Synthesis of small protein domains by automated flow chemistry

The most fundamental topological units of proteins are their autonomously folded domains. The rapid and reliable chemical synthesis of domains in the range of 5-10 kDa in size, remains a challenge. Their bacterial expression is cumbersome, especially when chemical changes, post-translational modifications or the incorporation of non-natural residues are involved. Here, we report an in-house flow-chemistry-based synthetic method that enables one-step, fully automated synthesis of small protein domains without native chemical ligation. Our improved protocol is more efficient, using only 3 equivalent reagents and small amounts of organic solvents (6 ml per cycle) at a scale of 36 mu mol. First, we tested the limits of our system with oligotuftins of increasing length, H-(TKPKG)k-NH2 (4 <= k <= 16), a polypeptide composed of five residue long repeats. Second, four very different small single domain proteins were selected, each representing one specific 3D-fold type. Z amyloid binding affibody 3, Z(A beta 3), is of alpha-fold, while SRC homology 3 domain (SH3) is a representative of the all beta-stand fold. Bovine pancreatic trypsin inhibitor (BPTI) is an example of alpha + beta, while human ubiquitin (UBI) is that of the alpha/beta type domains. Our protocol was developed by optimizing concentration, flow rate, solvent composition, coupling time and reaction temperature allowing the overnight chain assembly (e.g. 7.4-8.6 hours) of a protein such as BPTI. Our smart peptide chemistry in flow (SPF) protocols are versatile and can be successfully applied to produce not only small protein modules, but also their chemical variants such as foldamers, or chimeras in moderate yields providing the synthetic background for current academic and specific pharmaceutical research. The smart peptide chemistry in flow method, or SPF for short, has been fine-tuned to enable the efficient, rapid, cost-effective and more environmentally friendly synthesis of small domain proteins, each representing a basic fold type.