in black and white
Main menu
Share a book About us Home
Biology Business Chemistry Computers Culture Economics Fiction Games Guide History Management Mathematical Medicine Mental Fitnes Physics Psychology Scince Sport Technics

biopharmaceuticals biochemistry and biotecnology - Walsh G.

Walsh G. biopharmaceuticals biochemistry and biotecnology - John Wiley & Sons, 2003. - 572 p.
ISBN 0-470-84327-6
Download (direct link): biochemistryandbiotechnology2003.pdf
Previous << 1 .. 65 66 67 68 69 70 < 71 > 72 73 74 75 76 77 .. 292 >> Next

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 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 re-folding of the protein, a high percentage of which will generally fold into its native, biologically active, conformation.
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 which facilitates 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 3.7). 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 3.7)
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. Chaperones are themselves proteins which 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 therapeutically useful proteins. Many such proteins, when produced naturally in the body, are glycosylated (Table 3.11). However, the lack of the carbohydrate component of some glycoproteins has little, if any, negative influence upon their biological activity. The unglycosylated form of interleukin-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 lipopolysaccharide (LPS) molecules. The pyrogenic nature of LPS (see later) renders essential its
Figure 3.7. 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
Table 3.11. 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
Most interleukins (interleukin-1 being an important exception)
Interferon-b and -g (most interferon-as are unglycosylated)
Colony stimulating factors Tumour necrosis factors
Gonadotrophins (follicle stimulating hormone, luteinizing hormone and human chorionic gonadotrophin)
Blood factors (e.g. factors VII, VIII and IX)
Tissue plasminogen activator
Intact monoclonal antibodies
removal from the product stream. Fortunately, several commonly employed downstream processing procedures achieve such a separation without any great difficulty.
Expression of recombinant proteins in animal cell culture systems
Technical advances facilitating genetic manipulation of animal cells now allow routine production of therapeutic proteins in such systems. The major advantage of these systems is their ability to carry out post-translational modification of the protein product. As a result, many biopharmaceuticals that are naturally glycosylated are now produced in animal cell lines (Table 3.9). Chinese hamster ovary (CHO) and baby hamster kidney (BHK) cells have become particularly popular in this regard.
Previous << 1 .. 65 66 67 68 69 70 < 71 > 72 73 74 75 76 77 .. 292 >> Next