Recombinant Protein Production

The theory and application of this fluorescence method have been discussed in detail by LePecq and others (3,8). The assay requires that there is sufficient ionic strength to minimize ionic binding (e.g., O.IM sodium chloride), that the pH is 4-10, that no heavy metals are present, that the fluorescence is not enhanced on binding to other excipients (e.g., proteins) and that at least portions of the nucleic acids are not complexed. These requirements can usually he met when dealing with recombinant products in some cases the samples must he manipulated to create the appropriate conditions. In the intercalative method of dye binding, proteins rarely interfere with the assay, and procedures have been developed to remove the few interferences they may cause (e.g., the use of heparin or enzymatic digestion of the protein 9). 

It overcomes the problem of source availability. Many proteins of therapeutic potential are produced naturally in the body in minute quantities. Examples include interferons (Chapter 8), interleukins (Chapter 9) and colony-stimulating factors (CSFs Chapter 10). This rendered impractical their direct extraction from native source material in quantities sufficient to meet likely clinical demand. Recombinant production (Chapters 3 and 5) allows the manufacture of any protein in whatever quantity it is required. 

Despite the undoubted advantages of recombinant production, it remains the case that many protein-based products extracted directly from native source material remain on the market. In certain circumstances, direct extraction of native source material can prove equally/more attractive than recombinant production. This may be for an economic reason if, for example, the protein is produced in very large quantities by the native source and is easy to extract/purify, e.g. human serum albumin (HSA Chapter 12). Also, some blood factor preparations purified from donor blood actually contain several different blood factors and, hence, can be used to treat several haemophilia patient types. Recombinant blood factor preparations, on the other hand, contain but a single blood factor and, hence, can be used to treat only one haemophilia type (Chapter 12). 

Serum is a complex fluid, containing a variety of native proteins. This renders purification of the recombinant product more complex. 

Targeted extracellular recombinant production generally results in low-level extracellular accumulation of the desired protein (often in the milligram per litre range). Extracellular production simplifies subsequent downstream processing, as discussed later in this chapter. 

Two features of recombinant production in particular can impact very significantly upon the approach subsequently taken to purify the recombinant product inclusion body formation and the incorporation of purification tags. The processes of inclusion body formation, recovery and recombinant protein renaturation have been considered in Chapter 5. Once the recombinant protein has been refolded, additional purification (if required) follows traditional lines. 

Intact plants are also suitable for cost-effective production of recombinant pharmaceutical proteins providing products free of contaminations with endotoxins or human pathogens. Plants generally promote the proper fold of foreign proteins and post-translational modifications that are somehow similar to those of mammalian systems. Also, production of vaccine candidates in cereal seeds allows antigen protection from proteolysis which ensures their stability for a long period of time. 

FFeart disease, particularly clogged blood vessels, is also treated with recombinant products. Alteplase (Activase ), a human recombinant protein that breaks down fibrin, is used immediately after a stroke or heart attack to break down platelet-trapping clots in small blood vessels of the heart or brain and thus improve the patient s chances for recovery. Purified enzymes from bacteria are used for the same purposes. Abciximab (ReoPro ), a monoclonal antibody to... 

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