Protein production and purification

Abstract

In selecting a method to produce a recombinant protein, a researcher is faced with a bewildering array of choices as to where to start. To facilitate decision-making, we describe a consensus 'what to try first' strategy based on our collective analysis of the expression and purification of over 10,000 different proteins. This review presents methods that could be applied at the outset of any project, a prioritized list of alternate strategies and a list of pitfalls that trip many new investigators.

Figure 1

Solubility as a function of construct length. Fraction of successful purifications and structure determinations as a function of protein length (data from New York Structural GenomiX Research Center). Dotted line, fraction of cloned targets resulting in successful large-scale purifications. Dashed line, fraction of soluble clones (those that express soluble protein at a 1-ml scale) that yield pure protein at large scale. Solid line, fraction of purified proteins resulting in successful crystal structure determinations. There are relatively few targets with lengths greater than 800 amino acids, so these fractions have been extrapolated and are shown in gray.

Figure 2

Gel filtration profiles. Representative good (left) and bad (right) gel-filtration profiles of two different proteins purified on an ÄKTAxpress system using a HiLoad Superdex 200 column (GE Healthcare).