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With recent advances in recombinant expression technology, researchers can make just about any protein of interest. But it is not always a simple pursuit, especially for complicated endeavors such as incorporating spectroscopic probes or making membrane proteins. Stephen Kent of the University of Chicago is one of the champions of the somewhat overlooked but quite useful alternative techniques of chemical protein synthesis.

Solid phase peptide synthesis (SPPS) is used to chemically synthesize peptides of less than 50 amino acid residues. To assemble larger proteins, native chemical ligation (NCL) is an established method to stitch together synthetic peptide pieces. It exploits the highly specific reaction between an N-terminal cysteine and an α-thioester, and transpires in that most gentle solvent of all, water. Recently, by using a water-compatible solid support upon which to assemble a protein, Kent and his M.D.-Ph.D. student Erik Johnson showed that it is possible to carry out ligation, folding and functional assays, all while the protein is still attached to the support.

Kent, Johnson and colleagues discovered ideal properties for performing support-based NCL in a poly(ethyleneoxide)-based commercially available resin, the water-compatible superpermeable organic combinatorial chemistry (SPOCC) resin. Using SPOCC and NCL, they synthesized the small protein EETI-II, a trypsin inhibitor found in squash seeds. Because SPOCC is compatible with water, EETI-II could be folded on the resin. Even more interestingly, EETI-II retained its trypsin-inhibiting activity while it was still attached to the support. Because SPOCC is highly permeable, large molecules such as proteins can diffuse in and out without much trouble, facilitating functional assays.

Kent acknowledges that the most obvious practical application for this method would be to perform combinatorial protein synthesis, such as assembling one-bead–one-compound libraries for functional studies. But he is more engaged in exploring the intriguing properties of various resins for improving the efficiency of support-based NCL to demonstrate its often straightforward application in comparison to recombinant expression. “We are much more interested in [applying NCL] to make larger proteins, especially membrane proteins, for which we can keep the polypeptide chain well-solvated and available for reaction until we want to put it in a lipid bilayer,” says Kent. “We are more interested in being able to recover proteins in functional form rather than assay them directly on the resin.”