溫度與溶氧量對不同亞硫酸基轉移酵素在大腸桿菌體大量表達的影響

Abstract

鼠肝亞硫酸基轉移酵素基因經載入於大腸桿菌，大量表達後，源同一個 cDNA可得到兩種不同型態的酵素。酵素的活性與兩型酵素表達型態的比例 ，受整個大腸桿菌培養條件影響。β型酵素在低溶氧量的情況下（37℃ ，100rpm）表達，最高比例可達約68%，而α型酵素的活性在總產量計算 下，變化較少。因為只有α型酵素含有PAP，我們推測PAP產量和酵素的結 合性可能受到細菌生長速度及溫度的調控，進而影響到酵素的活性。為探 討PAP與酵素結合的關係及如何影響到酵素的表達，我們將K65ER68G與C66 S兩個突變株做了跟野生型相同的培養。在K65ER68G亞硫酸基轉移酵素大 量表達後，得到了完全的β型酵素，所以推測在此序列中確實為與PAP結 合的區域，而溫度越高，表現的總活性也提昇；另一個突變種C66S亞硫酸 基轉移酵素，則得到大部份的α型酵素，但依然有部份β型酵素的存在， 所以與PAP關係不明確。純化後的蛋白質，以SDS-PAGE電泳分析，分子量 大小與野生株相同，以Native-PAGE的電泳分析，顯示出在蛋白質構型上 ，突變種K65ER68G亞硫酸基轉移酵素跟野生種有差異性，而推測溝型上的 改變對輔因子PAP會有不的結合方式，而C66S亞硫酸基轉移酵素構型上相 似於α型亞硫酸基轉移酵素。我們偵測了其對各種輔因子與受質的Km和 Vmax值；在轉移反應方面：對PAP的Km，K65ER68G比野生種高500倍，表示 對PAP的親和力降低，而對於整個催化反應上，會較易對PAPS有較大的結 合性，而將亞硫酸基轉移給受質；而C66S大都為α型亞硫酸基轉移酵素， 己含有足量PAP。而對受質PNPS和2-naphthol值則跟野生種相近；而在生 理反應方面：K65ER68G在PAP的Km上和野生種相近，而對PNPS的Km值，K65 ER68G與C66S較野生種大，約為15倍與30倍，Vmax值相似。 Two forms recombinant sulfotransferase were obtained after a single cDNA being expressed in Escherischia coli (E. coli)[8]. We have found that condition of cell growth affects both the ratio of the two forms of enzymes and the amount of total enzyme activity. Lower oxygen supply produces higher ratio of β- enzyme. At 37℃, 68% and 18% ofβ form of recombinant sulfotransferase were obtained at 100rpm and 300rpm, respectively. Total amount of α form activity is relatively stable at different level of oxygen supply. We sus To study the effect of PAP binding on the formation of the two forms of recombinant enzyme, two mutants, K65ER68G and C66S, have been expressed at the same condition as that of wild type in E. coli. The region of mutation are known important for PAP binding. Km and Vmax of mutants have been determined K65ER68G mutant give an unusually high Km(PAP) and unable to bind PAP tightly as that of wild type. On the contrary, C66S give mainly α-form enzyme which contains a tightly bind PAP.