Cargando…

Design, Development and Optimization of a Functional Mammalian Cell-Free Protein Synthesis Platform

In this paper, we describe the stepwise development of a cell-free protein synthesis (CFPS) platform derived from cultured Chinese hamster ovary (CHO) cells. We provide a retrospective summary of the design challenges we faced, and the optimized methods developed for the cultivation of cells and the...

Descripción completa

Detalles Bibliográficos
Autores principales: Heide, Chiara, Buldum, Gizem, Moya-Ramirez, Ignacio, Ces, Oscar, Kontoravdi, Cleo, Polizzi, Karen M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7884609/
https://www.ncbi.nlm.nih.gov/pubmed/33604330
http://dx.doi.org/10.3389/fbioe.2020.604091
_version_ 1783651440306159616
author Heide, Chiara
Buldum, Gizem
Moya-Ramirez, Ignacio
Ces, Oscar
Kontoravdi, Cleo
Polizzi, Karen M.
author_facet Heide, Chiara
Buldum, Gizem
Moya-Ramirez, Ignacio
Ces, Oscar
Kontoravdi, Cleo
Polizzi, Karen M.
author_sort Heide, Chiara
collection PubMed
description In this paper, we describe the stepwise development of a cell-free protein synthesis (CFPS) platform derived from cultured Chinese hamster ovary (CHO) cells. We provide a retrospective summary of the design challenges we faced, and the optimized methods developed for the cultivation of cells and the preparation of translationally active lysates. To overcome low yields, we developed procedures to supplement two accessory proteins, GADD34 and K3L, into the reaction to prevent deactivation of the translational machinery by phosphorylation. We compared different strategies for implementing these accessory proteins including two variants of the GADD34 protein to understand the potential trade-offs between yield and ease of implementation. Addition of the accessory proteins increased yield of turbo Green Fluorescent Protein (tGFP) by up to 100-fold depending on which workflow was used. Using our optimized protocols as a guideline, users can successfully develop their own functional CHO CFPS system, allowing for broader application of mammalian CFPS.
format Online
Article
Text
id pubmed-7884609
institution National Center for Biotechnology Information
language English
publishDate 2021
publisher Frontiers Media S.A.
record_format MEDLINE/PubMed
spelling pubmed-78846092021-02-17 Design, Development and Optimization of a Functional Mammalian Cell-Free Protein Synthesis Platform Heide, Chiara Buldum, Gizem Moya-Ramirez, Ignacio Ces, Oscar Kontoravdi, Cleo Polizzi, Karen M. Front Bioeng Biotechnol Bioengineering and Biotechnology In this paper, we describe the stepwise development of a cell-free protein synthesis (CFPS) platform derived from cultured Chinese hamster ovary (CHO) cells. We provide a retrospective summary of the design challenges we faced, and the optimized methods developed for the cultivation of cells and the preparation of translationally active lysates. To overcome low yields, we developed procedures to supplement two accessory proteins, GADD34 and K3L, into the reaction to prevent deactivation of the translational machinery by phosphorylation. We compared different strategies for implementing these accessory proteins including two variants of the GADD34 protein to understand the potential trade-offs between yield and ease of implementation. Addition of the accessory proteins increased yield of turbo Green Fluorescent Protein (tGFP) by up to 100-fold depending on which workflow was used. Using our optimized protocols as a guideline, users can successfully develop their own functional CHO CFPS system, allowing for broader application of mammalian CFPS. Frontiers Media S.A. 2021-02-02 /pmc/articles/PMC7884609/ /pubmed/33604330 http://dx.doi.org/10.3389/fbioe.2020.604091 Text en Copyright © 2021 Heide, Buldum, Moya-Ramirez, Ces, Kontoravdi and Polizzi. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Bioengineering and Biotechnology
Heide, Chiara
Buldum, Gizem
Moya-Ramirez, Ignacio
Ces, Oscar
Kontoravdi, Cleo
Polizzi, Karen M.
Design, Development and Optimization of a Functional Mammalian Cell-Free Protein Synthesis Platform
title Design, Development and Optimization of a Functional Mammalian Cell-Free Protein Synthesis Platform
title_full Design, Development and Optimization of a Functional Mammalian Cell-Free Protein Synthesis Platform
title_fullStr Design, Development and Optimization of a Functional Mammalian Cell-Free Protein Synthesis Platform
title_full_unstemmed Design, Development and Optimization of a Functional Mammalian Cell-Free Protein Synthesis Platform
title_short Design, Development and Optimization of a Functional Mammalian Cell-Free Protein Synthesis Platform
title_sort design, development and optimization of a functional mammalian cell-free protein synthesis platform
topic Bioengineering and Biotechnology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7884609/
https://www.ncbi.nlm.nih.gov/pubmed/33604330
http://dx.doi.org/10.3389/fbioe.2020.604091
work_keys_str_mv AT heidechiara designdevelopmentandoptimizationofafunctionalmammaliancellfreeproteinsynthesisplatform
AT buldumgizem designdevelopmentandoptimizationofafunctionalmammaliancellfreeproteinsynthesisplatform
AT moyaramirezignacio designdevelopmentandoptimizationofafunctionalmammaliancellfreeproteinsynthesisplatform
AT cesoscar designdevelopmentandoptimizationofafunctionalmammaliancellfreeproteinsynthesisplatform
AT kontoravdicleo designdevelopmentandoptimizationofafunctionalmammaliancellfreeproteinsynthesisplatform
AT polizzikarenm designdevelopmentandoptimizationofafunctionalmammaliancellfreeproteinsynthesisplatform