Abstract
水稻第一型類金屬硫蛋白(OsMT1)利用pET32b(+)表現載體，成功地在大腸桿菌BL21(DE3)中以融合蛋白形式高量的表現。在SDS-PAGE上可以明顯觀察到轉殖株經1 mM isopropylthio-b-D-galactoside (IPTG) 誘發之後15分鐘後即開始出現蛋白，在2小時內隨著時間增加。用親和性色層分離法所純化的蛋白後，在原子吸收光譜中有鋅的訊號。此一融合蛋白尚待進一步的處理，以蛋白內切酉每 或化學劑將附合部分thioredoxin和Tag切除，再純化，然後測定每一個金屬硫蛋白分子是否如預期的結合六個Zn離子。至於理論上每一個水稻金屬硫蛋白分子含有12個半胱胺酸殘基，亦尚待驗證。若然，此蛋白可正式稱之為金屬硫蛋白。本論文試以Pichiapastoris為宿主來表現米酒糖化酵素。Pichia在發酵培養時，其細胞密度比一般的酵母菌高，而且會分泌表現蛋白，有便於純化。利用聚合酵素連鎖反應，將米酒糖化酵素的基因cDNA擴增，並連接在表現載體pPICZa上，再將建成的載體轉殖於Pichia(GS115)。用聚合酵素連鎖反應篩選轉殖株，以反轉錄酵素聚合酵素連鎖反應(RT-PCR)確定轉殖株細胞中出現此酵素的mRNA其序列並可確認，但是在培養液中測不到酵素及其活性，亦無此酵素蛋白的累積。若干解釋，尚待排除。Rice metallothionein-like gene(OsMT1) was highly expressed inE. coli as a fusion protein with thioredoxinIts cDNA wasgenerated from rice root cDNA library, amplified by polymerasechain reaction (PCR)and linked to thioredoxin cDNA plus a tagsequence via pET32b(+) vector. This chimeric sequence wasexpressed in E. coli BL21(DE3)after 1mM IPTG induction. A purefusion protein was obtained to a final concentration of 406 mg/ml by His-Bind chromotography and its yield was 609mg/100mlculture. The gene product of OsMT1 is characterized to be zinc-binding and cysteine-rich, the notation used heretofore asmetallothionein-like can be officialized as metallothionein.The expression and secretion of Rhizopus oryzae glucoamylase(GA) were studied in the yeast Pichia pastoris, a high levelexpression host for recombinant protein. The cDNA of Rhizopusoryzae glucoamylase was placed under the control of P. pastorisalcohol oxidase (AOX1) promoter. A transformant integrated witha single copy expression cassette to the chromosome produced nodemonstratable glucoamylase when the cells were grown withmethanol as the carbon source. However, mRNA of glucoamylasecould be detected as amplified by RT-PCR. Cause for thisfailure is discussed.