- 期刊
水稻懸浮細胞缺糖處理之細胞內及細胞外的蛋白酶活性分析
Characterization of the Proteases Activities in Rice Suspension Cultured Cells and Medium during Sucrose Starvation
摘要
已知利用水稻α-amylase基因啟動子受細胞缺糖處理時大量誘導表現的特性，來生產外源蛋白質並分泌到培養基中，證明是優良的外源蛋白質表現系統之一；但缺糖的水稻細胞內或培養基中，是否也會誘導某一特定的蛋白酶產生，因而減少外源蛋白質的產量，乃本研究之探討目的。為瞭解水稻懸浮培養細胞，細胞內及分泌至培養基中的蛋白酶表現，將水稻懸浮細胞培養於含糖(+S)及缺糖(-S)培養基1-5d，收集細胞及液體培養基後萃取蛋白質進行電泳分析。發現含糖培養細胞的總蛋白質含量比缺糖培養細胞多，且含糖及缺糖培養細胞均有30 kDa及37kDa二群主要的蛋白質出現；另外，缺糖處理的細胞在培養2天後，開始誘導一群外泌性的45 kDa蛋白質表現，且隨著培養天數的增加而增加表現量；在1-2天內則短暫性的誘導90 kDa蛋白質表現。經由膠體內蛋白酶活性分析結果，含糖培養的細胞內幾乎無法偵測到明顯的蛋白酶活性的表現，而培養基中則有少許的50、60及110 kDa的蛋白酶活性表現。缺糖培養的細胞內則會大量誘導一群60-70 kDa的蛋白酶活性表現，而分泌至培養基則是屬於一群約110 kDa的蛋白酶。利用不同蛋白酶抑制劑的分析結果，發現處理E-64及Leupeptin能抑制缺糖細胞誘導蛋白酶活性的表現，而DTPA則能抑制分泌至培養基之蛋白酶活性，因此推測缺糖培養的細胞中誘導表現的這群60-70 kDa蛋白酶屬於半胱胺酸蛋白酶，分泌至培養基中的110kDa蛋白酶則屬於另外一種蛋白酶，金屬蛋白酶。
並列摘要
It has been known that rice suspension cell culture was one of the powerful systems for production of a large amount of recombinant proteins, under the control of α-amylase gene promoter induced by sucrose starvation. In this study, our aim is to identify if there resides a specific protease to be induced accumulation in rice cells or secreted into medium during sucrose depletion, resulting in reduction of foreign proteins production. In order to verify the protease expression in rice cultured cells and in the medium, after rice cells were alternately grown in sucrose-containing (+S) or sucrose-free (-S) medium for 1 to 5 d, total proteins were extracted from the cultured cells or collected directly from the medium. When cells were cultured in +S medium, the expression levels of total proteins were higher than those in -S medium. There were 2 groups of secretoy proteins, 30 and 37 kDa, detected in +S and -S media. Moreover, a 45 kDa protein was induced after 2 d of sucrose starvation, and gradually increased with incubation time. A 90 kDa was also induced in 1-2 d in the -S medium, but not in +S medium. Based on the results of in-gel protease activity assay, there was barely detectable activity in +S cells. However, few protease activities were detected at the positions of 50, 60 and 110 kDa in +S medium. When cells were cultured in -S medium, a significant protease activities were induced with molecular mass of approximately 60-70 kDa in cells and 110 kDa in the medium, and were found inhibited by E-64, Leu and DTPA, respectively. Based on protease inhibitors analyzses, results suggest that the expression of protease activities reside at 60-70 kDa in the -S cells induced by sucrose starvation is cysteine proteinase, and the protein with 110 kDa that secreted into the -S medium is another type of protease, metalloproteinase.