Largest Chemically Made Protein

With a protein synthesis approach that they first used and reported last year for making a small model protein, researchers now have assembled large protein analogs from easily modified components.

The technique, kinetically controlled ligation (KCL), was developed by chemistry professor Stephen B. H. Kent of the University of Chicago and coworkers, who have now extended its applicability to the synthesis of a HIV protease analog (Angew. Chem. Int. Ed. 2007, 46, 1667). "To the best of our knowledge, the 203-amino-acid HIV protease covalent dimer is the protein with the largest linear polypeptide chain ever prepared by total chemical synthesis," Kent says.

The technique could make it easier to create protein analogs modified virtually at will for studies of protein function and mechanism and as drug candidates.

Kent believes the largest protein made previously by chemical synthesis was a 166-amino-acid erythropoiesis protein that his group created in 2003. That protein was made sequentially—working linearly from one end to the other—using an earlier technique, native chemical ligation (NCL). The power of KCL is that it makes it possible to synthesize proteins convergently—assembling large pieces that are then linked to one another—which is much more efficient and more practical than sequential synthesis.

Kent and graduate student Vladimir Torbeev created the 22-kD HIV protease molecule by synthesizing four peptide segments via stepwise solid-phase synthesis, using KCL to combine the segments into two large fragments, and then using NCL to combine the fragments.

The product is a single polypeptide chain that folds to form an HIV protease analog with the full enzymatic activity of the native version. The synthesis "illustrates the potential of the convergent synthetic strategy for making larger, more complex protein targets," Kent says.

"The work is a tour de force in protein chemical synthesis that showcases the state of the art in peptide ligation technology," comments Tom W. Muir, head of a protein engineering group at Rockefeller University.