Recombinant expression systems

Bacterial systems

E. coli grows fast and produces high yields, and is the workhorse for proteins that do not need modification after assembly. It cannot glycosylate, and it often deposits over-expressed protein in insoluble inclusion bodies that must be dissolved and refolded — a step that has to be validated, because incorrectly refolded protein is chemically correct and functionally wrong.

Bacterial hosts also introduce endotoxin, a component of the bacterial outer membrane, which is why endotoxin testing accompanies bacterially expressed products.

Yeast and mammalian systems

Yeast can perform some post-translational modification and secretes protein into the medium, simplifying recovery. Mammalian cells produce human-like glycosylation and correct folding for complex proteins, at considerably greater cost and lower yield.

A glycoprotein needs a system capable of glycosylating it. The molecule is not merely a chain in that case; the attached sugars are part of it.

Purification

Whatever the host, the product must be separated from everything else the cell made. Purification typically combines several chromatographic steps exploiting different properties — charge, size, specific affinity — because no single step removes everything.