Construction of a shuttle vector for the overexpression of recombinant proteins in Actinobacillus succinogenes

Pil Kim; Maris Laivenieks; James McKinlay; Claire Vieille; J. Gregory Zeikus

doi:10.1016/j.plasmid.2003.11.003

Construction of a shuttle vector for the overexpression of recombinant proteins in Actinobacillus succinogenes

Pil Kim
, Maris Laivenieks
, James McKinlay
, Claire Vieille
, J. Gregory Zeikus

Dept. of Biotechnology

Michigan State University

Research output: Contribution to journal › Article › peer-review

31 Scopus citations

Abstract

To express foreign proteins in Actinobacillus succinogenes, a shuttle vector was constructed based on the Actinobacillus pleuropneumoniae-Escherichia coli shuttle vector, pGZRS-19. We demonstrated that A. succinogenes is transformed by electroporation at reasonably high efficiency, that pGZRS-19 is stable in A. succinogenes, and that the ampicillin resistance gene carried by pGZRS-19 is expressed in A. succinogenes. Three steps were then required to develop our A. succinogenes-E. coli shuttle vector. (i) The constitutively expressed A. succinogenes phosphoenolpyruvate carboxykinase gene, pckA, was cloned and sequenced. (ii) Its promoter region and ribosome-binding site were subcloned into pGZRS-19. (iii) Finally, the ColE1 origin of replication was added to the vector to increase its stability in E. coli. High levels of A. succinogenes phosphoenolpyruvate carboxykinase, E. coli NADP-dependent malic enzyme, and Bacillus subtilis NAD-dependent malic enzyme activities detected in recombinant A. succinogenes strains confirmed that A. succinogenes and foreign proteins could be expressed in A. succinogenes under control of the A. succinogenes pckA promoter carried by pLGZ920. A. succinogenes is sensitive to chloramphenicol and tetracycline. Although not expressed from their own promoters, the Tn9 chloramphenicol and the Tn10 tetracycline resistance genes are expressed under control of the pckA promoter, and they can be used as additional selection markers in A. succinogenes.

Original language	English
Pages (from-to)	108-115
Number of pages	8
Journal	Plasmid
Volume	51
Issue number	2
DOIs	https://doi.org/10.1016/j.plasmid.2003.11.003
State	Published - Mar 2004

Bibliographical note

Funding Information:
This research was supported by a grant from NSF (# BES-0224596), and by a Michigan State University REF grant. P. Kim was partly supported by the Korea Science and Engineering Foundation (2001 postdoc fellowship 2nd half). We thank Felicia Codrea for constructing plasmid pLGZ-920. We express our sincere gratitude to Drs. Suzan West (University of Wisconsin, Madison, WI) and Stefan Schwarz (Institut für Tierzucht und Tierverhalten der Bundesforschungsantalt für Landwirtschaft, Celle, Germany) for providing us with plasmids pGZRS-19, pGZRS-30 (Dr. West), and UB214 (Dr. Schwarz).

Keywords

Actinobacillus succinogenes
Expression
Promoter
Shuttle vector

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10.1016/j.plasmid.2003.11.003

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title = "Construction of a shuttle vector for the overexpression of recombinant proteins in Actinobacillus succinogenes",

abstract = "To express foreign proteins in Actinobacillus succinogenes, a shuttle vector was constructed based on the Actinobacillus pleuropneumoniae-Escherichia coli shuttle vector, pGZRS-19. We demonstrated that A. succinogenes is transformed by electroporation at reasonably high efficiency, that pGZRS-19 is stable in A. succinogenes, and that the ampicillin resistance gene carried by pGZRS-19 is expressed in A. succinogenes. Three steps were then required to develop our A. succinogenes-E. coli shuttle vector. (i) The constitutively expressed A. succinogenes phosphoenolpyruvate carboxykinase gene, pckA, was cloned and sequenced. (ii) Its promoter region and ribosome-binding site were subcloned into pGZRS-19. (iii) Finally, the ColE1 origin of replication was added to the vector to increase its stability in E. coli. High levels of A. succinogenes phosphoenolpyruvate carboxykinase, E. coli NADP-dependent malic enzyme, and Bacillus subtilis NAD-dependent malic enzyme activities detected in recombinant A. succinogenes strains confirmed that A. succinogenes and foreign proteins could be expressed in A. succinogenes under control of the A. succinogenes pckA promoter carried by pLGZ920. A. succinogenes is sensitive to chloramphenicol and tetracycline. Although not expressed from their own promoters, the Tn9 chloramphenicol and the Tn10 tetracycline resistance genes are expressed under control of the pckA promoter, and they can be used as additional selection markers in A. succinogenes.",

keywords = "Actinobacillus succinogenes, Expression, Promoter, Shuttle vector",

author = "Pil Kim and Maris Laivenieks and James McKinlay and Claire Vieille and Zeikus, \{J. Gregory\}",

note = "Funding Information: This research was supported by a grant from NSF (\# BES-0224596), and by a Michigan State University REF grant. P. Kim was partly supported by the Korea Science and Engineering Foundation (2001 postdoc fellowship 2nd half). We thank Felicia Codrea for constructing plasmid pLGZ-920. We express our sincere gratitude to Drs. Suzan West (University of Wisconsin, Madison, WI) and Stefan Schwarz (Institut f{\"u}r Tierzucht und Tierverhalten der Bundesforschungsantalt f{\"u}r Landwirtschaft, Celle, Germany) for providing us with plasmids pGZRS-19, pGZRS-30 (Dr. West), and UB214 (Dr. Schwarz).",

year = "2004",

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doi = "10.1016/j.plasmid.2003.11.003",

language = "English",

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AU - Kim, Pil

AU - Laivenieks, Maris

AU - McKinlay, James

AU - Vieille, Claire

AU - Zeikus, J. Gregory

N1 - Funding Information: This research was supported by a grant from NSF (# BES-0224596), and by a Michigan State University REF grant. P. Kim was partly supported by the Korea Science and Engineering Foundation (2001 postdoc fellowship 2nd half). We thank Felicia Codrea for constructing plasmid pLGZ-920. We express our sincere gratitude to Drs. Suzan West (University of Wisconsin, Madison, WI) and Stefan Schwarz (Institut für Tierzucht und Tierverhalten der Bundesforschungsantalt für Landwirtschaft, Celle, Germany) for providing us with plasmids pGZRS-19, pGZRS-30 (Dr. West), and UB214 (Dr. Schwarz).

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N2 - To express foreign proteins in Actinobacillus succinogenes, a shuttle vector was constructed based on the Actinobacillus pleuropneumoniae-Escherichia coli shuttle vector, pGZRS-19. We demonstrated that A. succinogenes is transformed by electroporation at reasonably high efficiency, that pGZRS-19 is stable in A. succinogenes, and that the ampicillin resistance gene carried by pGZRS-19 is expressed in A. succinogenes. Three steps were then required to develop our A. succinogenes-E. coli shuttle vector. (i) The constitutively expressed A. succinogenes phosphoenolpyruvate carboxykinase gene, pckA, was cloned and sequenced. (ii) Its promoter region and ribosome-binding site were subcloned into pGZRS-19. (iii) Finally, the ColE1 origin of replication was added to the vector to increase its stability in E. coli. High levels of A. succinogenes phosphoenolpyruvate carboxykinase, E. coli NADP-dependent malic enzyme, and Bacillus subtilis NAD-dependent malic enzyme activities detected in recombinant A. succinogenes strains confirmed that A. succinogenes and foreign proteins could be expressed in A. succinogenes under control of the A. succinogenes pckA promoter carried by pLGZ920. A. succinogenes is sensitive to chloramphenicol and tetracycline. Although not expressed from their own promoters, the Tn9 chloramphenicol and the Tn10 tetracycline resistance genes are expressed under control of the pckA promoter, and they can be used as additional selection markers in A. succinogenes.

AB - To express foreign proteins in Actinobacillus succinogenes, a shuttle vector was constructed based on the Actinobacillus pleuropneumoniae-Escherichia coli shuttle vector, pGZRS-19. We demonstrated that A. succinogenes is transformed by electroporation at reasonably high efficiency, that pGZRS-19 is stable in A. succinogenes, and that the ampicillin resistance gene carried by pGZRS-19 is expressed in A. succinogenes. Three steps were then required to develop our A. succinogenes-E. coli shuttle vector. (i) The constitutively expressed A. succinogenes phosphoenolpyruvate carboxykinase gene, pckA, was cloned and sequenced. (ii) Its promoter region and ribosome-binding site were subcloned into pGZRS-19. (iii) Finally, the ColE1 origin of replication was added to the vector to increase its stability in E. coli. High levels of A. succinogenes phosphoenolpyruvate carboxykinase, E. coli NADP-dependent malic enzyme, and Bacillus subtilis NAD-dependent malic enzyme activities detected in recombinant A. succinogenes strains confirmed that A. succinogenes and foreign proteins could be expressed in A. succinogenes under control of the A. succinogenes pckA promoter carried by pLGZ920. A. succinogenes is sensitive to chloramphenicol and tetracycline. Although not expressed from their own promoters, the Tn9 chloramphenicol and the Tn10 tetracycline resistance genes are expressed under control of the pckA promoter, and they can be used as additional selection markers in A. succinogenes.

KW - Actinobacillus succinogenes

KW - Expression

KW - Promoter

KW - Shuttle vector

UR - https://www.scopus.com/pages/publications/1442299192

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DO - 10.1016/j.plasmid.2003.11.003

M3 - Article

C2 - 15003707

AN - SCOPUS:1442299192

SN - 0147-619X

VL - 51

SP - 108

EP - 115

JO - Plasmid

JF - Plasmid

IS - 2

ER -

Construction of a shuttle vector for the overexpression of recombinant proteins in Actinobacillus succinogenes

Abstract

Bibliographical note

Keywords

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