Sporamin在酵母菌表現系統中的生化特性及活性之研究

Abstract

本實驗將全長的甘藷sporamin A cDNA (TIA)序列接入酵母菌表現載體pYE8內，送入酵母菌KC1(Mata trp1 pep4)中，由GAPDH?動子來驅動TIA持續表現sporamin-related polypeptides。在帶有TIA::pYE8的酵母菌轉型株中，會合成二個分子量各為30.5kD及29.5kD的sporamin-related polypeptides。由於sporamin-related polypeptides和sporamin一樣具有trypsin inhibitor活性，所以可利用trypsin-sepharose 4B管柱進行親和管柱層析法來純化sporamin-related polypeptides。將純化出的sporamin-related polypeptides進一步作其N端胺基酸序列分析，可得知其N端胺基酸序列為LPTTHEPASSETP，相當於先驅蛋白質（preprosporamin）的第30個到第42個的胺基酸序列，此結果證實這些sporamin-related polypeptides在酵母菌轉型株中合成後需經processing的過程；同時，經由DIG Glycan Detection Kit的檢測，可發現在酵母菌轉型株合成的30.5kD sporamin-related polypeptide有醣基化（glycosylation）的現象。若用化學活性分析來比較酵母菌轉型株合成的sporamin-related polypeptides和大腸桿菌轉型株合成的GST-sporamin融合蛋白質所具的trypsin inhibitor活性強弱時，前者的trypsin inhibitor活性較後者強，推測可能是酵母菌表現的sporamin-related polypeptides經過醣基化後，有提供蛋白質活性的效果。

A full-length sporamin A cDNA (TIA) fragment was ligated into an expression vector pYE8, and transformed into yeast KC1 strain (Mata trp1 pep4) to constitutively express sporamin-related polypeptides under the control of GAPDH promotor. The transformed yeast cells containing TIA::pYE8 vector produced two sporamin-related polypeptides with M.r. of 30.5kD and 29.5kD respectively, which were proved having trypsin inhibitory activity. The sporamin-related polypeptides were purified through trypsin-sepharose 4B affiny chomatography. Sequencing results of these proteins revealed that the N terminal 13 residues of both proteins were LPTTHEPASSETP, which was corresponding to the 30th to 42nd amino acid residues of preprosporamin. These results indicated that these sporamin-related polypeptides were under post-translational processing in the transformed yeast cells. The 30.5kD sporamin-related polypeptide was shown to be glycosylated using DIG Glycan Detection Kit. Interestingly, it was found that the trypsin inhibitory activities of these sporamin-related polypeptides generated in transformed yeast cells were obviously higher than the fusion protein generated in transformed E. coli cells. Therefore, it was postulated that glycosylation could be a factor affecting the trypsin inhibitory activity of sporamin.