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

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

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

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 of the protein (see below).

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

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

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

preprotein is maintained in an extended (export-competent) state by interacting with the SecB chaperone. Subsequent to an interaction formed between the signal and the SecA ATPase, the preprotein-complex then associates with the SecYEG pathway. With repeated pushes of SecA, the preprotein is secreted through the translocon in an ATP-dependent manner, followed by removal of the signal peptide by signal peptidase before the mature protein is released to the periplasm [87, 89, 90].

A wide range of heterologous proteins including degradative enzymes [91, 92], human hormones, and growth factors [5, 25, 26] have been successfully expressed as secretory proteins in *E. coli*. Many of the secreted proteins were not only shown to be bioactive, but also confirmed by sequence determination to possess the correct structures, supporting that the signal peptides fused at the N-termini of the preproteins had been removed correctly during the process of secretion [5, 25]. These advantages, together with lower levels of protease complexity and activity [72], and the relatively more oxidative environment that may help proper folding and disulfide-linkage formation [93, 94], enable the periplasmic space to be consider as a reasonable and appropriate destination for the expression of recombinant secreted proteins. Later on, with the help of various genetic and/or biochemical manipulations [95], or even merely through improving the levels of the secreted proteins concerned, which was referred as the "self-driven approach" [95], interestingly, the target proteins might then to be allowed to leak out to the culture medium, a process termed excretion, which is essentially caused by non-specific leakage of periplasmic proteins (see below).

## **4.2 Excretory production of target proteins in** *E. coli*

In the mid-1980s, researchers from different groups discovered that heterologous proteins expressed and secreted to the periplasm of *E. coli* might also be further excreted to the culture supernatant [22, 79]. For example, the development of sensitive screening assays, e.g., the Congo red plate assay (**Figure 5**), helped to confirm that the detection of a recombinant endoglucanase (Eng) encoded by the *cenA* gene of a Gram-positive bacterium, *Cellulomonas fimi* [77, 91, 96], in the culture supernatant of its *E. coli* host was due to a new phenomenon, excretion (extracellular production), rather than from cell lysis.

Efficient expression regulatory elements such as the strong promoters including *tac*, pL, and T7 [97, 98], the consensus ribosome binding site [99], the coding sequence for the potent OmpA signal [100], an effective inducible system, e.g., the *lac* operator/repressor system for transcriptional regulation [101, 102], etc., which are carried on a stable and high-copy number vector, e.g., pUC18 [103], have been made available to improve not only secretory, but also excretory expression of a wide variety of proteins in *E. coli*. The achievement of this research milestone was well exemplified by the development of an efficient protocol for extracellular production of EGF [104]. In early attempts to express EGF as a secretory protein in *E. coli*, the relatively weak *phoA* promoter was employed to perform transcription of the *egf* gene and the less efficient PhoA signal peptide to direct EGF for secretion. Despite the demonstration of EGF secretion, the EGF detected in the periplasm was only at a considerably low level of 2.4 mg L<sup>−</sup><sup>1</sup> [21]. However, when the *tac* promoter and the *ompA* leader sequence were employed to facilitate EGF expression and secretion, respectively, it was shown that the level of excreted, but not secreted, EGF was markedly improved in *E. coli* cells [80](**Figure 6**). Moreover, further improvements in EGF expression resulted in a dramatic increase in the yield of excreted EGF [26, 95, 104, 105]. Similar trends were observed in increasing the levels of excretory production of other heterologous proteins, e.g., *C. fimi* cellulases Eng [91, 106, 107] and exoglucanase, as well as bFGF [6].

**37**

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

**4.3 Difficulties in implementing an effective excretory process and potential** 

*E. coli transformants expressing excreted Eng. Making use of a convenient and sensitive agar plate assay based on the ability of Eng to degrade carboxymethylcellulose (CMC), and the affinity of the dye Congo red for long molecules of CMC, E. coli cells excreting Eng were detected. In this assay, CMC molecules are degraded randomly by Eng to generate oligomers of the substrate. These oligomers, especially those with fewer than five sugar units, bind poorly to Congo red. Hence, after staining with Congo red and destaining with salt solution,* 

The findings described above support the view that excretion is a promising approach for recombinant production of heterologous proteins in *E. coli*. However, it has been shown that not all naturally secreted proteins may be expressed using excretion, despite using efficient transcriptional and translational controls, as well as effective secretion signal. For example, using the same regulatory elements

EGF peptide in *E. coli* [26, 95, 104, 105], attempts to produce authentic bFGF (146 residues) by excretion in *E. coli* were unsuccessful [5]. One might wonder whether the discrepancy between the results of EGF and bFGF excretion was due to the marked difference between their molecular sizes. However, cellulases such as Eng and Exg, which possess large mature forms comprising 418 aa [91] and 443 aa [108], respectively, have been shown to be efficiently produced by excretion in *E. coli* [92, 107, 109]. Therefore, in addition to the molecular size of a heterologous protein which might have some effect on the efficiency of excretory production of the protein (see below), it appears that other factor(s), which may be associated to either the protein itself or the host (or both), play a crucial role in determining whether a protein may be expressed as a secretory/excretory product or not. A major hurdle for excretory production of heterologous products is the dramatic cell death during enhanced expression of the preproteins—the fusion precursors formed between signal peptides and target proteins. A model designated "Saturated Translocation" was proposed to explain the phenomenon of cell lethality resulting from hyper-expression of the preproteins [110]. According to the model, when a preprotein exceeded a tolerable level, it would saturate the capacity of the SecYEG pathway and interfere with its normal function in exporting endogenous proteins. These functional disorders resulted finally in cell death [110]. The model also explained why heterologous proteins of different sizes, which were undergoing secretory expression, would trigger rapid cell death (**Figure 7**) if the presence of their preproteins had exceeded their individual allowable thresholds, the "Critical Values (CV)." A CV is defined as the largest quotient between an intracellular

) of excretion of the 53-amino acid (aa)

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

**solutions**

**Figure 5.**

which enabled a high level (325 mg L<sup>−</sup><sup>1</sup>

*clear zones where CMC has been hydrolyzed by Eng are revealed [110].*

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

#### **Figure 5.**

*The Universe of Escherichia coli*

proteins (see below).

**4.2 Excretory production of target proteins in** *E. coli*

(extracellular production), rather than from cell lysis.

periplasm was only at a considerably low level of 2.4 mg L<sup>−</sup><sup>1</sup>

Eng [91, 106, 107] and exoglucanase, as well as bFGF [6].

preprotein is maintained in an extended (export-competent) state by interacting with the SecB chaperone. Subsequent to an interaction formed between the signal and the SecA ATPase, the preprotein-complex then associates with the SecYEG pathway. With repeated pushes of SecA, the preprotein is secreted through the translocon in an ATP-dependent manner, followed by removal of the signal peptide by signal peptidase before the mature protein is released to the periplasm [87, 89, 90].

A wide range of heterologous proteins including degradative enzymes [91, 92], human hormones, and growth factors [5, 25, 26] have been successfully expressed as secretory proteins in *E. coli*. Many of the secreted proteins were not only shown to be bioactive, but also confirmed by sequence determination to possess the correct structures, supporting that the signal peptides fused at the N-termini of the preproteins had been removed correctly during the process of secretion [5, 25]. These advantages, together with lower levels of protease complexity and activity [72], and the relatively more oxidative environment that may help proper folding and disulfide-linkage formation [93, 94], enable the periplasmic space to be consider as a reasonable and appropriate destination for the expression of recombinant secreted proteins. Later on, with the help of various genetic and/or biochemical manipulations [95], or even merely through improving the levels of the secreted proteins concerned, which was referred as the "self-driven approach" [95], interestingly, the target proteins might then to be allowed to leak out to the culture medium, a process termed excretion, which is essentially caused by non-specific leakage of periplasmic

In the mid-1980s, researchers from different groups discovered that heterologous proteins expressed and secreted to the periplasm of *E. coli* might also be further excreted to the culture supernatant [22, 79]. For example, the development of sensitive screening assays, e.g., the Congo red plate assay (**Figure 5**), helped to confirm that the detection of a recombinant endoglucanase (Eng) encoded by the *cenA* gene of a Gram-positive bacterium, *Cellulomonas fimi* [77, 91, 96], in the culture supernatant of its *E. coli* host was due to a new phenomenon, excretion

Efficient expression regulatory elements such as the strong promoters including *tac*, pL, and T7 [97, 98], the consensus ribosome binding site [99], the coding sequence for the potent OmpA signal [100], an effective inducible system, e.g., the *lac* operator/repressor system for transcriptional regulation [101, 102], etc., which are carried on a stable and high-copy number vector, e.g., pUC18 [103], have been made available to improve not only secretory, but also excretory expression of a wide variety of proteins in *E. coli*. The achievement of this research milestone was well exemplified by the development of an efficient protocol for extracellular production of EGF [104]. In early attempts to express EGF as a secretory protein in *E. coli*, the relatively weak *phoA* promoter was employed to perform transcription of the *egf* gene and the less efficient PhoA signal peptide to direct EGF for secretion. Despite the demonstration of EGF secretion, the EGF detected in the

the *tac* promoter and the *ompA* leader sequence were employed to facilitate EGF expression and secretion, respectively, it was shown that the level of excreted, but not secreted, EGF was markedly improved in *E. coli* cells [80](**Figure 6**). Moreover, further improvements in EGF expression resulted in a dramatic increase in the yield of excreted EGF [26, 95, 104, 105]. Similar trends were observed in increasing the levels of excretory production of other heterologous proteins, e.g., *C. fimi* cellulases

[21]. However, when

**36**

*E. coli transformants expressing excreted Eng. Making use of a convenient and sensitive agar plate assay based on the ability of Eng to degrade carboxymethylcellulose (CMC), and the affinity of the dye Congo red for long molecules of CMC, E. coli cells excreting Eng were detected. In this assay, CMC molecules are degraded randomly by Eng to generate oligomers of the substrate. These oligomers, especially those with fewer than five sugar units, bind poorly to Congo red. Hence, after staining with Congo red and destaining with salt solution, clear zones where CMC has been hydrolyzed by Eng are revealed [110].*

#### **4.3 Difficulties in implementing an effective excretory process and potential solutions**

The findings described above support the view that excretion is a promising approach for recombinant production of heterologous proteins in *E. coli*. However, it has been shown that not all naturally secreted proteins may be expressed using excretion, despite using efficient transcriptional and translational controls, as well as effective secretion signal. For example, using the same regulatory elements which enabled a high level (325 mg L<sup>−</sup><sup>1</sup> ) of excretion of the 53-amino acid (aa) EGF peptide in *E. coli* [26, 95, 104, 105], attempts to produce authentic bFGF (146 residues) by excretion in *E. coli* were unsuccessful [5]. One might wonder whether the discrepancy between the results of EGF and bFGF excretion was due to the marked difference between their molecular sizes. However, cellulases such as Eng and Exg, which possess large mature forms comprising 418 aa [91] and 443 aa [108], respectively, have been shown to be efficiently produced by excretion in *E. coli* [92, 107, 109]. Therefore, in addition to the molecular size of a heterologous protein which might have some effect on the efficiency of excretory production of the protein (see below), it appears that other factor(s), which may be associated to either the protein itself or the host (or both), play a crucial role in determining whether a protein may be expressed as a secretory/excretory product or not.

A major hurdle for excretory production of heterologous products is the dramatic cell death during enhanced expression of the preproteins—the fusion precursors formed between signal peptides and target proteins. A model designated "Saturated Translocation" was proposed to explain the phenomenon of cell lethality resulting from hyper-expression of the preproteins [110]. According to the model, when a preprotein exceeded a tolerable level, it would saturate the capacity of the SecYEG pathway and interfere with its normal function in exporting endogenous proteins. These functional disorders resulted finally in cell death [110]. The model also explained why heterologous proteins of different sizes, which were undergoing secretory expression, would trigger rapid cell death (**Figure 7**) if the presence of their preproteins had exceeded their individual allowable thresholds, the "Critical Values (CV)." A CV is defined as the largest quotient between an intracellular

#### **Figure 6.**

*Excretory production of EGF in E. coli. Protein concentrations and enzymatic activities detected in different subcellular compartments: cytoplasm, periplasm, and culture supernatant of E. coli transformants expressing EGF are shown. The samples prepared for various measurements were taken from the E. coli cell culture grown for 12 (A) and 24 h (B) after IPTG induction. The culture conditions employed were as described previously [80]. The results show that the EGF activities detected in the culture supernatant samples were the highest in all three compartments. However, beta-galactosidase activity was undetectable in the supernatant samples, supporting the conclusion that EGF activities detected in the supernatant samples resulted from excretion rather than from cell lysis.*

preprotein and its secreted mature counterpart that was tolerable by the host cells [92]. Deletion of the signal peptide from its mature partner, despite the possibility of incurring the formation of inclusion bodies [48], interestingly it would help avoid the onset of the deadly effect resulting from an efficiently expressed secretory protein [110]. The results clearly indicate that the bottleneck of secretory/excretory production of a heterologous protein is at the stage of secretion.

Different approaches have been attempted to attain or even improve the CV, and hence the maximum production of a secretory heterologous protein on a per cell basis. Since cell death results from hyper-expression, strategies of optimizing, rather than maximizing, protein expression, e.g., less efficient promoters [92, 107, 111] and start codon [92], as well as defined minimal media and sub-optimal cultivation conditions [26] have been employed and shown to provide beneficial effects. More encouragingly, excretory production of Exg was

**39**

**Acknowledgements**

13142580CLIL07W011 and 1617219-0.

pathway [109].

*done as described previously [111].*

**Figure 7.**

**5. Conclusions**

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

markedly enhanced when the level of Phage shock protein A (PspA) was elevated in the same host [109]. In the presence of additional PspA, the CV of secretory Exg was shown to be markedly increased from 20/80 to 45/55 [109]. Presumably, PspA helped the host cells to maintain membrane integrity and an energized membrane [112–114], which was readily equipped to cope with the "stress," the presence of secretory Pre-Exg, by efficiently transporting it through the SecYEG

*Time-course experiments on extracellular expression of EGF and Exg in E. coli. (A) Measurements of EGF activities detected in the culture medium ( ) and viable cell counts ( ) of E. coli transformants harboring plasmid pWKW2 are shown (unpublished results). (B) Measurements of Exg activities detected in the culture medium ( ) and viable cell counts ( ) of E. coli transformants harboring plasmid tacIQpar8cex are shown. One unit of Exg activity in hydrolyzing p-nitrophenyl-β-D-cellobioside is defined as one nmol of p-nitrophenol produced per min. The growth conditions and IPTG induction of the cultures were* 

Since the advent of recombinant DNA technology in the 1970s, *E. coli* has been the most favorable host choice for the expression of heterologous proteins. Strategies including both intracellular and secretory methods have been designed for the expression of proteins of interest. Despite possessing an outer membrane, a wide variety of naturally secreted proteins including hormones, factors, and degradative enzymes have also been shown to be produced as extracellular (excreted) products in *E. coli*. In undertaking both intracellular and secretory/ excretory protein expression, a fusion approach is commonly adopted. Affinity tag proteins including β-galactosidase, glutathione *S*-transferase, and 6xHis-tag have been employed to form fusion precursors with desired proteins. To enable separation between the tag and target proteins, a protease cleavage site is required to be placed between the two proteins. However, on the one hand, it may be difficult to achieve the exact processing result through proteolytic cleavage. On the other hand, it is cost-ineffective to implement proteolytic cleavage on a large scale. Fusions of target proteins with inteins and secretion signal peptides have presented a practical approach to protein cleavages in cells without relying on the use of external proteases. It has been well demonstrated that using both methods of protein fusion, target proteins possessing the exactly processed sequences are obtainable through

This study was funded by RGC project: GRF16101515 and Research Contracts:

autocatalytic or signal peptidase cleavages in vivo in *E. coli*.

*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 7.**

*The Universe of Escherichia coli*

**38**

**Figure 6.**

*than from cell lysis.*

preprotein and its secreted mature counterpart that was tolerable by the host cells [92]. Deletion of the signal peptide from its mature partner, despite the possibility of incurring the formation of inclusion bodies [48], interestingly it would help avoid the onset of the deadly effect resulting from an efficiently expressed secretory protein [110]. The results clearly indicate that the bottleneck of secretory/excretory

*Excretory production of EGF in E. coli. Protein concentrations and enzymatic activities detected in different subcellular compartments: cytoplasm, periplasm, and culture supernatant of E. coli transformants expressing EGF are shown. The samples prepared for various measurements were taken from the E. coli cell culture grown for 12 (A) and 24 h (B) after IPTG induction. The culture conditions employed were as described previously [80]. The results show that the EGF activities detected in the culture supernatant samples were the highest in all three compartments. However, beta-galactosidase activity was undetectable in the supernatant samples, supporting the conclusion that EGF activities detected in the supernatant samples resulted from excretion rather* 

Different approaches have been attempted to attain or even improve the CV, and hence the maximum production of a secretory heterologous protein on a per cell basis. Since cell death results from hyper-expression, strategies of optimizing, rather than maximizing, protein expression, e.g., less efficient promoters [92, 107, 111] and start codon [92], as well as defined minimal media and sub-optimal cultivation conditions [26] have been employed and shown to provide beneficial effects. More encouragingly, excretory production of Exg was

production of a heterologous protein is at the stage of secretion.

*Time-course experiments on extracellular expression of EGF and Exg in E. coli. (A) Measurements of EGF activities detected in the culture medium ( ) and viable cell counts ( ) of E. coli transformants harboring plasmid pWKW2 are shown (unpublished results). (B) Measurements of Exg activities detected in the culture medium ( ) and viable cell counts ( ) of E. coli transformants harboring plasmid tacIQpar8cex are shown. One unit of Exg activity in hydrolyzing p-nitrophenyl-β-D-cellobioside is defined as one nmol of p-nitrophenol produced per min. The growth conditions and IPTG induction of the cultures were done as described previously [111].*

markedly enhanced when the level of Phage shock protein A (PspA) was elevated in the same host [109]. In the presence of additional PspA, the CV of secretory Exg was shown to be markedly increased from 20/80 to 45/55 [109]. Presumably, PspA helped the host cells to maintain membrane integrity and an energized membrane [112–114], which was readily equipped to cope with the "stress," the presence of secretory Pre-Exg, by efficiently transporting it through the SecYEG pathway [109].

#### **5. Conclusions**

Since the advent of recombinant DNA technology in the 1970s, *E. coli* has been the most favorable host choice for the expression of heterologous proteins. Strategies including both intracellular and secretory methods have been designed for the expression of proteins of interest. Despite possessing an outer membrane, a wide variety of naturally secreted proteins including hormones, factors, and degradative enzymes have also been shown to be produced as extracellular (excreted) products in *E. coli*. In undertaking both intracellular and secretory/ excretory protein expression, a fusion approach is commonly adopted. Affinity tag proteins including β-galactosidase, glutathione *S*-transferase, and 6xHis-tag have been employed to form fusion precursors with desired proteins. To enable separation between the tag and target proteins, a protease cleavage site is required to be placed between the two proteins. However, on the one hand, it may be difficult to achieve the exact processing result through proteolytic cleavage. On the other hand, it is cost-ineffective to implement proteolytic cleavage on a large scale. Fusions of target proteins with inteins and secretion signal peptides have presented a practical approach to protein cleavages in cells without relying on the use of external proteases. It has been well demonstrated that using both methods of protein fusion, target proteins possessing the exactly processed sequences are obtainable through autocatalytic or signal peptidase cleavages in vivo in *E. coli*.

#### **Acknowledgements**

This study was funded by RGC project: GRF16101515 and Research Contracts: 13142580CLIL07W011 and 1617219-0.

*The Universe of Escherichia coli*
