摘要
FactorC是鲎血细胞中的一种丝氨酸蛋白酶原 ,因其能以高亲和力结合脂多糖 (LPS) ,在医药产品的内毒素检测中起着重要的作用 .以往研究表明处于N端的 3个Sushi结构域对FactorC的LPS结合活性起着关键作用 ,而FactorCN端的其他 3个结构域 ,包括Cys rich结构域、EGF like结构域和Lectin like结构域对LPS结合活性的影响尚不清楚 .利用Bac to Bac昆虫细胞表达系统可使rFactorC及其 4个截短的片段rCES12 3L ,rCES12 3,rS12 3L和rS12 3获得表达 ,并且可采用亲和层析柱将这 5个重组表达产物纯化 .通过测定 5个重组表达产物的LPS结合活性及抑菌活性 ,可以确定FactorC的LPS结合位点位于S12 3区域 .尽管Cys rich结构域、EGF like结构域和Lectin like结构域中不存在LPS结合位点 ,但当这 3个结构域同时存在时 ,可提高FactorC或CES12 3L的LPS结合能力 ,因此rCES12 3L具有与rFactorC非常相近的LPS结合能力 .实验结果表明 ,rCES12 3L在昆虫细胞中的表达量比rFactorC高出 4倍 ,预示出rCES12
Factor C is a serine protease enzymogen presented in the circulating blood cell of horseshoe crab. Owing to its extreme sensitivity to endotoxin, Factor C (FC) plays an important role in pyrogen-detection in pharmaceutical products. Previous study has shown that three Sushi domains (S123) in the N-terminal of FC are critical for its LPS-recognition activity. The effect of three other domains in N-terminal of FC, namely Cys-rich, EGF-like and Lectin-like, on its LPS-neutralization function was not clear. The recombinant FC and its four truncated fragments were expressed using Bac-to-Bac baculovirus expression system in Tn cells. The five recombinant peptides were then purified and tested for the LPS-binding activity and bactericidal activity in vitro. The experiments showed that the LPS-binding site of Factor C resides in the S123 region. Even though Cys-rich, EGF-like and Lectin-like domains do not harbor LPS-binding site, the presence of all three domains can improve LPS-binding activity of S123. Recombinant peptide rCES123L containing Cys-rich, EGF-like, S123, and Lectin-like domains exhibits almost the same LPS-binding activity as that of the full-length parental FC. Provided the fact that rCES123L has 4-fold higher production yield than that of recombinant FC in Tn cells, this peptide has a potential in biotechnology and pharmaceutical application for LPS-neutralization.
出处
《生物化学与生物物理进展》
SCIE
CAS
CSCD
北大核心
2004年第8期736-740,共5页
Progress In Biochemistry and Biophysics
