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Construction of new genetic tools as alternatives for protein overexpression in Escherichia coli and pseudomonas aeruginosa

Background: Pseudomonas protein expression in E. coli is known to be a setback due to signifi cant genetic variation and absence of several genetic elements in E. coli for regulation and activation of Pseudomonas proteins. Modifi cations in promoter/repressor system and shuttle plasmid maintenance h...

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Main Authors: Wong, Chee Fah, Raja Abd. Rahman, Raja Noor Zaliha, Basri, Mahiran, Salleh, Abu Bakar
Format: Article
Language:English
Published: National Institute of Genetic Engineering and Biotechnology 2017
Online Access:http://psasir.upm.edu.my/id/eprint/61210/1/Construction%20of%20new%20genetic%20tools%20as%20alternatives%20for%20protein%20overexpression%20in%20Escherichia%20coli%20and%20pseudomonas%20aeruginosa.pdf
http://psasir.upm.edu.my/id/eprint/61210/
http://www.ijbiotech.com/article_64935_5e2328fa7d3233d15c267587e483e931.pdf
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spelling my.upm.eprints.612102018-09-12T07:11:28Z http://psasir.upm.edu.my/id/eprint/61210/ Construction of new genetic tools as alternatives for protein overexpression in Escherichia coli and pseudomonas aeruginosa Wong, Chee Fah Raja Abd. Rahman, Raja Noor Zaliha Basri, Mahiran Salleh, Abu Bakar Background: Pseudomonas protein expression in E. coli is known to be a setback due to signifi cant genetic variation and absence of several genetic elements in E. coli for regulation and activation of Pseudomonas proteins. Modifi cations in promoter/repressor system and shuttle plasmid maintenance have made the expression of stable and active Pseudomonas protein possible in both Pseudomonas sp. and E. coli. Objectives: Construction of shuttle expression vectors for regulation and overexpression of Pseudomonas proteins in Pseudomonas sp. and E. coli. Materials and Methods: Pseudomonas-Escherichia shuttle expression vectors, pCon2(3), pCon2(3)-Kan and pCon2(3)-Zeo as well as E. coli expression vectors of pCon4 and pCon5 were constructed from pUCP19-, pSS213-, pSTBlue-1- and pPICZαAbased vectors. Protein overexpression was measured using elastase strain K as passenger enzyme in elastinolytic activity assay. Results: The integration of two series of IPTG inducible expression cassettes in pCon2(3), pCon2(3)-Kan and pCon2(3)- Zeo, each carrying an E. coli lac-operon based promoter, Plac, and a tightly regulated T7(A1/O4/O3) promoter/repressor system was performed to facilitate overexpression study of the organic solvent-tolerant elastase strain K. These constructs have demonstrated an elastinolytic fold of as high as 1464.4 % in comparison to other published constructs. pCon4 and pCon5, on the other hand, are series of pCon2(3)-derived vectors harboring expression cassettes controlled by PT7(A1/O4/O3) promoter, which conferred tight regulation and repression of basal expression due to existence of respective double operator sites, O3 and O4, and lacIq. Conclusions: The constructs off ered remarkable assistance for overexpression of heterogeneous genes in Pseudomonas sp.and E. coli for downstream applications such as in industries and structural biology study. National Institute of Genetic Engineering and Biotechnology 2017 Article PeerReviewed text en http://psasir.upm.edu.my/id/eprint/61210/1/Construction%20of%20new%20genetic%20tools%20as%20alternatives%20for%20protein%20overexpression%20in%20Escherichia%20coli%20and%20pseudomonas%20aeruginosa.pdf Wong, Chee Fah and Raja Abd. Rahman, Raja Noor Zaliha and Basri, Mahiran and Salleh, Abu Bakar (2017) Construction of new genetic tools as alternatives for protein overexpression in Escherichia coli and pseudomonas aeruginosa. Iranian Journal of Biotechnology, 15 (3). art. no. e1524. 194 - 200. ISSN 1728-3043; ESSN: 2322-2921 http://www.ijbiotech.com/article_64935_5e2328fa7d3233d15c267587e483e931.pdf 10.15171/ijb.1524
institution Universiti Putra Malaysia
building UPM Library
collection Institutional Repository
continent Asia
country Malaysia
content_provider Universiti Putra Malaysia
content_source UPM Institutional Repository
url_provider http://psasir.upm.edu.my/
language English
description Background: Pseudomonas protein expression in E. coli is known to be a setback due to signifi cant genetic variation and absence of several genetic elements in E. coli for regulation and activation of Pseudomonas proteins. Modifi cations in promoter/repressor system and shuttle plasmid maintenance have made the expression of stable and active Pseudomonas protein possible in both Pseudomonas sp. and E. coli. Objectives: Construction of shuttle expression vectors for regulation and overexpression of Pseudomonas proteins in Pseudomonas sp. and E. coli. Materials and Methods: Pseudomonas-Escherichia shuttle expression vectors, pCon2(3), pCon2(3)-Kan and pCon2(3)-Zeo as well as E. coli expression vectors of pCon4 and pCon5 were constructed from pUCP19-, pSS213-, pSTBlue-1- and pPICZαAbased vectors. Protein overexpression was measured using elastase strain K as passenger enzyme in elastinolytic activity assay. Results: The integration of two series of IPTG inducible expression cassettes in pCon2(3), pCon2(3)-Kan and pCon2(3)- Zeo, each carrying an E. coli lac-operon based promoter, Plac, and a tightly regulated T7(A1/O4/O3) promoter/repressor system was performed to facilitate overexpression study of the organic solvent-tolerant elastase strain K. These constructs have demonstrated an elastinolytic fold of as high as 1464.4 % in comparison to other published constructs. pCon4 and pCon5, on the other hand, are series of pCon2(3)-derived vectors harboring expression cassettes controlled by PT7(A1/O4/O3) promoter, which conferred tight regulation and repression of basal expression due to existence of respective double operator sites, O3 and O4, and lacIq. Conclusions: The constructs off ered remarkable assistance for overexpression of heterogeneous genes in Pseudomonas sp.and E. coli for downstream applications such as in industries and structural biology study.
format Article
author Wong, Chee Fah
Raja Abd. Rahman, Raja Noor Zaliha
Basri, Mahiran
Salleh, Abu Bakar
spellingShingle Wong, Chee Fah
Raja Abd. Rahman, Raja Noor Zaliha
Basri, Mahiran
Salleh, Abu Bakar
Construction of new genetic tools as alternatives for protein overexpression in Escherichia coli and pseudomonas aeruginosa
author_facet Wong, Chee Fah
Raja Abd. Rahman, Raja Noor Zaliha
Basri, Mahiran
Salleh, Abu Bakar
author_sort Wong, Chee Fah
title Construction of new genetic tools as alternatives for protein overexpression in Escherichia coli and pseudomonas aeruginosa
title_short Construction of new genetic tools as alternatives for protein overexpression in Escherichia coli and pseudomonas aeruginosa
title_full Construction of new genetic tools as alternatives for protein overexpression in Escherichia coli and pseudomonas aeruginosa
title_fullStr Construction of new genetic tools as alternatives for protein overexpression in Escherichia coli and pseudomonas aeruginosa
title_full_unstemmed Construction of new genetic tools as alternatives for protein overexpression in Escherichia coli and pseudomonas aeruginosa
title_sort construction of new genetic tools as alternatives for protein overexpression in escherichia coli and pseudomonas aeruginosa
publisher National Institute of Genetic Engineering and Biotechnology
publishDate 2017
url http://psasir.upm.edu.my/id/eprint/61210/1/Construction%20of%20new%20genetic%20tools%20as%20alternatives%20for%20protein%20overexpression%20in%20Escherichia%20coli%20and%20pseudomonas%20aeruginosa.pdf
http://psasir.upm.edu.my/id/eprint/61210/
http://www.ijbiotech.com/article_64935_5e2328fa7d3233d15c267587e483e931.pdf
_version_ 1643837528573214720
score 13.211869

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