Selective Purification of Recombinant Neuroactive Peptides Using the Flagellar Type III Secretion System

ABSTRACT The structure, assembly, and function of the bacterial flagellum involves about 60 different proteins, many of which are selectively secreted via a specific type III secretion system (T3SS) (J. Frye et al., J. Bacteriol. 188:2233–2243, 2006). The T3SS is reported to secrete proteins at rate...

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Autores principales:	Hanna M. Singer, Marc Erhardt, Andrew M. Steiner, Min-Min Zhang, Doju Yoshikami, Grzegorz Bulaj, Baldomero M. Olivera, Kelly T. Hughes
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Lenguaje:	EN
Publicado:	American Society for Microbiology 2012
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/832a20cba56b447caad0757b36667181
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spelling	oai:doaj.org-article:832a20cba56b447caad0757b366671812021-11-15T15:39:02ZSelective Purification of Recombinant Neuroactive Peptides Using the Flagellar Type III Secretion System10.1128/mBio.00115-122150-7511https://doaj.org/article/832a20cba56b447caad0757b366671812012-07-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00115-12https://doaj.org/toc/2150-7511ABSTRACT The structure, assembly, and function of the bacterial flagellum involves about 60 different proteins, many of which are selectively secreted via a specific type III secretion system (T3SS) (J. Frye et al., J. Bacteriol. 188:2233–2243, 2006). The T3SS is reported to secrete proteins at rates of up to 10,000 amino acid residues per second. In this work, we showed that the flagellar T3SS of Salmonella enterica serovar Typhimurium could be manipulated to export recombinant nonflagellar proteins through the flagellum and into the surrounding medium. We translationally fused various neuroactive peptides and proteins from snails, spiders, snakes, sea anemone, and bacteria to the flagellar secretion substrate FlgM. We found that all tested peptides of various sizes were secreted via the bacterial flagellar T3SS. We subsequently purified the recombinant μ-conotoxin SIIIA (rSIIIA) from Conus striatus by affinity chromatography and confirmed that T3SS-derived rSIIIA inhibited mammalian voltage-gated sodium channel NaV1.2 comparably to chemically synthesized SIIIA. IMPORTANCE Manipulation of the flagellar secretion system bypasses the problems of inclusion body formation and cellular degradation that occur during conventional recombinant protein expression. This work serves as a proof of principle for the use of engineered bacterial cells for rapid purification of recombinant neuroactive peptides and proteins by exploiting secretion via the well-characterized flagellator type III secretion system.Hanna M. SingerMarc ErhardtAndrew M. SteinerMin-Min ZhangDoju YoshikamiGrzegorz BulajBaldomero M. OliveraKelly T. HughesAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 3, Iss 3 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Hanna M. Singer Marc Erhardt Andrew M. Steiner Min-Min Zhang Doju Yoshikami Grzegorz Bulaj Baldomero M. Olivera Kelly T. Hughes Selective Purification of Recombinant Neuroactive Peptides Using the Flagellar Type III Secretion System
description	ABSTRACT The structure, assembly, and function of the bacterial flagellum involves about 60 different proteins, many of which are selectively secreted via a specific type III secretion system (T3SS) (J. Frye et al., J. Bacteriol. 188:2233–2243, 2006). The T3SS is reported to secrete proteins at rates of up to 10,000 amino acid residues per second. In this work, we showed that the flagellar T3SS of Salmonella enterica serovar Typhimurium could be manipulated to export recombinant nonflagellar proteins through the flagellum and into the surrounding medium. We translationally fused various neuroactive peptides and proteins from snails, spiders, snakes, sea anemone, and bacteria to the flagellar secretion substrate FlgM. We found that all tested peptides of various sizes were secreted via the bacterial flagellar T3SS. We subsequently purified the recombinant μ-conotoxin SIIIA (rSIIIA) from Conus striatus by affinity chromatography and confirmed that T3SS-derived rSIIIA inhibited mammalian voltage-gated sodium channel NaV1.2 comparably to chemically synthesized SIIIA. IMPORTANCE Manipulation of the flagellar secretion system bypasses the problems of inclusion body formation and cellular degradation that occur during conventional recombinant protein expression. This work serves as a proof of principle for the use of engineered bacterial cells for rapid purification of recombinant neuroactive peptides and proteins by exploiting secretion via the well-characterized flagellator type III secretion system.
format	article
author	Hanna M. Singer Marc Erhardt Andrew M. Steiner Min-Min Zhang Doju Yoshikami Grzegorz Bulaj Baldomero M. Olivera Kelly T. Hughes
author_facet	Hanna M. Singer Marc Erhardt Andrew M. Steiner Min-Min Zhang Doju Yoshikami Grzegorz Bulaj Baldomero M. Olivera Kelly T. Hughes
author_sort	Hanna M. Singer
title	Selective Purification of Recombinant Neuroactive Peptides Using the Flagellar Type III Secretion System
title_short	Selective Purification of Recombinant Neuroactive Peptides Using the Flagellar Type III Secretion System
title_full	Selective Purification of Recombinant Neuroactive Peptides Using the Flagellar Type III Secretion System
title_fullStr	Selective Purification of Recombinant Neuroactive Peptides Using the Flagellar Type III Secretion System
title_full_unstemmed	Selective Purification of Recombinant Neuroactive Peptides Using the Flagellar Type III Secretion System
title_sort	selective purification of recombinant neuroactive peptides using the flagellar type iii secretion system
publisher	American Society for Microbiology
publishDate	2012
url	https://doaj.org/article/832a20cba56b447caad0757b36667181
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Selective Purification of Recombinant Neuroactive Peptides Using the Flagellar Type III Secretion System

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