Tong ZZ, Jacobsen MT*
The scale of total chemical synthesis of proteins has increased significantly over the last 25 years. However, one major challenge in synthesizing proteins is the tedious HPLC purification of synthetic intermediates, which becomes more difficult as the target length increases. Often, one difficult peptide segment complicates the HPLC purification of a target, ultimately thwarting the project. In this presentation, we provide a solution to this chromatographic dilemma. We demonstrate how the judicious application of optimized NCL (native chemical ligation) reactants, termed pullover peptides, can simplify the purification of challenging NCL reactions. We rationally designed two model peptides, ThiP (thioester pullover) and CysP (Cys pullover), and then evaluated their chromatographic effects on model peptides. Pullover peptides are added to completed NCL reactions to cause HPLC shifts in unreacted (non-ligated) peptides.
Using a ThiP pullover peptide, we streamlined the synthesis of the 74-amino acid human C5a protein. C5a is a pro-inflammatory cytokine, especially associated with acute lung injury, which has been intractable to specific antibody therapies. Our initial synthesis of C5a was complicated by ligation at a poorly-reactive Thr thioester and co-elution of reactant and product during final HPLC purification. Using optimized conditions with a pullover peptide, we achieved synthesis and purification of multiple C5a proteins including wild-type, mutant, Biotin-labeled, and Fluorescent-labeled versions. All proteins were then refolded and characterized with respect to recombinantly-expressed protein: the wild-type and labeled proteins behaved indistinguishably from recombinant protein, while the designed mutant was functionally inactive. Pullover peptides are straightforward to prepare and implement, and they can be used generally to simplify HPLC purification in total chemical protein synthesis.