CoachAn expression vector places the cloned coding sequence under a strong, controllable promoter of the host (the lac or T7 promoter in E. coli, induced by a sugar analogue), with a ribosome-binding site, a terminator, and often a tag — six histidines, a small protein — fused to the product for purification on a column. Bacteria make grams per litre of a simple protein (insulin, growth hormone, enzymes) but cannot glycosylate or fold complex mammalian proteins; yeast adds sugars of its own kind; insect and mammalian cells (Chinese hamster ovary cells are the industry’s standard) make antibodies and clotting factors folded and glycosylated as in a human. A monoclonal antibody is made by a hybridoma — an antibody-producing B cell fused to an immortal myeloma cell (Köhler and Milstein, 1975) — or, now, by cloning the antibody genes into a mammalian expression line, where the mouse framework can be replaced by human sequence to avoid immune rejection (Chapter 16).
Examples
Example 6.7 (Insulin)
Human insulin is two chains, A (21 residues) and B (30), joined by disulfide bonds, cut in the cell from a single proinsulin precursor. The first process expressed the two chains separately in E. coli, each fused to -galactosidase, cleaved them off chemically and joined them in vitro; later processes express proinsulin and cleave it with the enzymes the pancreas uses. Since 1996 the sequence itself has been altered: swapping or adding a residue or two gives analogues that dissociate faster (for a meal) or precipitate at the injection site and release over a day. A protein that took eight tonnes of glands per kilogram is now made in a fermenter, identical to the human one or better than it.