A study on large scale production, improvement of function, and utilization of recombinant cystatins

重组半胱氨酸蛋白酶抑制剂的规模化生产、功能改良及利用研究

基本信息

批准号：
06671872
负责人：
SAITOH Eiichi
金额：
$ 1.34万
依托单位：
The Nippon Dental University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06671872/
关键词：
Human Salivary Cystatin Cysteine Proteinase Inhibitor Periodontal Disease Chimeric Gene PCR (Polymerase Chain Reaction)Gingipain Recombinant cystatins Design and Production of Cystatin Molecules シスタチンカテプシンリコンビナントプロテイン PCR増幅キメラシ

项目摘要

1. To study the structural and functional relationship and the therapeutic potentials of salivary cystatins, we have established an E.coli system enabling a high level of expression and secretion of cystatins. The cDNAs encoding the precursors (141 residues) of cystatin S,cystatin SA and cystatin SN were expressed in E.coli JM109 with isopropyl-beta-thiogalactoside induction. The cystatins expressed in E.coli cells were detected by anti-cystatin S antibody and purified from the periplasmic fractions prepared by cold osmotic-shock treatment. The amino acid sequences of recombinant cystatins were determined by automated gas-phase Edman degradation. The precursor proteins were post-translationally secreted and assembled in the space between cytoplasmic membrane and outer membrane of E.coli cells as the mature cystatins (121 residues). A mutation (-18R - W) in the signal of cystatin S reduced its accumulation in the periplasmic space remarkably. The replacement of the amino (N-) terminal t … More hird residue of the signal of cystatin SA (-18W) and of cystatin SN (-18Q) by arginine facilitated the translocation of the cystatins across the cytoplasmic membrane. Cystatin SN lacking the N-terminal 17 residues and two molecular forms of cystatin SA lacking, respectively, the N-terminal 4 residues (WSPQ) and 6 residues (WSPQEE) were occasionally purified, suggesting that cystatins SA and SN have been under-gone further proteolysis by proteinases other than signal peptidase I of E.coli. Recombinant cystatins produced by the system showed virtually the same inhibitory properties for papain, ficin, and cathepsins (B,C,and H). Recombinant cystatins S and S (117R-W) did not inhibit Arg-gingipain or Lysgingipain from Porphyromonas gingivalis, however, the recombinant proteins inhibited the growth of P.gingivalis.2. The chimeric genes consisting of three exonic units from the genes of cystatin S and cystatin C,S1C2C3 (exon 1 of cystatin S - exon 2 of cystatin C - exon 3 of cystatin C), S1C2S3 (exon 1 of cystatin S - exon 2 of cystatin C - exon 3 of cystatin S) and S1S2C3 (exon 1 of cystatin S - exon 2 of cystatin S - exon 3 of cystatin C) were made by the polymerase chain reaction (PCR). The genes were expressed in E.coli JM109 cells with isopropyl-beta-thiogalactoside induction. Three chimeric proteins (S1C2C3, S1C2S3 and S1S2C3) were purified by column chromatography from periplasmic fractions of E.coli. Two chimeric proteins, S1C2C3 and S1C2S3, were found to be strong inhibitors for papain, ficin, cathepsin C and cathepsin H.The inhibitory activity of the chimeras for cathepsin B was extremely low level to compare with that of cystatin C.The inhibitory spectrums of S1C2C3 and S1C2S3 are different from those of cystatin S and cystatin C.The findings suggest that it is possible to design and produce artificial cystatins with new properties by exchanging exonic units of natural cystatins. Less

1。为了研究唾液囊蛋白的结构和功能关系以及唾液性囊肿的治疗潜力，我们建立了一个大肠杆菌系统，实现了伴半胱氨酸蛋白酶的高度表达和分泌。用异丙利-Beta-Beta-thioogalactoside诱导在大肠杆菌JM109中表达了编码抑制蛋白S，半胱氨酸蛋白酶SA和胱抑素SN的前体（141个保留）的cDNA。通过抗囊蛋白抗体检测到在大肠杆菌细胞中表达的囊肿，并从通过冷渗透浪冲处理制备的周质级分中纯化。通过自动气相EDMAN降解确定重组半胱氨酸蛋白酶的氨基酸序列。前体蛋白在后分泌后分泌，并在大肠杆菌细胞的细胞质膜和外膜之间的空间中聚集，作为成熟的囊蛋白（121个残留物）。胱抑素S信号中的突变（-18r -w）显着降低了其在周质空间中的积累。用精氨酸替换了伴半胱氨酸蛋白SA（-18W）和胱抑素SN（-18q）信号的氨基（N-）末端T…通过精氨酸的信号保留了更多的Hirt hirt tentiewention。 Cystatin SN缺乏N末端17个残留物和两种分子形式的Cystatin SA缺乏分别缺乏N端4残留物（WSPQ）（WSPQ）和6个残留物（WSPQEE）（WSPQEE）（WSPQEE），这表明cystatins sa和sn的蛋白酶蛋白酶蛋白酶均被其他信号pepteli e E. coli o.Coli I.Coli I.Coli of E.Coli提供了不足的蛋白质解。由系统产生的重组囊蛋白显示出对木瓜蛋白酶，乳脂蛋白和组织蛋白酶（B，C和H）的抑制作用几乎相同的抑制特性。重组囊蛋白S和S（117R-W）并未抑制卟啉单胞菌中的Arg-gyipain或lysgipain，但是，重组蛋白抑制了gingivalis.2的生长。嵌合基因由三个外显蛋白S和胱抑素C，S1C2C3的基因组成的外显子单位（伴半胱氨酸蛋白酶C）的Cystatin c-外显子3的外显子2-外显子3的基因组成，S1C2S3，cystatin s -Exont c -exstatin C -exstat c -extin 1 cystatin c -existin c -existin c -existin c -existin c -existin c -exins c -exins c -exins c cyst and cystr c Cyson c cys 2 cyst andon c Cys cys 2 cyson的1 cys 2 cys 2 cys 2 cys 2胱抑素S-外显蛋白C的外显蛋白S-外显子C）由聚合酶链反应（PCR）制成。这些基因在E.Coli JM109细胞中用异丙基-Beta-thiogalactoside诱导表达。三种嵌合蛋白（S1C2C3，S1C2S3和S1S2C3）通过圆柱色谱法纯化了E.Coli的周质级分。发现两种嵌合蛋白S1C2C3和S1C2S3是对木瓜蛋白酶，菲环蛋白，组织蛋白酶C和组织蛋白酶H的强抑制剂。嵌合体对组织蛋白酶B的抑制作用非常低，与c.s1c2c3和s1c2c3和s1c2c3的抑制光谱相比，与c.的抑制作用非常低。 Cystatin C.研究结果表明，可以通过交换天然胱抑素的外显子单元来设计和生产具有新特性的人造胱抑素。较少的