Structure study reveals active and inactive conformations of protein kinase C B1

A human cell-based recombinant protein expression system is advantageous for large scale protein expression and purification as it provides native and nearly ideal conditions for correct folding and post-translational modification of protein products. Recently, we developed an affinity tag system using llama single-domain antibodies (a.k.a. nanobodies) generated against the fluorescent proteins YFP and mCherry. These nanobodies are highly efficient, with high binding capacity while exhibiting binding affinities that are down to the sub-nanomolar Kd, serving as ideal affinity-matrices for routine protein expression and purification in a mammalian system. These mCherry-tag and YFP-tag systems have enabled us to successfully express and purify numerous recombinant human proteins with productive yields and high quality. Here, we utilize the YFP-tag system to express and purify recombinant full-length human Protein Kinase C βI (PKCβI) using HEK293F cells. We have determined structures of purified PKCβI protein using X-ray crystallography that define two distinct conformations of the enzyme, providing structural insights behind their inhibition and activation mechanisms.