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.

中文翻译：

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结核分枝杆菌重组蛋白酶MarP在毕赤酵母中的表达及其对人单核细胞的影响

客观的

分枝杆菌耐酸蛋白酶 (MarP) 是一种膜相关的丝氨酸蛋白酶，参与巨噬细胞中结核分枝杆菌的存活；在这里，我们在酵母毕赤酵母中产生了 MarP，并研究了它在巨噬细胞免疫调节中的作用。

结果

构建了含有全长或部分 MarP 序列的毕赤酵母载体。选择 GS115 克隆，并通过 PCR 评估 AOX1 基因座的同源重组。通过镍亲和层析纯化蛋白质，并在人单核细胞中测试其对细胞因子谱的影响。只有缺乏跨膜结构域的部分 MarP 蛋白 (121–397 aa) 成功表达为 N-糖基化蛋白水解活性蛋白酶。用 MarP 体外刺激 THP-1 细胞促进了 TNF-α 和 IL-10 的释放。

结论

分枝杆菌MarP在巴斯德毕赤酵母中成功表达，并且能够在体外释放细胞因子。