Production and purification of recombinants of mouse MASP-2 and sMAP
Production and purification of recombinants of mouse MASP-2 and sMAP
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DOI：
10.1179/096805105225006704
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发表时间：
2005-01-01
期刊：
影响因子：
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通讯作者：
Fujita, T
中科院分区：
文献类型：
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作者：
Iwaki, D;Fujita, T
小甘露糖结合凝集素（MBL）相关蛋白（sMAP）是凝集素补体途径中MBL和MBL相关丝氨酸蛋白酶（MASP）组成的复合物的组分。sMAP是MASP-2的截短形式，其通过从单个结构MASP-2基因的选择性剪接产生。在MBL-MASP复合物活化后，MASP-2切割补体C4，但缺乏丝氨酸蛋白酶结构域的sMAP的作用尚不清楚。为了阐明sMAP在凝集素途径活化中的作用，我们产生了sMAP基因缺陷型小鼠，其也是MASP-2缺陷型。在这项研究中，我们产生和纯化小鼠重组sMAP（rsMAP）和rMASP-2使用果蝇表达系统的缺陷小鼠的重建试验。在初步实验中，这些纯化的重组体能够重建MBL-MASP-sMAP复合物，并且向缺陷血清中添加rMASP-2恢复了MBL-MASP复合物的C4切割活性。根据这些数据，本研究中产生的rsMAP和rMASP-2似乎可用于缺陷小鼠的分析。
Small mannose-binding lectin (MBL)-associated protein (sMAP) is a component of the complex consisting of MBL and MBL-associated serine proteases (MASPs) in the lectin complement pathway. sMAP is a truncated form of MASP-2, which is generated by an alternative splicing from a single structural MASP-2 gene. Upon activation of the MBL-MASPs complex, MASP-2 cleaves the complement C4, but the role of sMAP which lacks the serine protease domain is not clear. To clarify the role of sMAP in activation of the lectin pathway, we have generated sMAP-gene deficient mice which are also deficient for MASP-2. In this study, we generated and purified mouse recombinant sMAP (rsMAP) and rMASP-2 using the Drosophila expression system for the reconstitution assay of the deficient mice. In preliminary experiments, these purified recombinants were able to reconstitute the MBL-MASPs-sMAP complexes and the addition of rMASP-2 to deficient serum restored the C4 cleavage activity of the MBL-MASPs complex. From these data, rsMAP and rMASP-2 generated in this study seem to be useful in analysis of the deficient mice.