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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...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Chemical Society
2022
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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. |
format | Online Article Text |
id | pubmed-9872166 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | American Chemical Society |
record_format | MEDLINE/PubMed |
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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