在大腸桿菌表現水稻第一族低分子量熱休克蛋白質之生理功能探討

Abstract

本研究以Oshsp16.9 cDNA為材料建構四轉殖質體，分別為pUC（只含pUC8表現載體）、pUC-FL（pUC8表現載體可表現全長的水稻第一族低分子量熱休克蛋白質Oshsp16.9 cDNA）、pUC-C108（pUC8表現載體可表現Oshsp16.9 cDNA C端108個胺基酸的DNA片段）及pUC-N78（pUC8表現載體可表現Oshsp16.9 cDNA N端78個胺基酸的DNA片段）。經轉型至JM-109型大腸桿菌後，其中pUC-FL及pUC-C108均能穩定的經IPTG誘導而表現外來蛋白質且能被Oshsp16.9抗體所辨識，但是pUC-N78卻無法表現。pUC-FL及pUC-C108二轉殖大腸桿菌於自然狀態下均會聚合形成高分子複合體，而其中pUC-FL的複合體經電子顯微鏡觀察為直徑約18?35 nm的球體狀構造，估計分子量界於220?240-kDa間並含有約12個Oshsp16.9單元體。 轉殖大腸桿菌在經IPTG誘導後於50℃的熱耐性測定，結果顯示pUC-FL細胞在高溫下的存活率較pUC-C108細胞的存活率為高，而若以高溫存活率半衰期比較，分別為70分鐘及22分鐘。大腸桿菌總蛋白質於55℃熱處理30分鐘的蛋白質熱穩定性測定，結果顯示pUC-FL轉殖大腸桿菌在經IPTG誘導後的總蛋白質熱變性程度可由43％降至18％，而JM-109、pUC及pUC-C108細胞於IPTG誘導前後的總蛋白質熱變性程度卻一直維持在50％左右。為尋找Oshsp16.9熱休克蛋白質在大腸桿菌中可能保護的對象及探討交互作用的型式，因此利用免疫沉澱的技術進行分析。結果顯示經IPTG誘導的pUC-FL細胞總蛋白質經45℃熱處理15分鐘後，其中有15個特殊蛋白質條帶與Oshsp16.9共同沉澱下來；而這些蛋白質均不會出現在未加熱處理的pUC-FL細胞總蛋白質之免疫沉澱分析結果中，至於這些蛋白質與Oshsp16.9的相互關係，則有待進一步研究。

Four constructed plasmids were transformed into E. coli, including pUC (contains pUC8 vector only), pUC-FL (rice class I low molecular mass heat shock protein Oshsp16.9 cDNA ORF inserted into pUC8), pUC-C108 (DNA fragment containing C-terminal 108 amino acids of Oshsp16.9 cDNA ORF inserted into pUC8) and pUC-N78 (DNA fragment containing N-terminal 78 amino acids of Oshsp16.9 cDNA ORF inserted into pUC8). Foreign proteins expressed in pUC-FL and pUC-C108 cells with IPTG induction is stable and could be recognized by Oshspl6.9 antibody. Both foreign proteins expressed in pUC-FL and pUC-C108 cells formed high molecular weight native complexes. The native complex of pUC-FL cell had a globular form examined by electron microscopy, with a diameter between 18?35 nm and a molecular weight range of 220?240-kDa, with approximately 12 subunits. Studies on cell thermotolerance of transformants that were induced by IPTG at 50℃ showed that pUC-FL cells have a higher survivability compared to that of pUC-C108 cells, with LT50 of 70 minutes and 22 minutes, respectively. Studies on protein thermostability induced by IPTG showed that only 18% of pUC-FL total protein denatured at 55℃ for 30 minutes incubation. While the total protein of JM-109, pUC-C108, and pUC cells denatured up to 50% with or without IPTG induction. We used the immunoprecipitation technique to find whether Oshsp16.9 will interact with E. coli proteins under those stress conditions. Our results showed that, among the pUC-FL cell total proteins which were incubated at 45℃ for 15 minutes, 15 proteins co-precipitated with Oshspl6.9. These proteins did not co-precipitated with Oshspl6.9 without heat treatment. The interaction of the Oshsp16.9 and those co-precipitated proteins requires further investigation.