(666h) Robust Cell-Free Production of Active Recombinant Human Phosphoproteins

Protein phosphorylation is a global regulator of cellular activity with approximately one-third of mammalian proteins being phosphorylated. Biochemical understanding of these proteins has been fundamentally limited by our technological inability to manufacture useful quantities of homogenous, site-specific phosphoserylated human proteins. To overcome these barriers, we present the use of cell-free protein synthesis (CFPS) for the co-translational incorporation phosphoserine (Sep) into human kinases. We first demonstrate the ability to produce 492 ± 54 μg mL^-1 superfolder green fluorescent protein (sfGFP) containing a single Sep (Sep-sfGFP) site-specifically incorporated, with a suppression efficiency of ~80%, confirmed by quantitative mass spectrometry. By adopting a semi-continuous method, where passive diffusion enables substrate replenishment and byproduct removal, we prolonged the CFPS reaction duration 4-fold and increased Sep-sfGFP to 800 μg mL^-1. With these advances at hand, we then set out to demonstrate the production of an active human kinase. We observed the synthesis of human full-length, doubly phosphserylated protein at yields of 269 ± 28 μg mL^-1. Notably, we observed a significant (>1,000-fold) improvement in protein yields over published in vivo bacterial expression of active, doubly-phosphoserylated kinase. This improvement comes from the fact that CFPS lacks viability constraints, thus allowing the production of phosphoproteins without toxicity effects resulting from orthogonal translation systems necessary for Sep incorporation in vivo. Our results demonstrate an important proof of concept for the synthesis of human proteins with proper post-translation modifications using cell-free systems. Further, it opens the way to new rapid and robust methods for producing phosphoserylated proteins in vitro.