Construction of New Genetic Tools as Alternatives for Protein Overexpression in Escherichia coli and Pseudomonas aeruginosa
protein expression in is known to be a setback due to significant genetic variation and absence of several genetic elements in for regulation and activation of proteins. Modifications in promoter/repressor system and shuttle plasmid maintenance have made the expression of stable and active protein p...
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Published in | Iranian journal of biotechnology Vol. 15; no. 3; pp. 194 - 200 |
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Main Authors | , , , |
Format | Journal Article |
Language | English |
Published |
Iran
National Institute of Genetic Engineering and Biotechnology
27.09.2017
|
Subjects | |
Online Access | Get full text |
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Summary: | protein expression in
is known to be a setback due to significant genetic variation and absence of several genetic elements in
for regulation and activation of
proteins. Modifications in promoter/repressor system and shuttle plasmid maintenance have made the expression of stable and active
protein possible in both
sp. and
.
Construction of shuttle expression vectors for regulation and overexpression of
proteins in
sp. and
.
shuttle expression vectors, pCon2(3), pCon2(3)-Kan and pCon2(3)-Zeo as well as
expression vectors of pCon4 and pCon5 were constructed from pUCP19-, pSS213-, pSTBlue-1- and pPICZαA-based vectors. Protein overexpression was measured using elastase strain K as passenger enzyme in elastinolytic activity assay.
The integration of two series of IPTG inducible expression cassettes in pCon2(3), pCon2(3)-Kan and pCon2(3)-Zeo, each carrying an
lac-operon based promoter, Plac, and a tightly regulated T7
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 P
promoter, which conferred tight regulation and repression of basal expression due to existence of respective double operator sites, O3 and O4, and lacI
.
The constructs offered remarkable assistance for overexpression of heterogeneous genes in
sp. and
for downstream applications such as in industries and structural biology study. |
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ISSN: | 1728-3043 2322-2921 |
DOI: | 10.15171/ijb.1524 |