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New alkane-responsive expression vectors for Escherichia coli and pseudomonas
T H Smits1, M A Seeger, B Witholt
1Institute of Biotechnology, Zürich, 8093, Switzerland.
Plasmid
|September 6, 2001
Summary
New expression vectors utilizing the PalkB promoter from Pseudomonas putida enable high-level protein production in Escherichia coli and Pseudomonas species. These vectors achieve significant expression, exceeding 10% of total cell protein.
Area of Science:
- Molecular Biology
- Microbial Biotechnology
- Gene Expression Systems
Background:
- The PalkB promoter from Pseudomonas putida GPo1 (oleovorans) is responsive to alkanes.
- Developing robust expression vectors is crucial for microbial biotechnology applications.
Purpose of the Study:
- To engineer and evaluate novel Escherichia coli and Pseudomonas expression vectors based on the PalkB promoter.
- To assess the induction efficiency and protein expression levels in various bacterial hosts.
Main Methods:
- Construction of expression vectors (pKKPalk, pCom8, pCom9, pCom10) utilizing the PalkB promoter.
- Testing vector performance in multiple Escherichia coli strains, Pseudomonas putida GPo12, and Pseudomonas fluorescens KOB2Delta1.
- Quantification of protein expression, specifically catechol-2,3-dioxygenase (XylE), as a percentage of total cell protein.
Main Results:
- High induction factors (100-2700) were achieved with pKKPalk in E. coli and pCom8 in Pseudomonas.
- Lower induction factors were observed for pCom8, pCom9, and pCom10 in E. coli.
- Achieved XylE expression levels exceeding 10% of total cellular protein in both E. coli and Pseudomonas.
Conclusions:
- The developed PalkB-based expression vectors are effective for high-level protein production in both E. coli and Pseudomonas.
- These vectors offer a valuable tool for microbial gene expression studies and biotechnological applications.
