoxin [54] might help improve the solubility of fusion products formed between the tags and target proteins (**Figure 2**). Given that a fusion product is expressed as a soluble and properly folded intermediate, and that it is readily purified using affinity chromatography and proteolytically processed at a recognition site engineered between the tag and target proteins, the frailty of this fusion approach is how the affinity tag may be removed from the target protein on condition that the latter till possesses the peptide sequence as stipulated. Thus, this approach may not be able to meet the stringent demand from therapeutic proteins of which any discrepancy found in their primary structures may result in undesirable side effects such as increased immunogenicity [33], reduced levels of stability and bioactivity [34, 55, 56], and worse still, greater tendency to promote malignancy. It is believed that target proteins bearing the authentic structures are as safe as their native counterparts in performing biological functions [57].

## **3. Intein-mediated expression of heterologous proteins**

#### **3.1 Inteins as fusion partners**

Since the first intein, or protein intron, was discovered in the late 1980s [58], over 600 putative intein genes have been discovered [59]. Being able to undergo autocatalytic cleavages of themselves from sequences flanking their two termini, the N- and C-exteins, the application of inteins to the development of *E. coli* expression platforms has revolutionized the production of recombinant proteins in two different facets. First, fusion proteins formed between inteins and target proteins may undergo auto-cleavage activities in the cytoplasm of *E. coli* [5, 6, 60–63]. Second, despite taking place intracellularly, the detached target proteins possess the requisite structures, e.g., the authentic N-terminal sequences which are the same as those of their native counterparts [5, 6, 60–63].

### **3.2 Autocatalytic cleavages of intein-target fusion proteins: through an in vitro method**

In the early days of exploiting the application of inteins to protein expression, fusion precursors formed among three components, comprising an N-terminal protein tag, a common example being a chitin-binding domain (CBD; [64]), a central intein, and a C-terminal target protein (CBD-I-TP), were frequently expressed as biologically inactive inclusion bodies in the cytoplasm of *E. coli* [65–67]. Subsequent to denaturation

**33**

*(A) may also result in incorrectly folded proteins.*

**Figure 3.**

*Escherichia coli: A Versatile Platform for Recombinant Protein Expression*

expected to result in a substantial loss of bioactive target proteins.

**3.3 Autocatalytic cleavages of intein-target fusion proteins: in vivo**

and renaturation of the protein aggregates [67], the renatured precursors comprising CBD and the target proteins, e.g., Cre recombinase, α-1-antitrypsin, human epidermal growth factor [67] (**Figure 2**), collected in a chitin column was cleaved [64] by modulating the environmental conditions to release the target proteins [67] (**Figure 3**). Being expressed as inclusion bodies, as discussed in Section 2.3, it is unlikely that the renatured CBD-I-TP molecules would all be bound to the chitin matrix or be correctly refolded. Therefore, the above described intein-mediated expression process working in conjunction with an in vitro autocatalytic cleavage protocol is

Despite the inducibility of self-cleavages of inteins by modulating the environmental conditions [68], the exact mechanisms regarding how the induction works is not clear. Recent findings have shown that the ability of an intein element in fusion proteins to undergo self-cleavages appears to be dependent upon the presence of a pair of "well-matched" heterologous "exteins." If this condition is fulfilled, autocatalytic cleavages might take place at the two terminal junctions where the intein is fused with the two exteins (**Figure 3**). It was demonstrated that when human epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) were precisely fused at the Nand C-termini, respectively, of the *Sce* VMA intein, auto-cleavage processing occurred [5] (**Figure 2**). Both EGF and bFGF were retrieved and shown to share not only authentic structures, but also potent bioactivities with their native counterparts [5].

*Processing of precursors formed between inteins and target proteins. The processing may be achieved by one of the following two methods: (A) induced in vitro cleavage; (B) in vivo cleavage. To attain a soluble precursor protein for processing in (A), the insoluble precursor protein express in the host cells is first required to be denatured and renatured. The solubilized precursor is then adsorbed onto an affinity column containing, e.g., chitin resin; changes of the conditions in the column may result in self-cleavage and hence the detachment of the target protein. In (B), the self-cleavage reaction takes place automatically in the host without extra input of effort. In the figure, hatched box ( ) denotes affinity tag, white box ( ) denotes intein, dotted box ( ) denotes target protein and affinity resin is denoted by . The correctly folded affinity tag, intein, and target protein are denoted as , and, respectively. However, the in vitro cleavage process shown in* 

*DOI: http://dx.doi.org/10.5772/intechopen.82276*

#### *Escherichia coli: A Versatile Platform for Recombinant Protein Expression DOI: http://dx.doi.org/10.5772/intechopen.82276*

and renaturation of the protein aggregates [67], the renatured precursors comprising CBD and the target proteins, e.g., Cre recombinase, α-1-antitrypsin, human epidermal growth factor [67] (**Figure 2**), collected in a chitin column was cleaved [64] by modulating the environmental conditions to release the target proteins [67] (**Figure 3**).

Being expressed as inclusion bodies, as discussed in Section 2.3, it is unlikely that the renatured CBD-I-TP molecules would all be bound to the chitin matrix or be correctly refolded. Therefore, the above described intein-mediated expression process working in conjunction with an in vitro autocatalytic cleavage protocol is expected to result in a substantial loss of bioactive target proteins.

#### **3.3 Autocatalytic cleavages of intein-target fusion proteins: in vivo**

Despite the inducibility of self-cleavages of inteins by modulating the environmental conditions [68], the exact mechanisms regarding how the induction works is not clear. Recent findings have shown that the ability of an intein element in fusion proteins to undergo self-cleavages appears to be dependent upon the presence of a pair of "well-matched" heterologous "exteins." If this condition is fulfilled, autocatalytic cleavages might take place at the two terminal junctions where the intein is fused with the two exteins (**Figure 3**). It was demonstrated that when human epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) were precisely fused at the Nand C-termini, respectively, of the *Sce* VMA intein, auto-cleavage processing occurred [5] (**Figure 2**). Both EGF and bFGF were retrieved and shown to share not only authentic structures, but also potent bioactivities with their native counterparts [5].

#### **Figure 3.**

*The Universe of Escherichia coli*

β-D-1-thiogalactopyranoside (IPTG); (5) a codon-optimized gene sequence [46, 47]; and (6) an efficient plasmid maintenance system. These various methods have been commonly applied, either individually or in conjunction with a fusion

Although high yields of products may result from the application of above mentioned expression approaches, oftentimes, the products present themselves as insoluble inclusion bodies or aggregates. Unfortunately, these inclusion bodies are composed of denatured and misfolded proteins, which are functionally inactive [48, 49]. Due to the rearrangements of disulfide bridges in the aggregates, despite going through the processes of denaturation and renaturation, the target proteins

Fusion of a target protein to an affinity tag presents a viable approach to not only the purification of the final product, but also the preservation of the product as a soluble protein. It has been shown that protein tags such as maltose-binding protein [50], glutathione S-transferase [51], small ubiquitin modifying protein [52, 53], and thioredoxin [54] might help improve the solubility of fusion products formed between the tags and target proteins (**Figure 2**). Given that a fusion product is expressed as a soluble and properly folded intermediate, and that it is readily purified using affinity chromatography and proteolytically processed at a recognition site engineered between the tag and target proteins, the frailty of this fusion approach is how the affinity tag may be removed from the target protein on condition that the latter till possesses the peptide sequence as stipulated. Thus, this approach may not be able to meet the stringent demand from therapeutic proteins of which any discrepancy found in their primary structures may result in undesirable side effects such as increased immunogenicity [33], reduced levels of stability and bioactivity [34, 55, 56], and worse still, greater tendency to promote malignancy. It is believed that target proteins bearing the authentic structures are as safe

approach, to achieving efficient expression of target proteins in *E. coli*.

are unlikely able to regain their functional activities [48].

as their native counterparts in performing biological functions [57].

Since the first intein, or protein intron, was discovered in the late 1980s [58], over 600 putative intein genes have been discovered [59]. Being able to undergo autocatalytic cleavages of themselves from sequences flanking their two termini, the N- and C-exteins, the application of inteins to the development of *E. coli* expression platforms has revolutionized the production of recombinant proteins in two different facets. First, fusion proteins formed between inteins and target proteins may undergo auto-cleavage activities in the cytoplasm of *E. coli* [5, 6, 60–63]. Second, despite taking place intracellularly, the detached target proteins possess the requisite structures, e.g., the authentic N-terminal sequences which are the same as

**3.2 Autocatalytic cleavages of intein-target fusion proteins: through an in vitro** 

In the early days of exploiting the application of inteins to protein expression, fusion precursors formed among three components, comprising an N-terminal protein tag, a common example being a chitin-binding domain (CBD; [64]), a central intein, and a C-terminal target protein (CBD-I-TP), were frequently expressed as biologically inactive inclusion bodies in the cytoplasm of *E. coli* [65–67]. Subsequent to denaturation

**3. Intein-mediated expression of heterologous proteins**

**3.1 Inteins as fusion partners**

those of their native counterparts [5, 6, 60–63].

**32**

**method**

*Processing of precursors formed between inteins and target proteins. The processing may be achieved by one of the following two methods: (A) induced in vitro cleavage; (B) in vivo cleavage. To attain a soluble precursor protein for processing in (A), the insoluble precursor protein express in the host cells is first required to be denatured and renatured. The solubilized precursor is then adsorbed onto an affinity column containing, e.g., chitin resin; changes of the conditions in the column may result in self-cleavage and hence the detachment of the target protein. In (B), the self-cleavage reaction takes place automatically in the host without extra input of effort. In the figure, hatched box ( ) denotes affinity tag, white box ( ) denotes intein, dotted box ( ) denotes target protein and affinity resin is denoted by . The correctly folded affinity tag, intein, and target protein are denoted as , and, respectively. However, the in vitro cleavage process shown in (A) may also result in incorrectly folded proteins.*

Moreover, since EGF was fused to the OmpA signal peptide (OmpA) in the abovementioned work (**Figure 2**), the EGF-VMA-bFGF fusion was also shown to be secretory and both EGF and bFGF were finally detected to be present in the culture medium of their *E. coli* host [5]. Interestingly, when EGF was absent in the fusion, thus leaving the formation of OmpA-VMA-bFGF, and when the positions of EGF and bFGF in the fusion were switched, thus leading to the expression of OmpAbFGF-VMA-EGF, neither of the two precursors resulted in successful self-cleavages to yield authentic bFGF as the final product [5]. The results support the idea that not only the presence of a matched pair of exteins, but also their relative position in the fusion is important in effecting autocatalytic cleavages of the extein from their intein fusion partner.

Another noteworthy observation from the above work is the soluble nature of the fusion precursor, EGF-VMA-bFGF. This unusual condition, which contrasts markedly with the results of insoluble aggregates reported previously [64], has facilitated auto-cleavages of the fusion precursor to undergo self-cleavages directly in the cytoplasm, thereby avoiding the involvement of a time-consuming and ineffective process of denaturation and renaturation, followed by the extra time and effort spent on implementing the in vitro cleavage operation (**Figure 3**).

The in vivo autocatalytic processing approach introduced above may also be extended for use in the co-expression of other target proteins [60] (**Figure 4**). Moreover, through a combined protocol of gene amplification and refined fed-batch fermentation, the EGF-VMA-bFGF fusion has been upgraded to result in an expression of EGF-VMA-bFGF-VMA-bFGF as the precursor in *E. coli* [6]. Despite 92% bigger in size than EGF-VMA-bFGF, which was shown to have a mass of 73 kDa [5], EGF-VMA-bFGF-VMA-bFGF was found to be expressed as a soluble protein, which was still able to undergo autocatalytic cleavages to result in authentic and bioactive

#### **Figure 4.**

*Analysis of the CBD-DnaB-IFN precursor by polyacrylamide gel electrophoresis. Cell lysate samples were prepared from E. coli transformants harboring plasmid pTWIN-CBD-DnaB-IFN grown in 2×YT medium before and after 4 h of IPTG induction. Cell pellets were lysed by a chemical lysis protocol as described previously [5]. Lanes containing soluble proteins retrieved from the clear lysate (CL) and insoluble proteins retrieved from cell debris (Deb) were resolved as shown. The products CBD-DnaB ( ) and IFN ( ) resulting from autocatalytic cleavages of CBD-DnaB-IFN are denoted.*

**35**

*Escherichia coli: A Versatile Platform for Recombinant Protein Expression*

bFGF as the final product [6, 61–63]. In addition, fermentative production of EGF-VMA-bFGF-VMA-bFGF resulted in a dramatic improvement in the yield bFGF,

that resulting from the processing of EGF-VMA-bFGF expressed previously [5].

**4. Expression of heterologous proteins across the inner membrane of** 

The approach of secretory expression of heterologous proteins stemmed from the work of W. Gilbert's group, which employed the N-terminal 23 amino acid leader sequence of the *E. coli* penicillinase [69], to direct secretion of eukaryotic proteins, using rat proinsulin as the model protein, to the periplasmic space of *E. coli* in the late 1970s and early 1980s [69–72]. The secreted proinsulin was not only shown to possess an authentic structure, with the cleavage of the signal peptide done precisely [71], but also shown to be more stable than its cytoplasmic counterparts fused to defective signal sequences [72]. Over the next few years, different eukaryotic proteins, e.g., EGF [21], human interferon-α [73], hirudin [22], human growth hormone [23], and human granulocyte-macrophage colony stimulating factor [24], were also successfully expressed though secretion using various bacterial

Meanwhile, *E. coli* mutants that were able to leak endogenous enzymes from the periplasm were isolated [74, 75]. The results suggested that heterologous proteins might also leak from the periplasm to the culture medium in *E. coli*. As expected, a few years later, heterologous proteins including bacterial endoglucanases [76, 77], a penicillinase of an alkalphilic *Bacillus* [78], as well as human proteins, such as β-endorphin [79], EGF, parathyroid hormone, and interleukin-6 [80], were expressed as extracel-

Not all proteins, e.g., the cytoplasmic enzyme—β-galactosidase, may be possibly expressed as secreted or excreted products in *E. coli*, despite their fusions to secretory proteins [81, 82] or directly to the signal peptides of these proteins [83]. Intracellular proteins do not appear to possess a molecular structure that is compatible with the SecYEG pathway, the major translocation machinery located in the inner membrane for protein transport [70, 82, 84–86]. On the other hand, when a naturally secreted target protein, e.g., EGF, is fused to a signal peptide, it may end up as a mature protein in either the periplasm [21] or the culture medium [80] of *E. coli* cells, depending essentially upon the efficiencies of expression and secretion

In *E. coli*, several protein export systems, including the SecYFG (a trimeric complex comprising three polypeptides: SecY, SecE and SecG), Tat (twin-arginine translocation), and SRP (signal recognition particle) pathways which are embedded in the inner or cytoplasmic membrane, are responsible for the transport of proteins from the cytoplasm to the periplasm [86–89] (**Figure 1**). Among them, the SecYEG translocon is a general, conserved, and essential pathway which is found in both prokaryotic and eukaryotic cells [85, 86]. Being the major protein transport system, over 90% of the translocated proteins are secreted through the SecYEG pathway

To enable proteins to be secreted using the SecYEG translocon, they are required to be expressed first as preproteins, which are fused at their N-termini with a short (commonly less than 24 amino acids) signal peptide [87]. In the cytoplasm, a

lular products using either wildtype or leaky *E. coli* strains as hosts.

**4.1 Secretory expression of target proteins in** *E. coli*

of cell culture [6], which was over 2.4 times higher than

*DOI: http://dx.doi.org/10.5772/intechopen.82276*

signal peptides in *E. coli* (**Figure 2**).

of the protein (see below).

[87, 88] (**Figure 1**).

amounting to 610 mg L<sup>−</sup><sup>1</sup>

*E. coli*

*The Universe of Escherichia coli*

intein fusion partner.

Moreover, since EGF was fused to the OmpA signal peptide (OmpA) in the abovementioned work (**Figure 2**), the EGF-VMA-bFGF fusion was also shown to be secretory and both EGF and bFGF were finally detected to be present in the culture medium of their *E. coli* host [5]. Interestingly, when EGF was absent in the fusion, thus leaving the formation of OmpA-VMA-bFGF, and when the positions of EGF and bFGF in the fusion were switched, thus leading to the expression of OmpAbFGF-VMA-EGF, neither of the two precursors resulted in successful self-cleavages to yield authentic bFGF as the final product [5]. The results support the idea that not only the presence of a matched pair of exteins, but also their relative position in the fusion is important in effecting autocatalytic cleavages of the extein from their

Another noteworthy observation from the above work is the soluble nature of the fusion precursor, EGF-VMA-bFGF. This unusual condition, which contrasts markedly with the results of insoluble aggregates reported previously [64], has facilitated auto-cleavages of the fusion precursor to undergo self-cleavages directly in the cytoplasm, thereby avoiding the involvement of a time-consuming and ineffective process of denaturation and renaturation, followed by the extra time and

The in vivo autocatalytic processing approach introduced above may also be extended for use in the co-expression of other target proteins [60] (**Figure 4**). Moreover, through a combined protocol of gene amplification and refined fed-batch fermentation, the EGF-VMA-bFGF fusion has been upgraded to result in an expression of EGF-VMA-bFGF-VMA-bFGF as the precursor in *E. coli* [6]. Despite 92% bigger in size than EGF-VMA-bFGF, which was shown to have a mass of 73 kDa [5], EGF-VMA-bFGF-VMA-bFGF was found to be expressed as a soluble protein, which was still able to undergo autocatalytic cleavages to result in authentic and bioactive

*Analysis of the CBD-DnaB-IFN precursor by polyacrylamide gel electrophoresis. Cell lysate samples were prepared from E. coli transformants harboring plasmid pTWIN-CBD-DnaB-IFN grown in 2×YT medium before and after 4 h of IPTG induction. Cell pellets were lysed by a chemical lysis protocol as described previously [5]. Lanes containing soluble proteins retrieved from the clear lysate (CL) and insoluble proteins retrieved from cell debris (Deb) were resolved as shown. The products CBD-DnaB ( ) and IFN ( ) resulting* 

*from autocatalytic cleavages of CBD-DnaB-IFN are denoted.*

effort spent on implementing the in vitro cleavage operation (**Figure 3**).

**34**

**Figure 4.**

bFGF as the final product [6, 61–63]. In addition, fermentative production of EGF-VMA-bFGF-VMA-bFGF resulted in a dramatic improvement in the yield bFGF, amounting to 610 mg L<sup>−</sup><sup>1</sup> of cell culture [6], which was over 2.4 times higher than that resulting from the processing of EGF-VMA-bFGF expressed previously [5].
