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Purification and characterization of recombinant human cathepsin E
Advances in Experimental Medicine and Biology
|January 1, 1995
Summary
Recombinant human cathepsin E was successfully purified from Pichia pastoris. The enzyme was found to be a mature dimer and underwent N-linked glycosylation.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Expression
Background:
- Human cathepsin E is an aspartic protease with potential roles in various physiological and pathological processes.
- Efficient expression and purification of recombinant cathepsin E are crucial for its functional and structural characterization.
Purpose of the Study:
- To express and purify biologically active human cathepsin E in the methylotrophic yeast Pichia pastoris.
- To characterize the molecular properties and post-translational modifications of the recombinant enzyme.
Main Methods:
- Recombinant human cathepsin E expression in Pichia pastoris.
- Three-step column chromatography (TSKgel Phenyl-5PW, Toyopearl HW55S, TSKgel DEAE-5PW) for purification.
- Western blot analysis and endoglycosidase-H digestion for glycosylation analysis.
Main Results:
- Purified recombinant human cathepsin E exhibited a molecular mass of approximately 82 kDa, indicating a mature dimer form.
- The amino-terminal sequence analysis confirmed the mature enzyme's start at Ile37.
- Endoglycosidase-H digestion and Western blot revealed N-linked high-mannose type glycosylation.
Conclusions:
- Pichia pastoris is a suitable host for expressing and purifying active human cathepsin E.
- The recombinant human cathepsin E undergoes N-linked glycosylation in Pichia pastoris, yielding a mature dimeric enzyme.