Escherichia coli as a source of recombinant therapeutic proteins

Many microorganisms represent attractive potential production systems for therapeutic proteins. They can usually be cultured in large quantities, inexpensively and in a short time, by standard

Pharmaceutical biotechnology: concepts and applications Gary Walsh © 2007 John Wiley & Sons, Ltd ISBN 978 0 470 01244 4 (HB) 978 0 470 01245 1 (PB)

Table 5.1 Expression systems which are/could potentially be used for the production of recombinant biopharmaceutical products

E. coli (and additional prokaryotic systems, e.g. bacilli) Yeast (particularly S. cerevisiae) Fungi (particularly aspergilli)

Animal cell culture (particularly CHO and BHK cell lines) Transgenic animals (focus thus far is upon sheep and goats) Plant-based expression systems (various) Insect cell culture systems methods of fermentation. Production facilities can be constructed in any world region, and the scale of production can be varied as required.

The expression of recombinant proteins in cells in which they do not naturally occur is termed heterologous protein production (Chapter 3). The first biopharmaceutical produced by genetic engineering to gain marketing approval (in 1982) was recombinant human insulin (tradename 'Humulin'), produced in E. coli. An example of a more recently approved biopharmaceutical that is produced in E. coli is that of Kepivance, a recombinant keratinocyte growth factor used to treat oral mucositis (Chapter 10). Many additional examples are provided in subsequent chapters. As a recombinant production system, E. coli displays a number of advantages. These include:

• E. coli has long served as the model system for studies relating to prokaryotic genetics. Its molecular biology is thus well characterized.

• High levels of expression of heterologous proteins can be achieved in recombinant E. coli (Table 5.3). Modern, high-expression promoters can routinely ensure that levels of expression of the recombinant protein reach up to 30 per cent total cellular protein.

• E. coli cells grow rapidly on relatively simple and inexpensive media, and the appropriate fermentation technology is well established.

These advantages, particularly its ease of genetic manipulation, rendered E. coli the primary biopharmaceutical production system for many years. However, E. coli also displays a number of drawbacks as a biopharmaceutical producer. These include:

Table 5.2 Some biopharmaceuticals currently on the market which are produced by genetic engineering in either E. coli or animal cells






E. coli, CHO




E. coli






E. coli




E. coli

Factor VIIa



E. coli


E. coli


E. coli

Table 5.3 Levels of expression of various biopharmaceuticals produced in recombinant E. coli cells

Biopharmaceutical Level of expression (% of total cellular protein)

IFN-y 25

Insulin 2G

TNF 15

a1-Antitrypsin 15

IL-2 1G

hGH 5

• heterologous proteins accumulate intracellularly;

• inability to undertake post-translational modifications (particularly glycosylation) of proteins;

• the presence of lipopolysaccharide (LPS) on its surface.

The vast bulk of proteins synthesized naturally by E. coli (i.e. its homologous proteins) are intracellular. Few are exported to the periplasmic space or released as true extracellular proteins. Heterologous proteins expressed in E. coli thus invariably accumulate in the cell cytoplasm. Intracellular protein production complicates downstream processing (relative to extracellular production) as:

• additional primary processing steps are required, i.e. cellular homogenization with subsequent removal of cell debris by centrifugation or filtration;

• more extensive chromatographic purification is required in order to separate the protein of interest from the several thousand additional homologous proteins produced by the E. coli cells.

An additional complication of high-level intracellular heterologous protein expression is inclusion body formation. Inclusion bodies (refractile bodies) are insoluble aggregates of partially folded heterologous product. Because of their dense nature, they are easily observed by dark-field microscopy. Presumably, when expressed at high levels, heterologous proteins overload the normal cellular protein-folding mechanisms. Under such circumstances, it would be likely that hydropho-bic patches normally hidden from the surrounding aqueous phase in fully folded proteins would remain exposed in the partially folded product. This, in turn, would promote aggregate formation via intermolecular hydrophobic interactions.

However, the formation of inclusion bodies displays one processing advantage: it facilitates the achievement of a significant degree of subsequent purification by a single centrifugation step. Because of their high density, inclusion bodies sediment even more rapidly than cell debris. Low-speed centrifugation thus facilitates the easy and selective collection of inclusion bodies directly after cellular homogenization. After collection, inclusion bodies are generally incubated with strong denaturants, such as detergents, solvents or urea. This promotes complete solubilization of the inclusion body (i.e. complete denaturation of the proteins therein). The denaturant is then removed by techniques such as dialysis or diafiltration. This facilitates refolding of the protein, a high percentage of which will generally fold into its native, biologically active, conformation.

Figure 5.1 High-level expression of a protein of interest in E. coli in soluble form by using the engineered 'thiofusion' expression system. Refer to text for specific details

Various attempts have been made to prevent inclusion body formation when expressing heterologous proteins in E. coli. Some studies have shown that a simple reduction in the temperature of bacterial growth (from 37 °C to 30 °C) can significantly decrease the incidence of inclusion body formation. Other studies have shown that expression of the protein of interest as a fusion partner with thioredoxin will eliminate inclusion body formation in most instances. Thioredoxin is a homologous E. coli protein, expressed at high levels. It is localized at the adhesion zones in E. coli and is a heat-stable protein. A plasmid vector has been engineered to facilitate expression of a fusion protein consisting of thioredoxin linked to the protein of interest via a short peptide sequence recognized by the protease enterokinase (Figure 5.1). The fusion protein is invariably expressed at high levels, while remaining in soluble form. Congregation at adhesion zones facilitates its selective release into the media by simple osmotic shock. This can greatly simplify its subsequent purification. After its release, the fusion protein is incubated with enterokinase, thus releasing the protein of interest (Figure 5.1).

Table 5.4 Proteins of actual or potential therapeutic use that are glycosylated when produced naturally in the body (or by hydridoma technology in the case of monoclonal antibodies). These proteins are discussed in detail in various subsequent chapters. See also Table 2.9

Most interleukins (IL-1 being an important exception) IFN-ß and -y (most IFN-as are unglycosylated) CSFs TNFs

Gonadotrophins (FSH, luteinizing hormone and hCG)

Blood factors (e.g. Factor VII, VIII and IX)

Thrombopoietin tPA


Intact monoclonal antibodies

An alternative means of reducing/potentially eliminating inclusion body accumulation entails the high-level co-expression of molecular chaperones along with the protein of interest. Chaper-ones are themselves proteins that promote proper and full folding of other proteins into their biologically active, native three-dimensional shape. They usually achieve this by transiently binding to the target protein during the early stages of its folding and guiding further folding by preventing/correcting the occurrence of improper hydrophobic associations.

The inability of prokaryotes such as E. coli to carry out post-translational modifications (particularly glycosylation) can limit their usefulness as production systems for some therapeuti-cally useful proteins. Many such proteins, when produced naturally in the body, are glycosylated (Table 5.4). However, the lack of the carbohydrate component of some glycoproteins has little, if any, negative influence upon their biological activity. The unglycosylated form of IL-2, for example, displays essentially identical biological activity to that of the native glycosylated molecule. In such cases, E. coli can serve as a satisfactory production system.

Another concern with regard to the use of E. coli is the presence on its surface of LPS molecules. The pyrogenic nature of LPS (Chapter 7) renders essential its removal from the product stream. Fortunately, several commonly employed downstream processing procedures achieve such a separation without any great difficulty.

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