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High level expression of genes cloned in phage lambda gt11
N G Stoker1, K A Grant, H M Dockrell
1Department of Clinical Sciences, London School of Hygiene and Tropical Medicine, U.K.
Gene
|May 15, 1989
Summary
New plasmid cloning vectors enable high-level expression of genes in Escherichia coli. These vectors enhance recombinant protein stability and expression, as demonstrated with Mycobacterium leprae protein.
Area of Science:
- Molecular Biology
- Biotechnology
- Protein Expression
Background:
- Lambda gt11 is a common system for gene cloning.
- High-level protein expression in Escherichia coli is crucial for research and biotechnology.
- Existing vectors may have limitations in expression efficiency and protein stability.
Purpose of the Study:
- To develop novel plasmid cloning vectors for high-level gene expression in Escherichia coli.
- To enhance the stability of recombinant proteins using a specific leader sequence.
- To facilitate the expression of genes originally cloned in lambda gt11.
Main Methods:
- Construction of plasmid vectors based on pEMBL and pUC.
- Utilizing the lac promoter for gene transcription.
- Altering the EcoRI site for compatible reading frame with lambda gt11 cloning.
- Fusion of cloned proteins to a 2-kDa Aparagine-rich leader sequence.
Main Results:
- Achieved high-level expression of Mycobacterium leprae 18-kDa protein.
- Recombinant protein constituted 15% of total cell protein.
- Expression levels reached 20 mg per litre of culture.
- Leader sequence improved protein stability without affecting immunological assays.
Conclusions:
- The developed plasmid vectors are effective for high-level recombinant protein expression in Escherichia coli.
- The Aparagine-rich leader sequence enhances protein stability.
- These vectors offer a valuable tool for expressing genes cloned in lambda gt11.