权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Biochemical analysis of the novel tumor suppressor gene product RECK

新型抑癌基因产物RECK的生化分析

基本信息

批准号：
08660101
负责人：
MAKI Masatoshi
金额：
$ 1.41万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08660101/
关键词：
tumor suppressor gene protease baculovirus monoclonal antibody

项目摘要

RECK is a novel tumor suppressor gene which reverses normally the activated-ras-transformed mouse fibroblast cells. In malignant transformed cells, its expression is suppressed. Forced exogenous expression of the gene in the cancer cells suppresses colony formation in soft agar and inhibits tumor metastasis. We characterized the gene product as follows :(1) Partial cDNA was expressed in E.coli, and the purified recombinant protein was subjected to immunize mice for preparation of monoclonal antibodies. Hybridomas were screened with ELISA method. (2) RECK cDNA was expressed in insect cells using baculovirus system. Western analysis using the monoclonal antibodies revealed that the molecular mass of the expressed protein was about 100 kDa. It agreed well with the value estimated from the amino acid sequence and suggested low glycosylation. (3) RECK contains signal sequence at N-terminus and hydrophobic sequence at C-terminus and is transported onto the membrane outside of the cell. Deletion of the C-terminal hydrophobic region caused secretion of RECK into the culture medium. We established a method of affinity purification of the secreted type of RECK by substituting the C-terminal sequence with a tag of histidine hexamer. (4) Potential reactive site residue (P1) of the Kazal domain of RECK is Asp and it suggests caspase-like proteases as target enzymes. RECK did not inhibit caspase-1. It did not interact with granzyme B,either.

RECK是一种新的肿瘤抑制基因，能正常逆转活化的ras转化的小鼠成纤维细胞。在恶性转化的细胞中，它的表达受到抑制。该基因在癌细胞中的强制外源表达抑制了软琼脂中的集落形成，并抑制了肿瘤转移。基因产物的鉴定如下：(1)在大肠杆菌中表达部分基因，纯化的重组蛋白免疫小鼠制备单抗。用ELISA法筛选杂交瘤。(2)利用杆状病毒系统在昆虫细胞中表达RECK基因。用单抗进行Western分析表明，表达蛋白的相对分子质量约为100 kDa。这与根据氨基酸序列估计的值很好地吻合，并表明糖基化程度较低。(3)RECK在N-末端含有信号序列，在C-末端含有疏水序列，并转运到细胞外膜上。C末端疏水区的缺失导致RECK分泌到培养基中。我们建立了一种将分泌型RECK的C端序列替换为组氨酸六聚体的亲和纯化方法。(4)RECK的Kazal结构域的潜在反应位点残基(P1)为Asp，提示Caspase样酶是RECK的靶酶。RECK不抑制caspase-1。它也不与颗粒酶B相互作用。