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Author Spotlight: The Production of Recombinant Proteins
Published on: June 30, 2023
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Development of efficient modules for recombinant protein expression and periplasmic localisation in Pseudomonas
Harshit Malhotra1, Braja Kishor Saha1, Prashant S Phale1
1Department of Biosciences and Bioengineering, Indian Institute of Technology-Bombay, Powai, Mumbai, 400076, India.
Protein Expression and Purification
|May 21, 2023
Summary
Pseudomonas bharatica CSV86T is a novel soil bacterium suitable for heterologous protein expression. Researchers developed expression systems using Pnah and Psal promoters for enhanced xenobiotic degradation applications.
Area of Science:
- Microbiology
- Biotechnology
- Metabolic Engineering
Background:
- Escherichia coli is a common host for protein expression but has limitations.
- Alternative hosts like Pseudomonas, Lactococcus, and Bacillus are being explored.
- Pseudomonas bharatica CSV86T, a soil isolate, shows potential due to its unique metabolic capabilities and eco-physiological traits.
Purpose of the Study:
- To develop and evaluate heterologous expression systems in Pseudomonas bharatica CSV86T.
- To engineer xenobiotic degradation pathways in this novel host.
- To assess the suitability of P. bharatica CSV86T for protein expression and metabolic engineering.
Main Methods:
- Selected Pnah and Psal promoters based on growth and naphthalene metabolism.
- Used 1-naphthol 2-hydroxylase (1NH) as a reporter gene to compare promoter strength.
- Expressed Carbaryl hydrolase (CH) under the Pnah promoter and utilized Tmd + Sp sequence for periplasmic translocation.
Main Results:
- Pnah promoter was identified as stronger and leakier than Psal.
- Recombinant CH was successfully expressed, translocated to the periplasm, purified, and showed similar kinetics to the native enzyme.
- The Tmd + Sp sequence facilitated periplasmic localization of the recombinant protein.
Conclusions:
- Pseudomonas bharatica CSV86T is a promising host for heterologous protein expression.
- The Pnah promoter and Tmd + Sp sequence are effective tools for overexpression and periplasmic localization.
- These advancements support applications in metabolic engineering and xenobiotic degradation.
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