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Updated: Nov 4, 2025
Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris
Published on: February 25, 2010
Expression of recombinant protease MarP from Mycobacterium tuberculosis in Pichia pastoris and its effect on human
Gerardo García-González1, Jorge Ángel Ascacio-Martínez2, Romel Hernández-Bello1
1Departamento de Microbiología, Facultad de Medicina y Hospital Universitario "Dr. José Eleuterio González", Universidad Autónoma de Nuevo León, Ave. Francisoco I. Madero y Dr. Eduardo Aguirre Pequeño s/n, Col. Mitras Centro, C.P. 64460, Monterrey, Nuevo León, Mexico.
Objective:
Mycobacterial acid-resistant protease (MarP) is a membrane-associated serine protease involved in the survival of Mycobacterium tuberculosis in macrophages; here we produced MarP in the yeast Pichia pastoris and study its involvement in macrophage immune modulation.
Results:
Pichia pastoris vectors, harboring a full-length or a partial sequence of MarP, were constructed. GS115 clones were selected, and homologous recombination at the AOX1 locus was assessed by PCR. Protein was purified by nickel affinity chromatography, and its effect on the cytokine profile was tested in human monocytes. Only the partial MarP protein (121-397 a.a.) lacking the transmembrane domain was successfully expressed as an N-glycosylated proteolytically active protease. In vitro stimulation of THP-1 cells with MarP promoted the release of TNF-α and IL-10.
Conclusion:
Mycobacterial MarP was successfully expressed in P. pastoris, and it is capable of cytokine release in vitro.
Insights
Mycobacterial acid-resistant protease (MarP) was successfully produced in Pichia pastoris. This protease modulates macrophage immune responses by promoting the release of key cytokines like TNF-α and IL-10.
Area of Science:
- Microbiology
- Immunology
- Protease research
Background:
- Mycobacterium tuberculosis survival in macrophages is facilitated by the membrane-associated serine protease, MarP.
- Understanding MarP's role in immune modulation is crucial for developing therapeutic strategies.
Purpose of the Study:
- To produce Mycobacterial acid-resistant protease (MarP) in Pichia pastoris.
- To investigate the effect of MarP on macrophage immune modulation.
Main Methods:
- Construction of Pichia pastoris vectors with MarP sequences.
- Expression and purification of MarP using nickel affinity chromatography.
- In vitro stimulation of human THP-1 cells with purified MarP.
Main Results:
- A partial, N-glycosylated, and proteolytically active MarP (121-397 a.a.) was successfully expressed.
- MarP stimulation induced the release of tumor necrosis factor-alpha (TNF-α) and interleukin-10 (IL-10) from THP-1 cells.
Conclusions:
- Mycobacterial MarP can be effectively expressed in Pichia pastoris.
- Expressed MarP demonstrates the ability to induce cytokine release in vitro, suggesting a role in immune modulation.
