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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 fun...

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Autores principales: Liu, Yan, Wu, Zihan, Wu, Damu, Gao, Ning, Lin, Jinzhong
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2022
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9872166/
https://www.ncbi.nlm.nih.gov/pubmed/36512506
http://dx.doi.org/10.1021/acssynbio.2c00416
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author Liu, Yan
Wu, Zihan
Wu, Damu
Gao, Ning
Lin, Jinzhong
author_facet Liu, Yan
Wu, Zihan
Wu, Damu
Gao, Ning
Lin, Jinzhong
author_sort Liu, Yan
collection PubMed
description [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.
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spelling pubmed-98721662023-01-25 Reconstitution of Multi-Protein Complexes through Ribozyme-Assisted Polycistronic Co-Expression Liu, Yan Wu, Zihan Wu, Damu Gao, Ning Lin, Jinzhong ACS Synth Biol [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. American Chemical Society 2022-12-13 /pmc/articles/PMC9872166/ /pubmed/36512506 http://dx.doi.org/10.1021/acssynbio.2c00416 Text en © 2022 The Authors. Published by American Chemical Society https://creativecommons.org/licenses/by-nc-nd/4.0/Permits non-commercial access and re-use, provided that author attribution and integrity are maintained; but does not permit creation of adaptations or other derivative works (https://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Liu, Yan
Wu, Zihan
Wu, Damu
Gao, Ning
Lin, Jinzhong
Reconstitution of Multi-Protein Complexes through Ribozyme-Assisted Polycistronic Co-Expression
title Reconstitution of Multi-Protein Complexes through Ribozyme-Assisted Polycistronic Co-Expression
title_full Reconstitution of Multi-Protein Complexes through Ribozyme-Assisted Polycistronic Co-Expression
title_fullStr Reconstitution of Multi-Protein Complexes through Ribozyme-Assisted Polycistronic Co-Expression
title_full_unstemmed Reconstitution of Multi-Protein Complexes through Ribozyme-Assisted Polycistronic Co-Expression
title_short Reconstitution of Multi-Protein Complexes through Ribozyme-Assisted Polycistronic Co-Expression
title_sort reconstitution of multi-protein complexes through ribozyme-assisted polycistronic co-expression
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9872166/
https://www.ncbi.nlm.nih.gov/pubmed/36512506
http://dx.doi.org/10.1021/acssynbio.2c00416
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