利用昆蟲細胞－桿狀病毒或大腸桿菌表達系統生產大鼠肝臟重鏈儲鐵蛋白同質聚合體所導致其蛋白質結構與功能上的差異

Structural and Functional Differences of Recombinant Rat Liver Heavy Chain Ferritin Homopolymers Produced in Insect Cell-Baculovirus and Escherichia coli Expression Systems

郭嘉信(Jia-Hsin Guo)

《先進工程學刊》 1卷1期 (2006/07) Pp. 15-20

https://doi.org/10.29948/JAE.200607.0003

摘要

每個由含T7表達載體的大腸桿菌系統JM109(DE3)所產生之基因重組大鼠肝臟重鏈儲鐵蛋白同質聚合體(rH-Ft-Pol-E)內部已含158±8原子的鐵，但由昆蟲桿狀病毒感染昆蟲Spodoptera frugiperda細胞系統表現的同質聚合體(rH-Ft-Pol-I)則未偵測到鐵原子。兩種來源的蛋白質都是由單－21kDa的次單元體組成；然而，非變性聚丙烯醯胺凝膠的等電點電泳上，rH-Ft-Pol-E可分離出3-4種不同等電點的蛋白質種類，而rH-Ft-Pol-I 只顯示一種；rH-Ft-Pol-E的pI值與羰基含量比rH-Ft-Pol-I高。由於大腸桿菌系統在表達同質聚合體時，逕行將鐵原子裝載入儲鐵蛋白質，可能因此造成蛋白質氧化（增加羰基含量），如以圓二色光譜儀檢測，發現rH-Ft-Pol-E的二級結構秩序比rH-Ft-Pol-I低；當使用血清藍胞漿素系統將鐵原子裝載入儲鐵蛋白質時，兩者也顯示不同的速率與容量。綜括而論，昆蟲細胞－桿狀病毒系統應該比大腸桿菌系統更適合用來生產較符合生理學特性的儲鐵蛋白質聚合體。

關鍵字

蛋白質表現 ； 儲鐵蛋白同質體 ； 鐵結合 ； 血清藍胞漿素

並列摘要

Each recombinant H chain rat liver ferritin homopolymers produced in Escherichia coli JM109(DE3) using a T7 expression vector (rH-Ft-Pol-E) contained 158±8 atoms of iron, but not one expressed in Spodoptera frugiperda cells infected with a recombinant baculovirus (rH-Ft-Pol-I). Both were made up of a 21 kDa subunit; however, the rH-Ft-Pol-E resolved into 3-4 species, whereas the rH-Ft-Pol-I showed only a single species upon a nondenaturing isoelectric focusing PAGE. All of the rH-Ft-Pol-E showed relatively greater pIs and carbonyl content than the rH-Ft-Pol-I. Evidence for protein oxidation (increased carbonyl content) from iron incorporation during expression of the homopolymer in the E. coli system also resulted in a decrease in ordered secondary structures, compared with the rH-Ft-Pol-I, as analyzed by circular dichroism. Different rates and extent of iron incorporation were also found between the rH-Ft-Pol-E and the rH-Ft-Pol-I using a ceruloplasmin loading system. Taken together, these results suggested that the insect cell-baculovirus system, instead of E. coli system, should be used for expression of ferritin H chain homopolymer to obtain physiologically-relevant ferritin.

並列關鍵字

protein expression ； ferritin homopolymer ； iron incorporation ； ceruloplasmin