Handbooks from Amersham Pharmacia Biotech, 2000. — 106 p. Edition AAThis handbook is intended for the general reader interested in the amplification and purification of recombinant proteins and for everyday use at the laboratory bench.
The growth in the use of recombinant proteins has increased greatly in recent years, as has the wealth of techniques and products used for their amplification and purification. The advantages of using a fusion protein to facilitate purification and detection of the recombinant proteins are now widely recognised. This handbook introduces the reader to the initial considerations to be made when deciding upon host, vector and use of a fusion or non-fusion protein and covers general guidelines for successful protein amplification. General advice is also given on harvesting and extraction, handling of inclusion bodies, tag removal and removal of unvanted salts and small molecules.
The more that is known about the characteristics of a protein, the more easily it can be isolated and purified. Consequently, fusion proteins are simple and convenient to work with and, for many applications, a single purification step, using a commercially available affinity chromatography column, is sufficient. This is clearly demonstrated in the specific chapters on the amplification, purification and detection of the two most common fusion proteins (GST and (His)6 tagged proteins) which include simple practical protocols for use in the laboratory. The handbook also gives suggestions for the successful purification of other fusion proteins by a single affinity chromatography step.
In situations where no fusion system is available, or when a higher degree of purity is required, a multi-step purification will be necessary. This can also become a straightforward task by following a Three Phase Purification Strategy reviewed in the final chapter.
