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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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2012-07-01
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| Series: | mBio |
| Online Access: | https://journals.asm.org/doi/10.1128/mBio.00115-12 |
| _version_ | 1818568751229960192 |
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| 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 |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-12-14T06:39:14Z |
| format | Article |
| id | doaj.art-832a20cba56b447caad0757b36667181 |
| institution | Directory Open Access Journal |
| issn | 2150-7511 |
| language | English |
| last_indexed | 2024-12-14T06:39:14Z |
| publishDate | 2012-07-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | mBio |
| spelling | doaj.art-832a20cba56b447caad0757b366671812022-12-21T23:13:16ZengAmerican Society for MicrobiologymBio2150-75112012-07-013310.1128/mBio.00115-12Selective Purification of Recombinant Neuroactive Peptides Using the Flagellar Type III Secretion SystemHanna M. Singer0Marc Erhardt1Andrew M. Steiner2Min-Min Zhang3Doju Yoshikami4Grzegorz Bulaj5Baldomero M. Olivera6Kelly T. Hughes7Dèpartement de Médecine, Université de Fribourg, Fribourg, SwitzerlandDèpartement de Médecine, Université de Fribourg, Fribourg, SwitzerlandDepartment of Medicinal Chemistry, University of Utah, Salt Lake City, Utah, USADepartment of Biology, University of Utah, Salt Lake City, Utah, USADepartment of Biology, University of Utah, Salt Lake City, Utah, USADepartment of Medicinal Chemistry, University of Utah, Salt Lake City, Utah, USADepartment of Biology, University of Utah, Salt Lake City, Utah, USADèpartement de Médecine, Université de Fribourg, Fribourg, SwitzerlandABSTRACT 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.https://journals.asm.org/doi/10.1128/mBio.00115-12 |
| spellingShingle | 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 mBio |
| title | 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_short | 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 |
| url | https://journals.asm.org/doi/10.1128/mBio.00115-12 |
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