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rBmTI-6, a Kunitz-BPTI domain protease inhibitor from the tick Boophilus microplus, its cloning, expression and
Sergio D Sasaki1, Aparecida S Tanaka
1Centro de Ciências Naturais e Humanas, UFABC, Rua Catequese 242, 09090-400 Santo André, SP, Brazil.
Abstract:
Boophilus microplus is a rich source of trypsin inhibitors, numerous Kunitz-BPTI (bovine pancreatic trypsin inhibitor) inhibitors have been described from larvae and eggs, named BmTIs. Among them, were characterized inhibitors for trypsin, human neutrophil elastase, human plasma kallikrein and plasmin. BmTIs elicited a protective immunological response against B. microplus infestation in cattle. However, only a small amount of purified natural BmTIs can be obtained from larvae and eggs by chromatographic methods, thus if BmTIs are to be used as vaccine antigens (immunogens) the production of recombinant BmTIs (rBmTIs) is essential. In this work we describe the cloning, expression, purification and characterization of rBmTI-6. rBmTI-6 is a three-headed Kunitz-BPTI inhibitor, expressed in the Pichia pastoris system. Although rBmTI-6 was processed by proteases and glycosylated during the expression process, these post-translational modifications did not alter the ability of rBmTI-6 to inhibit protease activity. Purified rBmTI-6 inhibited trypsin and plasmin.
Insights
Recombinant Kunitz-BPTI inhibitors from ticks (BmTIs) were produced to overcome natural supply limitations. The recombinant BmTI-6 effectively inhibited trypsin and plasmin, showing potential as a vaccine antigen.
Area of Science:
- Biochemistry
- Parasitology
- Immunology
Background:
- Boophilus microplus ticks are a source of Kunitz-BPTI inhibitors (BmTIs) with potential immunological benefits against tick infestation.
- Natural BmTIs are difficult to obtain in sufficient quantities for vaccine development.
- Recombinant production of BmTIs is crucial for their application as vaccine antigens.
Purpose of the Study:
- To clone, express, purify, and characterize a recombinant BmTI, designated rBmTI-6.
- To assess the protease inhibitory activity of rBmTI-6.
- To evaluate the potential of rBmTI-6 as a vaccine candidate against Boophilus microplus.
Main Methods:
- Cloning and expression of rBmTI-6 in the Pichia pastoris system.
- Purification of rBmTI-6 using chromatographic methods.
- Characterization of rBmTI-6, including assessment of post-translational modifications and protease inhibition assays.
Main Results:
- Recombinant BmTI-6 was successfully expressed and purified.
- Post-translational modifications (protease processing and glycosylation) occurred during expression but did not abolish inhibitory activity.
- Purified rBmTI-6 demonstrated inhibitory activity against trypsin and plasmin.
Conclusions:
- Recombinant production of BmTI-6 in Pichia pastoris is feasible.
- rBmTI-6 retains its protease inhibitory function despite post-translational modifications.
- rBmTI-6 shows promise as a potential vaccine antigen for controlling Boophilus microplus infestations.
