Reconstitution of Multi-Protein Complexes through Ribozyme-Assisted Polycistronic Co-Expression

[Image: see text] In living cells, proteins often exert their functions by interacting with other proteins forming protein complexes. Obtaining homogeneous samples of protein complexes with correct fold and stoichiometry is critical for its biochemical and biophysical characterization as well as functional investigation. Here, we developed a Ribozyme-Assisted Polycistronic co-expression system (pRAP) for heterologous co-production and in vivo assembly of multi-subunit complexes. In the pRAP system, a polycistronic mRNA transcript is co-transcriptionally converted into individual mono-cistrons in vivo. Each cistron can initiate translation with comparable efficiency, resulting in balanced production for all subunits, thus permitting faithful protein complex assembly. With pRAP polycistronic co-expression, we have successfully reconstituted large functional multi-subunit complexes involved in mammalian translation initiation. Our invention provides a valuable tool for studying the molecular mechanisms of biological processes.