Becerra S P, Palmer I, Kumar A, Steele F, Shiloach J, Notario V, Chader G J
Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 1993 Nov 5;268(31):23148-56.
Pigment epithelium-derived factor (PEDF) is a neurotrophic protein present in low amounts in conditioned medium of cultured fetal human retinal pigment epithelial cells. Recently, the PEDF cDNA has been cloned from a fetal human cDNA library, and its derived amino acid sequence identified it as a member of the serine protease inhibitor (serpin) supergene family (Steele, F. R., Chader, G. J., Johnson, L. V., and Tombran-Tink, J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 1526-1530). We have prepared recombinant expression constructs from the fetal human PEDF cDNA and obtained milligram amounts of biologically active PEDF from Escherichia coli. The full-length open reading frame (Met1-Pro418) and a truncated form (Asp44-Pro418) were used in our constructs. Induction from a vector containing the truncated PEDF version, named pEV-BH, produced a protein (BH) of expected size (M(r) 42,800) associated with inclusion bodies, which contained 25-40% of expressed protein. After solubilization, BH was highly purified by gel filtration and cation exchange chromatography. The NH2-terminal sequence of the purified protein matched that of the pEV-BH construct. We have conducted neurite outgrowth assays in a human retinoblastoma Y-79 cell culture system. Recombinant PEDF (BH) demonstrated neurotrophic activity, as reported for the native PEDF. Thus, unfolded and refolded in vitro BH retained a potent biological activity. In parallel experiments, protease inhibition assays were performed. Recombinant PEDF did not have an effect on trypsin, chymotrypsin, elastase, cathepsin G, endoproteinase Lys-C, endoproteinase Glu-C, or subtilisin activity, suggesting that inhibition of known serine proteases is not the biochemical pathway for the PEDF neutrophic activity.
色素上皮衍生因子（PEDF）是一种神经营养蛋白，在培养的人胎儿视网膜色素上皮细胞的条件培养基中含量较低。最近，PEDF cDNA已从人胎儿cDNA文库中克隆出来，其推导的氨基酸序列将其鉴定为丝氨酸蛋白酶抑制剂（serpin）超基因家族的成员（斯蒂尔，F.R.，查德，G.J.，约翰逊，L.V.，和汤布兰 - 廷克，J.（1993年）美国国家科学院院刊90，1526 - 1530）。我们从人胎儿PEDF cDNA制备了重组表达构建体，并从大肠杆菌中获得了毫克量的生物活性PEDF。我们的构建体中使用了全长开放阅读框（Met1 - Pro418）和截短形式（Asp44 - Pro418）。从含有截短的PEDF版本（名为pEV - BH）的载体诱导产生了预期大小（M(r) 42,800）的蛋白质（BH），其与包含体相关，包含体中含有25 - 40%的表达蛋白。溶解后，BH通过凝胶过滤和阳离子交换色谱法进行了高度纯化。纯化蛋白的NH2末端序列与pEV - BH构建体的序列匹配。我们在人视网膜母细胞瘤Y - 79细胞培养系统中进行了神经突生长试验。重组PEDF（BH）表现出神经营养活性，正如天然PEDF所报道的那样。因此，在体外展开和重折叠的BH保留了强大的生物活性。在平行实验中，进行了蛋白酶抑制试验。重组PEDF对胰蛋白酶、胰凝乳蛋白酶、弹性蛋白酶、组织蛋白酶G、内肽酶Lys - C、内肽酶Glu - C或枯草杆菌蛋白酶活性没有影响，这表明抑制已知的丝氨酸蛋白酶不是PEDF神经营养活性的生化途径。
