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Expression and characterization of a recombinant cysteine proteinase of Leishmania mexicana
S J Sanderson1, K G Pollock, J D Hilley
1Division of Infection and Immunity, Joseph Black Building, Institute of Biomedical and Life Sciences, University of Glasgow, University Avenue, Glasgow G12 8QQ, UK. s.sanderson@bio.gla.ac.uk
Abstract:
A major cysteine proteinase (CPB) of Leishmania mexicana, that is predominantly expressed in the form of the parasite that causes disease in mammals, has been overexpressed in Escherichia coli and purified from inclusion bodies to apparent homogeneity. The CPB enzyme, CPB2.8, was expressed as an inactive pro-form lacking the characteristic C-terminal extension (CPB2.8DeltaCTE). Pro-region processing was initiated during protein refolding and proceeded through several intermediate stages. Maximum enzyme activity accompanied removal of the entire pro-region. This was facilitated by acidification. Purified mature enzyme gave a single band on SDS/PAGE and gelatin SDS/PAGE gels, co-migrated with native enzyme in L. mexicana lysates, and had the same N-terminal sequence as the native enzyme. The procedure yielded >3.5 mg of active enzyme per litre of E. coli culture.
Insights
Researchers successfully overexpressed and purified a key Leishmania mexicana cysteine proteinase (CPB) in E. coli. This parasite enzyme, CPB2.8, was activated through refolding and acidification, yielding significant amounts of active protein.
Area of Science:
- Biochemistry
- Parasitology
- Molecular Biology
Background:
- Leishmania mexicana causes disease in mammals.
- Cysteine proteinases (CPBs) are crucial virulence factors in Leishmania.
- CPB2.8 is a major CPB predominantly expressed in the mammalian-infective stage.
Purpose of the Study:
- To overexpress and purify the Leishmania mexicana CPB2.8 enzyme in Escherichia coli.
- To characterize the processing and activation of the recombinant CPB2.8 enzyme.
Main Methods:
- Overexpression of CPB2.8 (lacking C-terminal extension) in E. coli.
- Purification of the enzyme from inclusion bodies.
- Protein refolding, pro-region processing, and activity assays.
- SDS-PAGE and gelatin SDS-PAGE for purity and activity assessment.
- N-terminal sequencing for enzyme identity confirmation.
Main Results:
- Active CPB2.8 enzyme was purified to apparent homogeneity from E. coli inclusion bodies.
- Pro-region processing and enzyme activation were achieved during protein refolding, facilitated by acidification.
- The purified mature enzyme exhibited characteristics of the native Leishmania mexicana CPB.
- The expression system yielded over 3.5 mg of active enzyme per liter of E. coli culture.
Conclusions:
- Recombinant expression of Leishmania mexicana CPB2.8 in E. coli is feasible.
- Acidification-mediated refolding effectively processes and activates the recombinant enzyme.
- This method provides a scalable source of active CPB2.8 for further research.