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

1.为了研究唾液半胱氨酸氨基转移酶的结构和功能关系及其治疗潜力，我们建立了一个高效表达和分泌半胱氨酸半胱氨酸氨基转移酶的大肠杆菌系统。在异丙基-β-硫代半乳糖苷诱导下，在E.ColiJM109中表达了编码胱抑素前体(141个残基)的基因S、胱抑素SA和胱抑素SN。用抗半胱氨酸半胱氨酸酶S抗体检测半胱氨酸半胱氨酸酶在大肠杆菌中的表达，并从冷渗透休克处理后的胞质中分离纯化半胱氨酸半胱氨酸酶。用气相自动Edman降解法测定重组半胱氨酸氨基转移酶的氨基酸序列。前体蛋白被翻译后分泌并组装在细胞质膜和外膜之间的空隙中，作为成熟的半胱氨酸蛋白(121个残基)。半胱氨酸氨基转移酶S信号的突变(-18R-W)显著减少了其在周质间隙中的积累。氨基(N-)末端t…的取代精氨酸使半胱氨酸蛋白SA(-18W)和半胱氨酸氨基转移酶SN(-18Q)信号中含有更多的Hird残基，促进了半胱氨酸氨基转移。Cystatin SN缺少N-末端17个残基和两种分子形式的Cystatin SA，N-末端4个残基(WSPQ)和6个残基(WSPQEE)偶尔会被提纯，这表明Cystatin SA和SN已经被大肠杆菌信号肽I酶以外的其他酶进一步进行了蛋白分解。该系统生产的重组半胱氨酸菌素对木瓜酶、无花果酶和组织蛋白酶(B、C和H)具有几乎相同的抑制特性。结论：1.重组半胱氨酸氨基转移酶S和S(117r-W)对牙龈卟啉单胞菌产生的牙周炎和牙周炎均无抑制作用，但对牙龈卟啉单胞菌的生长有抑制作用。用聚合酶链式反应方法构建了由3个外显子单元组成的嵌合基因：S1C2C3(Cystatin S基因的外显子1-Cystatin C的外显子2-Cystatin C的外显子3)、S1C2S3(Cystatin S的外显子1-Cystatin C的外显子2-Cystatin S的外显子3)和S1S2C3(Cystatin S的外显子1-Cystatin S的外显子2-Cystatin C的外显子3)。在异丙基-β-硫代半乳糖苷诱导下，这些基因在大肠杆菌JM109细胞中表达。用柱层析法从大肠杆菌周质组分中分离纯化了3种嵌合蛋白(S1C2C3、S1C2S3和S1S2C3)。两种嵌合蛋白S1C2C3和S1C2S3对木瓜酶、无花果蛋白、组织蛋白酶C和组织蛋白酶H都有很强的抑制作用。与胱抑素C相比，这两种嵌合体对组织蛋白酶B的抑制活性极低。S1C2C3和S1C2S3的抑制谱不同于胱抑素S和胱抑素C。这一结果表明，通过交换天然胱抑素的外显子单元来设计和生产具有新性质的人工胱抑素是可能的。较少

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