Expression of recombinant protease MarP from Mycobacterium tuberculosis in Pichia pastoris and its effect on human monocytes

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...

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Bibliographic Details
Published in:Biotechnology letters 2021-09, Vol.43 (9), p.1787-1798
Main Authors: García-González, Gerardo, Ascacio-Martínez, Jorge Ángel, Hernández-Bello, Romel, González, Gloria María, Palma-Nicolás, José Prisco
Format: Article
Language:English
Subjects:
Acid resistance
Affinity chromatography
Aldehyde Oxidase - genetics
Applied Microbiology
Bacterial Proteins - chemistry
Bacterial Proteins - genetics
Bacterial Proteins - metabolism
Biochemistry
Biomedical and Life Sciences
Biotechnology
Chromatography, Affinity
Cytokines
Fungal Proteins - genetics
Gene Expression Regulation - drug effects
Homologous Recombination
Homology
Humans
Immunomodulation
Interleukin 10
Interleukin-10 - metabolism
Life Sciences
Macrophages
Microbiology
Monocytes
Monocytes - cytology
Monocytes - drug effects
Monocytes - immunology
Mycobacterium tuberculosis
Mycobacterium tuberculosis - enzymology
Mycobacterium tuberculosis - genetics
Nickel
Original Research Paper
Pichia - genetics
Pichia - growth & development
Pichia - metabolism
Pichia pastoris
Protease
Protein Domains
Protein Engineering
Proteins
Serine
Serine Proteases - chemistry
Serine Proteases - genetics
Serine Proteases - metabolism
Serine Proteases - pharmacology
Serine proteinase
THP-1 Cells
Tuberculosis
Tumor Necrosis Factor-alpha - metabolism
Tumor necrosis factor-α
Yeasts
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Summary: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.
ISSN:0141-5492
1573-6776
DOI:10.1007/s10529-021-03149-3