Abstract
細胞骨骼中間絲蛋白 (cytoskeletal intermediate filament) 的氮端功能區部份在絲狀結構的形成與網狀系統的建立中扮演了很重要的角色, 而且它包含了多個磷酸激脢作用的位置 (phosphorylation site),先前的報導指出: 在細胞受到熱休克刺激 (heat shock) 時, 中間絲蛋白的磷酸化會明顯增加, 為了得知氮端功能區的結構形態, 以及證明是否其結構在經歷熱休克處理時會產生巨大變化而造成磷酸化的增加, 在本研究中, 選殖出中間絲蛋白氮端功能區的基因並在大腸桿菌中進行大量的異體表現 (heterologous expression), 所表現出來的蛋白質是包含了硫氧還蛋白 (thioredoxin) 的融合蛋白 (fusion protein), 分子量為 33,279道爾頓 (dalton), 選殖的蛋白經過組氨酸親和色譜層析法 (His-Bindaffinity chromatography) 的純化並以西式墨點法 (Western blotting)確認. 但因為嚴重蛋白質水解 (proteolysis) 的問題, 未能取得純化的氮端功能區, 因此轉而以整個融合蛋白的旋光圖譜 (circular dichorismspectra) 來分析其結構變化, 然而在圖譜中並沒有觀察到所預期因溫度變化而產生的結構變動, 所以蛋白質水解的問題仍須解決, 才能得到中間絲蛋白的氮端功能區, 並取得其結構資訊而對其功能有更進一步的了解.The head domains of cytoskeletal intermediate filaments (IFs)are considered to play an important role in IF assembly andnetwork formation. Previously, it has been shown that the headdomain of vimentin is the major target site of phosphorylation,and the degree of phosphorylation is increased in cellsexperiencing heat shock. In order to investigate theconformation of the head domain of vimentin and obtain firmlyevidence of the temperature-related conformation change, thehead domain has been cloned and heterologously expressed in E.coli by pET32/BL21(DE3) expression system. The resultingprotein contains a thioredoxin fusion which can increase thesolubility of the target protein and has a calculated molecularweight 33,279 dalton. The cloned protein has been purified byHis-Bond affinity column and identified by Western blotting.Protein degradation during the purification process made thedesired pure head domain difficult to achieve. The conformationof the fusion protein containing thioredoxin with head domainvimentin, instead of the pure head domain, was analyzed bycircular dichorism. However, the expected temperature-relatedconformation change was not observed clearly in the CD spectrumof the fusion protein. The problem of proteolysis should stillbe resolved to achieve the exact conformation of the headdomain.