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Heterologous expression and purification of encapsulins in Streptomyces coelicolor
In recent years a large number of encapsulin nanocompartment-encoding operons have been identified in bacterial and archaeal genomes. Encapsulin-encoding genes and operons from GC-rich Gram-positive bacteria, particularly of the phylum Actinobacteria, are often difficult to overexpress and purify in...
Autores principales: | , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Elsevier
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9309400/ https://www.ncbi.nlm.nih.gov/pubmed/35898614 http://dx.doi.org/10.1016/j.mex.2022.101787 |
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author | Andreas, Michael P. Giessen, Tobias W. |
author_facet | Andreas, Michael P. Giessen, Tobias W. |
author_sort | Andreas, Michael P. |
collection | PubMed |
description | In recent years a large number of encapsulin nanocompartment-encoding operons have been identified in bacterial and archaeal genomes. Encapsulin-encoding genes and operons from GC-rich Gram-positive bacteria, particularly of the phylum Actinobacteria, are often difficult to overexpress and purify in a soluble form using standard Escherichia coli expression systems. Here, we present a protocol to heterologously overexpress encapsulin nanocompartments and encapsulin-containing operons in Streptomyces coelicolor. Successful encapsulin production begins with the transfer of a Streptomyces expression plasmid, encoding the gene(s) of interest, via conjugation to the model actinobacterium S. coelicolor. After growing the conjugated S. coelicolor culture to the optimal optical density, protein production is induced by the addition of the inducer thiostrepton, followed by expression in liquid culture for 1–3 days. Cells are lysed and encapsulin proteins purified using ammonium sulfate precipitation and size exclusion chromatography. The method outlined in this protocol can be utilized to improve cargo loading and overall soluble expression of encapsulin systems when compared to expression in E. coli. • Clone an encapsulin-encoding gene or operon into a Streptomyces expression vector. • Transfer the Streptomyces expression vector to S. coelicolor via conjugation. • Heterologously express and purify empty or cargo-loaded encapsulins from S. coelicolor. |
format | Online Article Text |
id | pubmed-9309400 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Elsevier |
record_format | MEDLINE/PubMed |
spelling | pubmed-93094002022-07-26 Heterologous expression and purification of encapsulins in Streptomyces coelicolor Andreas, Michael P. Giessen, Tobias W. MethodsX Method Article In recent years a large number of encapsulin nanocompartment-encoding operons have been identified in bacterial and archaeal genomes. Encapsulin-encoding genes and operons from GC-rich Gram-positive bacteria, particularly of the phylum Actinobacteria, are often difficult to overexpress and purify in a soluble form using standard Escherichia coli expression systems. Here, we present a protocol to heterologously overexpress encapsulin nanocompartments and encapsulin-containing operons in Streptomyces coelicolor. Successful encapsulin production begins with the transfer of a Streptomyces expression plasmid, encoding the gene(s) of interest, via conjugation to the model actinobacterium S. coelicolor. After growing the conjugated S. coelicolor culture to the optimal optical density, protein production is induced by the addition of the inducer thiostrepton, followed by expression in liquid culture for 1–3 days. Cells are lysed and encapsulin proteins purified using ammonium sulfate precipitation and size exclusion chromatography. The method outlined in this protocol can be utilized to improve cargo loading and overall soluble expression of encapsulin systems when compared to expression in E. coli. • Clone an encapsulin-encoding gene or operon into a Streptomyces expression vector. • Transfer the Streptomyces expression vector to S. coelicolor via conjugation. • Heterologously express and purify empty or cargo-loaded encapsulins from S. coelicolor. Elsevier 2022-07-16 /pmc/articles/PMC9309400/ /pubmed/35898614 http://dx.doi.org/10.1016/j.mex.2022.101787 Text en © 2022 The Author(s) https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). |
spellingShingle | Method Article Andreas, Michael P. Giessen, Tobias W. Heterologous expression and purification of encapsulins in Streptomyces coelicolor |
title | Heterologous expression and purification of encapsulins in Streptomyces coelicolor |
title_full | Heterologous expression and purification of encapsulins in Streptomyces coelicolor |
title_fullStr | Heterologous expression and purification of encapsulins in Streptomyces coelicolor |
title_full_unstemmed | Heterologous expression and purification of encapsulins in Streptomyces coelicolor |
title_short | Heterologous expression and purification of encapsulins in Streptomyces coelicolor |
title_sort | heterologous expression and purification of encapsulins in streptomyces coelicolor |
topic | Method Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9309400/ https://www.ncbi.nlm.nih.gov/pubmed/35898614 http://dx.doi.org/10.1016/j.mex.2022.101787 |
work_keys_str_mv | AT andreasmichaelp heterologousexpressionandpurificationofencapsulinsinstreptomycescoelicolor AT giessentobiasw heterologousexpressionandpurificationofencapsulinsinstreptomycescoelicolor |