Recombinant production of de novo designed miniprotein using various fusion protein tags

Numerous designed miniprotein (4-12 kDa) binders have been developed and reported to exhibit high-affinity interactions with disease targets that show great promise as therapeutic and diagnostic candidates. Despite these advances, detailed methodologies for their production have not been thoroughly explored. We chose two de novo miniprotein binders, LCB1 and AHB2, as model proteins to study their production by recombinant technology using various fusion protein tags. We selected fusion tags, namely, Small Ubiquitin-like Modifier (SUMO), thioredoxin (Trx), super folder green fluorescent protein (GFP), PhoN (non-specific acid phosphatase) and StrepII-Hexahistidine (StrepII-6xHis) to evaluate them for level of expression and purification of miniproteins. Our results revealed that LCB1 and AHB2, when expressed with fusion protein tags SUMO (3.3 & 2.8 millimoles), GFP (2.8 & 2.8 millimoles), Trx (2.3 & 1.42 millimoles), and StrepII-6xHis (1.96 & 1.38 millimoles), led to high expression levels and high yields of purified protein from 1 l of bacterial culture. Restriction enzyme cloning leaves residual amino acids at the N-terminal end of the miniprotein. The effect of these residual amino acids on the receptor-miniprotein binding was evaluated by SPR and MD simulation.