The expression of recombinant proteins, especially using bacterial vectors and hosts, is a mature technology. With the appropriate cDNA and PCR methods, expression plasmids can be rapidly produced. Following sequence determination of the constructs, plasmids are transformed into expression hosts, single colonies picked, and fermentation performed. With E. coli, a 2-liter fermentation using complex media will generate ~ 50 to 80 g (wet weight of cells). Assuming modest protein expression (2% to 5% of the total cellular protein), between 100 and 300 mg of recombinant protein is available in the cells. The problem is, of course, how to isolate it in an active form. Soluble proteins can be recovered with good yields (>50%), and insoluble proteins, which must undergo a denaturation and folding cycle, can be recovered with more modest yields (5% to 20%). Hence, using small-scale fermentations and laboratory-scale processing equipment, proteins (or subdomains thereof) can usually be produced in sufficient quantities (10 to 100 mg) to initiate most studies including detailed structural determinations. Some strategies for achieving high-level expression of genes in E. coli have been reviewed by Markrides (1996) and Baneyx (1999) and are also discussed in Unit 5.24.

Protein purification proceeds following the approaches and methods discussed in the unit and elsewhere. The protein is characterized using various biophysical and biochemical methods which have also been detailed in the various Chapters of the book. The level of characterization depends on the final usage of the protein. It can be argued that characterization for structural determination requires the most rigorous approach, as micro chemical and physical heterogeneities can, for example, prevent protein crystallization. Characterization of therapeutic protein will also require rigor but more emphases will be placed on biochemical, immunological and functional testing. As the direction of arrows indicates (Figure 6.1.1) all the various stages are interdependent and there are always adjustments to be made based on the accumulation of information on the protein system being studied. Under ideal conditions, there is very high expression of biomedically important proteins such as HIV-1 Nef (NEF) and a Src homology-3 (SH3) domain of tyrosine kinase. These purified proteins form a complex the structure of which was solved by NMR (Grzesiek, et.al., 1996a).