Recombinant Retinal Pigment Epithelium-Derived Factor: Its Purification from Overexpressing Escherichia Coli and Survival Effect on Induced Apoptosis in Cerebellar Granule Neurons

基因重組網膜色素上皮細胞因子：由大腸桿菌中選殖表現及純化和其對於小腦顆粒神經元誘導性程式化死亡之救活效應

葉則祥(Tse-Hsiang Yeh) ； 蔡長祐(Chang-Youh Tsai) ； 李淑美(Shui-Mei Lee) ； 劉榮宏(Jorn-Hon Liu) ； 吳燕琴(Yan-Qiu Wu) ； R. Theodore Fletcher ； Takayuki Taniwaki ； S. Patricia Becerra ； Joan P. Schwartz ； Gerald J. Chader

《中華民國眼科醫學會雜誌》 34卷3期 (1995/09) Pp. 293-304

https://doi.org/10.30048/ACTASOS.199509.0001

摘要

網膜色素上皮因子最初純化自胎兒網膜色素上皮細胞之培養基，而為一具神經滋養性之蛋白質。因其在培養基中含量稀少，前人乃經由人類胎兒補體DNA基因庫建立基因重組的截斷架構（天門冬素^44－普羅林^418），以基因工程技術殖入表現於大腸菌中。經由引導含有此截斷架構之載體，我們自菌體之包含體得到一預期大小(42.8kDa)的蛋白質。溶解後，此蛋白質經陽離子交換色層分析和超過濾可被高度純化且在西方墨點試驗中被色素上皮因子抗體特定檢出。於神經突生長檢驗中，這基因重組色素上皮因子以促進Y-79視網膜胚母細胞瘤細胞株之分化來表現其神經滋養活性。因此這基因重組蛋白有類似天然色素上皮細胞因子的生物活性。網膜色素上皮因子也在源自中樞神經的小腦顆粒神經元上表現神經滋養救活作用；它可明顯增加活存神經元數目。進一步，我們在以低鉀引導小腦顆粒神經元發生程式化死亡的模式中，檢驗重組色素上皮因子的作用。我們發現其可經由抑制DNA斷裂來延緩細胞的程式化死亡。有鑑於細胞程式化死亡無論於發育中的神經系統（包括視網膜）和神經退化性疾病都很常見，於該系統，尤其是視網膜，細胞程式化死亡的發生及其受存活因子所抑制的機轉的釐清就顯得非常值得。畢竟色素上皮細胞因子是感光細胞間質組成中唯一能引導Y-79細胞產生神經突分化的。

並列摘要

Pigment epithelium-derived factor (PEDF), first purified from the culture medium of retinal pigment epithelial cells of human fetus, is a neurotrophic protein. Since it is present in low amount in conditioned medium, a truncated recombinant expression construct (Asp^44-Pro^418), based on PEDF cDNA cloned from a fetal human cDNA library, has been expressed in Escherichia coil. By induction from a vector containing the truncated PEDF version, we produced a protein with expected size of 42.8kDa associated with inclusion bodies in the host. After solubilization, the protein was highly purified by cation exchange chromatography and ultrafiltration, and specifically recognized on Western transfers by polyclonal antibodies to PEDF. In neurite outgrowth assays, the recombinant PEDF (rPEDF) demonstrated neurotrophic activity by potentiating the differentiation of human Y-79 retinoblastoma cells as reported for the native PEDF. Thus, the recombinant protein retains a potent biological activity.
PEDF has shown to be a neurotrophic survival factor of the primary culture of neurons from the central nervous system, the cerebellar granule neurons (CGNs), by significantly increasing the number of viable neurons. To extend the study, we further tested the survival effect of rPEDF on a model of low K-induced apoptosis (programmed cell death) in CGN. rPEDF could delay the induced cell death by inhibiting DNA fragmentation. Given that apoptosis is extensively observed in the developing nervous system including the retina as well as in neurodegenerative diseases, it is worthwhile carrying out further studies on the underlying mechanisms of apoptosis and its suppression by survival factors such as PEDF in the nervous system, especially the retina where PEDF is the single known component in the interphotoreceptor matrix responsible for the induction of neurite outgrowth in Y-79 cells.

並列關鍵字

Pigment epithelium-derived factor PEDF ； neurotrophic factor ； apoptosis programmed cell death ； cell survival