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Expression, refolding, and purification of recombinant human granzyme B
Rikke H Lorentsen1, Charlotte H Fynbo, Hans C Thøgersen
1Department of Molecular Biology, University of Aarhus, Gustav Wieds Vej 10, DK-8000 Aarhus C, Denmark. rhl@boreanpharma.com
Protein Expression and Purification
|December 15, 2004
Summary
Recombinant Granzyme B (GrB) derivatives were engineered for specific fusion protein cleavage. These modified GrB enzymes demonstrate high specificity and efficiency in processing target proteins.
Area of Science:
- Biochemistry
- Molecular Biology
- Immunology
Background:
- Granzyme B (GrB) is a serine protease crucial for cytotoxic T-lymphocyte-mediated apoptosis.
- GrB cleaves specific substrates to induce programmed cell death.
Purpose of the Study:
- To explore the utility of engineered Granzyme B (GrB) derivatives as specific enzymes for fusion protein cleavage.
- To develop self-activating or factor-activated GrB variants for biotechnological applications.
Main Methods:
- Recombinant expression of two human GrB derivatives in Escherichia coli, including FXa-activated and self-activating forms.
- Purification using Ni2+-NTA agarose chromatography and refolding via a cyclic protocol.
- Characterization of enzymatic activity and specificity using fusion proteins with the GrB recognition sequence.
Main Results:
- Successfully expressed and refolded Granzyme B (GrB) derivatives with C-terminal hexa-histidine tags.
- Demonstrated efficient activation of pro-rGrB-H6 by FXa and autocatalytic processing of pro(IEPD)-rGrB-H6.
- Confirmed high specificity and efficiency of both wild-type rGrB-H6 and the C228F mutant in cleaving fusion proteins.
Conclusions:
- Engineered Granzyme B (GrB) derivatives are effective and specific enzymes for fusion protein processing.
- These modified GrB enzymes hold potential for applications requiring precise protein cleavage.