biostudies-literatureLiu YShanghai Municipal Education CommissionChina Postdoctoral Science FoundationNational Natural Science Foundation of ChinaShanghai Institute for Advanced Study, Zhejiang University136-143https://www.ebi.ac.uk/biostudies/studies/S-EPMC9872166biostudies-literatureUnknown12(1)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, weACS synthetic biologyReconstitution of Multi-Protein Complexes through Ribozyme-Assisted Polycistronic Co-Expression.PMC98721662021-01-07-0007-E00074322009872022M72080032130063SNZJU-SIAS-009Liu YWu ZGao NWu DLin JfalseReconstitution of Multi-Protein Complexes through Ribozyme-Assisted Polycistronic Co-Expression.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, we2023-01-01T00:00:00Z2023 Jan2025-04-04T13:07:51.069Z2025-04-04T13:07:51.069ZS-EPMC98721663651250610.1021/acssynbio.2c00416