Expression and refolding of full-length human TIMP-1.

Abstract

Structural analysis of any protein, whether by X-ray crystallography or NMR, requires a reliable source of large amounts of good quality protein. Most proteins are not sufficiently abundant in their natural state to be used as the primary source, and so it is essential for recombinant protein to be expressed in an appropriate system. In many cases the preferred expression system is E. coli for ease of handling, generally high yields and, where the protein is required for NMR, relatively inexpensive isotopic labeling. Although an E. coli expression has the potential to produce high yields of a protein, it can be found in an insoluble form in inclusion bodies. There are a number of ways in which this problem can be avoided, for example (1) careful choice of growth conditions can reduce the rate of protein production allowing the protein to remain in soluble form; (2) alternatively there are several expression systems that use secretion tags, either to the medium or the periplasmic space where conditions may maintain the protein of interest in solution (); or (3) another method utilises a low induction of a gene expressing bacteriocin release protein (BRP) which produces holes in the cell wall, expression of this gene at high levels will cause cell death, but at lower levels will induce a leakiness in the cells, allowing protein release into the media ().