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Original Article
Dina Mulyanti
Bandung Institute of Technology; Bandung Islamic University
Corresponding Author
License:
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Background: Nowadays, recombinant therapeutic proteins have been widely produced and consumed. For the safety and effectiveness of the protein production, an auto-inducible expression vector is required to replace inducer interference, which is uneconomic and could be harmful. In this research, an auto-inducible expression plasmid, pCAD2_sod, which was under dps (RpoS-dependent gene) promoter control, was modified to provide RpoS at earlier phase, hence accumulate more target protein by generated a new plasmid, pCAD2+_sod. pCAD2_sod had been constructed to automatically induce the expression of recombinant superoxide dismutase (SOD) from Staphylococcus equorum (rMnSODSeq) in the stationary growth phase of Escherichia coli. This work aimed to obtain pCAD2+_sod and determined the expression level of rMnSODSeq.
Method and Results: A synthetic rpoS coding region under rpoD promoter control (prpoD_rpoS) was inserted to pCAD2_sod and generated pCAD2+_sod. The rMnSODSeq (24.3 kDa) produced from pCAD2+_sod was ~1.5 fold higher at 37 °C and more intense at 43 °C compared to that produced from pCAD2_sod, likewise shifted to earlier phase (after 1 h of incubation), as shown in the SDS-PAGE. The dismutase activity was also retained after zymography assay. The mRNA level of rMnSODSeq from pCAD2+_sod, in all growth phases, gave a consistently lower Cq values compared to the one carried pCAD2_sod and so gave higher quantification values relative to rho reference gene as well.
Conclusions: The prpoD_rpoS insertion shifts and increases the rMnSODSeq production from stationary to exponential phase, either in the RNA or protein level. The pCAD2+_sod plasmid has good potential for further recombinant protein productions.
Keywords
auto-inducible, RpoS, dps promoter, superoxide dismutase
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CURRENT STATUS: Published
View the published article at Molecular Biology Reports.
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Editorial decision: Major Revisions Needed
On 15 Mar, 2021
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A new preprint design is coming!
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See the published version of this preprint at Molecular Biology Reports.
This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Original Article
Dina Mulyanti
Bandung Institute of Technology; Bandung Islamic University
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Published
View the published article at Molecular Biology Reports.
Version 1
Posted 08 Feb, 2021
No community comments so far
Editorial decision: Major Revisions Needed
On 15 Mar, 2021
Reviewers invited
Invitations sent on 09 Feb, 2021
Reviews received
Received 03 Feb, 2021
First submitted
On 17 Jan, 2021
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Molecular Biology
License:
This work is licensed under a CC BY 4.0 License. Read Full License
View the published article at Molecular Biology Reports.
Background: Nowadays, recombinant therapeutic proteins have been widely produced and consumed. For the safety and effectiveness of the protein production, an auto-inducible expression vector is required to replace inducer interference, which is uneconomic and could be harmful. In this research, an auto-inducible expression plasmid, pCAD2_sod, which was under dps (RpoS-dependent gene) promoter control, was modified to provide RpoS at earlier phase, hence accumulate more target protein by generated a new plasmid, pCAD2+_sod. pCAD2_sod had been constructed to automatically induce the expression of recombinant superoxide dismutase (SOD) from Staphylococcus equorum (rMnSODSeq) in the stationary growth phase of Escherichia coli. This work aimed to obtain pCAD2+_sod and determined the expression level of rMnSODSeq.
Method and Results: A synthetic rpoS coding region under rpoD promoter control (prpoD_rpoS) was inserted to pCAD2_sod and generated pCAD2+_sod. The rMnSODSeq (24.3 kDa) produced from pCAD2+_sod was ~1.5 fold higher at 37 °C and more intense at 43 °C compared to that produced from pCAD2_sod, likewise shifted to earlier phase (after 1 h of incubation), as shown in the SDS-PAGE. The dismutase activity was also retained after zymography assay. The mRNA level of rMnSODSeq from pCAD2+_sod, in all growth phases, gave a consistently lower Cq values compared to the one carried pCAD2_sod and so gave higher quantification values relative to rho reference gene as well.
Conclusions: The prpoD_rpoS insertion shifts and increases the rMnSODSeq production from stationary to exponential phase, either in the RNA or protein level. The pCAD2+_sod plasmid has good potential for further recombinant protein productions.
Figure 1
Figure 2
Figure 3
This is a list of supplementary files associated with this preprint. Click to download.
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