An improved procedure for breaking proteins in two is allowing chemists to create new proteins. Derek Macmillan and Jonathan Richardson from University College London, UK, have used their method to make a biologically active protein.

Cyanogen bromide is used to cleave a protein next to a methionine residue to give a protein fragment for NCL

Low reaction yields make creating large proteins by chemical synthesis unfeasible, yet not all proteins can be prepared easily by other routes. Native chemical ligation (NCL) is one technique that chemists can use to create these proteins by joining a small peptide containing a thioester to a protein fragment with an N-terminal cysteine. In this way troublesome proteins can be created without having to synthesise the whole protein from scratch.

The advantages of NCL are its 'versatility and reliability', explains Macmillan. 'The reaction is conducted in aqueous solution with no protecting groups, and the product has a native peptide bond,' he adds. 

But whilst the small peptides required for NCL can be made by peptide synthesis, preparing the large protein fragment is often more problematic and poses many of the same problems associated with creating whole proteins. Now, Macmillan and Richardson have optimised their procedure for generating these fragments. The starting, bacterially-derived protein is cleaved next to a methionine to give two fragments, one with the cysteine at its N-terminus. The cysteine-containing fragment is then isolated and combined with a synthetic peptide containing a thioester to create a whole, semi-synthetic protein. 

Using their optimised procedure Macmillan and Richardson were able to create a biologically active variant of erythropoietin, a hormone protein involved in red blood cell production which is used in the treatment of anaemia. Commenting on this success, Phil Dawson an expert in synthetic protein chemistry at The Scripps Research Institute, La Jolla, US, says that 'the functional complexity of large proteins can turn even the most straightforward procedure into a daunting challenge. The Macmillan lab has taken an important first step towards establishing a robust semi-synthesis for an important therapeutic target.' 

Russell Johnson