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

• 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

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.为了研究唾液半胱氨酸蛋白酶抑制剂的结构和功能关系以及治疗潜力，我们建立了能够高水平表达和分泌半胱氨酸蛋白酶抑制剂的大肠杆菌系统。在异丙基-β-硫代半乳糖苷诱导下，编码cystatin S、cystatin SA和cystatin SN的前体（141个残基）的cDNA在大肠杆菌JM 109中表达。大肠杆菌细胞中表达的半胱氨酸蛋白酶抑制剂通过抗半胱氨酸蛋白酶抑制剂S抗体检测，并从冷休克处理制备的周质组分中纯化。重组半胱氨酸蛋白酶抑制剂的氨基酸序列通过自动气相Edman降解测定。前体蛋白在大肠杆菌细胞内分泌后，在细胞质膜和外膜之间组装成成熟的半胱氨酸蛋白酶抑制剂（121个残基）。半胱氨酸蛋白酶抑制剂S信号中的突变（-18 R-W）显著减少了其在周质间隙中的积累。氨基（N-）末端的替换 ...更多信息 CystatinSA（-18W）和CystatinSN（-18Q）信号的第三个残基通过精氨酸促进Cystatins的跨膜转运。缺失N-末端17个残基的半胱氨酸蛋白酶抑制剂SN和缺失N-末端4个残基的半胱氨酸蛋白酶抑制剂SA（WSPQ）和缺失N-末端6个残基的半胱氨酸蛋白酶抑制剂SA（WSPQEE）的两种分子形式偶尔被纯化，这表明半胱氨酸蛋白酶抑制剂SA和SN已经被大肠杆菌的蛋白酶而不是信号肽酶I进一步蛋白水解。由该系统产生的重组半胱氨酸蛋白酶抑制剂对木瓜蛋白酶、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。重组半胱氨酸蛋白酶抑制剂S和S（117 R-W）对牙龈卟啉单胞菌的Arg-牙龈菌蛋白酶和Lysgingipain无抑制作用，但对牙龈卟啉单胞菌的生长有抑制作用. CystatinS和CystatinC基因的三个外显子单元组成的嵌合基因S1 C2 C3（胱抑素S的外显子1-胱抑素C的外显子2-胱抑素C的外显子3），S1C2S3（胱抑素S的外显子1-胱抑素C的外显子2-胱抑素S的外显子3）和S1 S2 C3（胱抑素S的外显子1-胱抑素S的外显子2-胱抑素C的外显子3）。在异丙基-β-硫代半乳糖苷诱导下，在大肠杆菌JM 109细胞中表达基因。三个嵌合蛋白（S1 C2 C3，S1 C2 S3和S1 S2 C3）通过柱层析从大肠杆菌的周质组分中纯化。发现两种嵌合蛋白S1 C2 C3和S1 C2 S3是木瓜蛋白酶，无花果蛋白酶，嵌合体对组织蛋白酶B的抑制活性极低，而对cystatin C的抑制活性较低。S1 C2 C3和S1 C2 S3的抑制谱与cystatin S和cystatin C的抑制谱不同。通过交换天然半胱氨酸蛋白酶抑制剂的外显子单元而获得具有新性质的半胱氨酸蛋白酶抑制剂。少

项目成果

E. Saitoh and S. Isemura: "Proteases involved in cancer" Michiya Suzuki and Takaki Hiwasa, 5 (1995)

E. Saitoh 和 S. Isemura：“与癌症有关的蛋白酶” Michiya Suzuki 和 Takaki Hiwasa，5 (1995)

Satoko Isemura: "Inhibitory activities of partially degraded salivary cystatins." Int.J.Biochem.Vol.26, No.2. (1994)

Satoko Isemura：“部分降解的唾液胱抑素的抑制活性。”

Eiichi Saitoh: "Production, purification and partial characterization of recombinant human salivary type cystatins." Proteases Involved in Cancer (M.Suzuki and T.Hiwasa, eds). 171-175 (1995)

Eiichi Saitoh：“重组人唾液型胱抑素的生产、纯化和部分表征。”

Masuro Shintani et.al.: "Genetic Polymorphisms of CST2 Locus Coding for Cystatin SA" Human Genetics. 94. 41-45 (1994)

Masuro Shintani 等人：“半胱氨酸蛋白酶抑制剂 SA 的 CST2 位点编码的遗传多态性”人类遗传学。

Eiichi Saitoh and Satoko Isemura: "Proceedings of Chiba International Symposium on Cancer" Takaki Hiwasa(in press), (1995)

Eiichi Saitoh 和 Satoko Isemura：《千叶国际癌症研讨会论文集》Takaki Hiwasa（印刷中），（1995 年）