Recombinant production of therapeutic proteins

The evaluation of any protein as a potential biopharmaceutical and its subsequent routine medical use are dependent upon the availability of sufficient quantities of the target protein. In most instances this is best achieved via production by recombinant means (i.e. via genetic engineering). In addition to facilitating the production of any protein in substantial quantities, recombinant-based production can have a number of additional advantages over direct extraction from a naturally producing source, as described in Chapter 1. Production of any protein via rDNA technology entails the initial identification and isolation of a DNA sequence coding for the target protein. This sequence can be direct genomic DNA, but mRNA coding for the protein of interest can also act as a starting point. In the latter approach, the mRNA is enzymatically 'reverse transcribed' into cDNA. If the target therapeutic protein is eukaryotic (which is invariably the case) then the genomic DNA will contain both coding (exon) and non-coding (intron) sequences (Figure 3.9), whereas the cDNA will be a reflection of the exons only.

The desired gene/cDNA is normally amplified, sequenced and then introduced into an expression vector that facilitates its introduction and expression (transcription and translation) in an appropriate producer cell type. All recombinant therapeutic proteins approved to date are produced in E. coli, S. cerevisiae or in animal cell lines (mainly CHO or BHK cells). The general

characteristics of these various producer cells and their advantages and disadvantages, along with factors taken into account when choosing one for biopharmaceutical production, are points considered in Chapter 5. In the remainder of this chapter we will review the basic molecular biology techniques that underpin the isolation, identification, cloning and expression of a target protein-encoding gene sequence. We will first overview the classical approach to cloning, which entails the generation of genomic libraries as described immediately below. We will then consider an alternative approach that has now come to the fore, and which is based upon the polymerase chain reaction (PCR) technique.

Diabetes 2

Diabetes 2

Diabetes is a disease that affects the way your body uses food. Normally, your body converts sugars, starches and other foods into a form of sugar called glucose. Your body uses glucose for fuel. The cells receive the glucose through the bloodstream. They then use insulin a hormone made by the pancreas to absorb the glucose, convert it into energy, and either use it or store it for later use. Learn more...

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