重組小鼠普昂蛋白質表現系統之建構與研究

Abstract

普昂蛋白質 (prion protein, PrP) 是一個藉 GPI-anchor 連接在膜上的醣蛋白質，由正常構形 (PrPC) 轉變為致病構形 (PrPSc) 的過程，被廣泛接受為引起普昂疾病的原因，但這個轉變過程仍不清楚，包括醣基化的影響以及物種屏障 (species barrier) 的成因，都未成定論。我們試著利用可進行轉譯後修飾作用的表現系統，例如進行醣基化，來表現重組 PrP。因此，本論文在 Escherichia coli 與 Pichia pastoris 中建立了重組小鼠 PrP 的表現系統。將 E. coli 中純化得到的重組蛋白質進行澱粉樣蛋白質纖維之培養。以 thioflavin T (ThT) 呈色法監控纖維的形成，再藉由穿透式電子顯微鏡 (Transmission electron microscope, TEM) 觀察纖維的形狀。基於合成胜肽系統推導出關於物種屏障的理論，而建構出三個突變蛋白質。依照金倉鼠 PrP 的序列，將小鼠重組 PrP 序列中 108, 111, 138 這三個位置的胺基酸換成 methionine。重組小鼠 PrP(23-230) 蛋白質的表現系統也同時建構在 P. pastoris 中，結果顯示由酵母菌產生的重組 PrP 也會存在不可溶的蛋白質中。

Prion protein (PrP) is a glycoprotein attached to plasma membrane via GPI-anchor. It is believed that the conversion from normal cellular PrP (PrPC) to pathogenic scrapie PrP (PrPSc) results in prion diseases. However, many issues still remained elusive, e.g. the effects of glycosylation and the factors that contributed to species barrier. We tried to express recombinant PrP in an expression system which was able to provide post-translational modifications, such as glycosylation. Therefore, recombinant wild type mouse PrP expression in Escherichia coli and Pichia pastoris were established. Recombinant wild type PrP purified from E. coli was used to form amyloid fibrils. The formation of the amyloid fibril was monitored by thioflavin T (ThT) assay. Transmission electron microscopy (TEM) was used to examine the morphology of the fibrils. Based on the theory for species barrier derived from synthetic peptide system established in previous study, three PrP(23-230) mutants were generated in an attempt to study the theory. The amino acids at positions of 108, 111, 138 in mouse PrP sequence were altered to methionine according to the PrP sequence of golden Syrian hamster. Recombinant mouse PrP(23-230) was also constructed for the expression in P. pastoris. It appeared that the recombinant PrP produced by yeast was also accumulated in the insoluble fraction.