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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. 为了研究唾液胱他汀的结构和功能关系以及治疗潜力，我们建立了一个高水平表达和分泌胱他汀的大肠杆菌系统。利用异丙基- β -硫代半乳糖苷诱导，在大肠杆菌JM109中表达了胱抑素S、胱抑素SA和胱抑素SN前体（141个残基）的cdna。用抗胱抑素S抗体检测大肠杆菌细胞中表达的胱抑素，并从冷渗透休克制备的质周组分中纯化。采用自动气相Edman降解法测定重组胱他汀类药物的氨基酸序列。前体蛋白在大肠杆菌胞质膜和外膜之间的空间中作为成熟的胱他汀类药物（121个残基）分泌和组装。胱抑素S信号的突变（- 18r - W）显著减少了其在质周间隙的积累。胱抑素SA （- 18w）和胱抑素SN （- 18q）信号的第三个残基被精氨酸取代，促进了胱抑素在细胞质膜上的易位。胱抑素SN缺失n端17个残基，胱抑素SA缺失两种分子形式，其中n端4个残基（WSPQ）和6个残基（WSPQEE）偶有纯化，提示胱抑素SA和胱抑素SN被大肠杆菌信号肽酶I以外的蛋白酶进一步水解。该系统产生的重组胱抑素对木瓜蛋白酶、无花果蛋白酶和组织蛋白酶（B、C和H）表现出几乎相同的抑制特性。重组胱抑素S和S （117R-W）对牙龈卟啉单胞菌Arg-gingipain和Lysgingipain没有抑制作用，但对牙龈卟啉单胞菌p .gingivalis的生长有抑制作用。用聚合酶链式反应（PCR）合成由胱抑素S和胱抑素C基因的3个外显子单元组成的嵌合基因，分别为S1C2C3（胱抑素S外显子1 -胱抑素C外显子2 -胱抑素C外显子3）、S1C2S3（胱抑素S外显子1 -胱抑素C外显子2 -胱抑素S外显子3）和S1S2C3（胱抑素S外显子1 -胱抑素S外显子2 -胱抑素C外显子3）。这些基因通过异丙基- β -硫代半乳糖苷诱导在大肠杆菌JM109细胞中表达。用柱层析法从大肠杆菌的质周组分中纯化了3种嵌合蛋白（S1C2C3、S1C2S3和S1S2C3）。两种嵌合蛋白S1C2C3和S1C2S3对木瓜蛋白酶、ficin、组织蛋白酶C和组织蛋白酶h具有较强的抑制作用，对组织蛋白酶B的抑制活性与胱抑素C相比极低，抑制谱与胱抑素S和胱抑素C的抑制谱不同，提示通过交换天然胱抑素的外显子单元，设计和制备具有新性能的人造胱抑素是可能的。少