SUMO蛋白酶Ulp1的固定化及其應用研究
發(fā)布時間：2018-08-05 19:32
【摘要】：SUMO融合表達系統(tǒng)廣泛應用于實驗室表達那些易于錯誤折疊、難溶或有毒性的蛋白,可以得到很好的表達效果。6*His是目前實驗室最為廣泛使用的融合標簽之一,但是由于其分子量很小,容易被目的蛋白包裹,若將SUMO標簽置于6*His與目的蛋白之間,可以很好的排除這種情況,使得掛柱效率大幅度提升。但是,某些分子量較小的蛋白由于大標簽SUMO的引入可能導致其功能喪失。SUMO蛋白酶Ulp1可以很好的解決這個問題,通過識別SUMO的三級結(jié)構(gòu),切割SUMO羧基端的GG-,從而得到目的蛋白,因此不會留下任何氨基酸殘基,也不會影響對目的蛋白進一步的研究。但是商業(yè)化的Ulp1價格昂貴,也有的實驗室自己表達純化Ulp1,我們發(fā)現(xiàn)Ulp1在經(jīng)過反復凍融之后很容易失活,即使長期保存在-80℃冰箱中,也存在失活的情況,從而大大增加了純化的人力與物力。本研究擬通過共價結(jié)合固定的方法,提高Ulp1的穩(wěn)定性,擴大它的應用范圍。本文主要從以下幾個方面展開研究工作:(1)在前人的研究中我們了解到Ulp1的活性位點是580位的半胱氨酸和514位的組氨酸,而賴氨酸殘基并不在它的活性位點上,因為我們考慮通過賴氨酸殘基或者是氨基末端將其固定在活性載體上。為此,我們制備了可靠的溴化氰活化的瓊脂糖,醛基化的瓊脂糖和N-羥基琥珀酰亞胺活化的瓊脂糖,并檢驗了三種beads可以很好的用來交聯(lián)蛋白。(2)在溴化氰活化的瓊脂糖,醛基化的瓊脂糖和N-羥基琥珀酰亞胺活化的瓊脂糖均可以有效的用來固定化蛋白前提下,我們對這三種beads固定化Ulp1蛋白做了研究,發(fā)現(xiàn)只有N-羥基琥珀酰亞胺活化的瓊脂糖可以在有效地共價結(jié)合Ulp1的同時不影響其活性,通過用BSA做標樣,BCA定量法和考馬斯亮藍法測定了固定化效率為1.68 mg/mL。(3)對固定化Ulp1的酶學性質(zhì)進行研究,結(jié)果表明:固定化Ulp1的pH適用范圍廣泛,可以從4.5-11.0,在11.0的時候,酶活略有減弱,和游離Ulp1基本相似。由于酶切體系是液體,固定化Ulp1是固態(tài),因此不利于混勻,為此在做切割速率試驗時,加入了游離酶3倍酶量的固定化酶,結(jié)果表明它們的切割速率一致,所以在使用的時候為了提高切割效率,可以通過增加固定化酶的用量。在對于小分子耐受試驗中,發(fā)現(xiàn)固定化和游離的Ulp1均可以耐受800 m M的氯化鈉、1 M的尿素、100 mM的二硫蘇糖醇、400 m M的咪唑,對于變性劑十二烷基硫酸鈉均不耐受。此外檢測了Ulp1對有機試劑的耐受,發(fā)現(xiàn)20%的乙醇均不影響固定化和游離Ulp1的活性,而15%DMSO會導致游離Ulp1失活,但不影響固定化Ulp1的活性。(4)對固定化Ulp1的穩(wěn)定性研究,結(jié)果表明:固定化Ulp1在熱穩(wěn)定性上得到了顯著的提高,在37℃處理24小時后,游離Ulp1已經(jīng)完全失活,但是固定化Ulp1仍有97%的活性;而30℃處理72小時后,游離Ulp1已經(jīng)完全失活,但并沒有降低固定化Ulp1的活性;同樣的25℃處理14天,固定化Ulp1仍有93%的活性,在4℃條件下保存50天,仍有活性。當用極端pH去處理Ulp1后,發(fā)現(xiàn)固定化Ulp1可以耐受4.5-10.5的極端pH處理,而Ulp1在pH 10.5處理12個小時后已表現(xiàn)出活性喪失。通過對其重復使用次數(shù)探究,發(fā)現(xiàn)固定化Ulp1在重復使用15次后仍保持90%的活性。(5)為了進一步擴大固定化Ulp1的使用范圍,為此構(gòu)建了快速純化系統(tǒng),可以在1個小時之內(nèi)獲得高純度且沒有標簽的目的蛋白,若配合使用96孔板,可以同時純化多種蛋白,為工業(yè)化奠定了基礎。
[Abstract]:SUMO fusion expression system is widely used in laboratory to express those proteins that are prone to error folding, insoluble or toxic..6*His is one of the most widely used fusion labels in the laboratory. But because of its small molecular weight, it is easy to be encapsulated by the eye protein. If the SUMO label is placed in 6*His and its purpose is placed. Between the proteins, it can be well eliminated and the efficiency of the column is greatly improved. However, some of the smaller molecular weight proteins, due to the introduction of the large label SUMO, may lead to the loss of the function of the.SUMO protease Ulp1, which can be a good solution to this problem. By identifying the three stage structure of the SUMO, the GG- of the SUMO carboxyl terminus is cut. The protein, therefore, does not leave any amino acid residues and does not affect further research on the target protein. But the commercialized Ulp1 is expensive and some laboratories themselves express the purified Ulp1. We found that Ulp1 is easily inactivated after repeated freezing and thawing. Even if the long term is at -80 centigrade refrigerators, there is also an inactivation situation. The purpose of this study is to improve the stability of Ulp1 and expand its application range by covalent and immobilized method. This paper mainly studies the following aspects: (1) in previous studies, we have learned that the active sites of Ulp1 are the cysteine and 514 - bit histidine, which are 580 bit and 514. The amino acid residue is not on its active site, because we consider immobilized on the active carrier through the lysine residue or the amino terminal. To this end, we have prepared a reliable cyanogen activated agarose, aldehyde agarose and N- hydroxy succinimide activated agarose, and tested that three kinds of beads can be good. (2) (2) in the presence of cyanogen activated agarose, aldehyde based agarose and N- hydroxy succinimide activated agarose can be used to immobilize protein effectively, we studied the three beads immobilized Ulp1 proteins, and found that only N- hydroxysuccinimide activated agarose could be effectively covalently covalent. Combining with Ulp1 without affecting its activity, the enzymology properties of immobilized Ulp1 were studied by BCA quantitative method and Coomassie brilliant blue method by using BSA as standard sample, BCA quantitative method and Coomassie brilliant blue method. The results showed that the pH of immobilized Ulp1 was widely used, and the enzyme activity was slightly weakened and free Ulp1 base could be reduced from 4.5-11.0, at 11. This is similar. As the enzyme cutting system is a liquid, immobilized Ulp1 is solid, so it is not conducive to mixing. Therefore, when the cutting rate test is done, the immobilized enzyme with 3 times the amount of free enzyme is added. The results show that the cutting rate is consistent, so in order to improve the cutting efficiency, the dosage of immobilized enzyme can be increased. In the small molecular tolerance test, it was found that both the immobilized and free Ulp1 can tolerate 800 m M of sodium chloride, 1 M urea, 100 mM two sulphose alcohol, 400 m M imidazole, and the tolerance to the denaturant twelve alkyl sulfate. Furthermore, the tolerance of Ulp1 to organic reagents and the occurrence of the present 20% ethanol do not affect the immobilization and free Ulp1 activity. 15%DMSO can cause the inactivation of free Ulp1, but does not affect the activity of the immobilized Ulp1. (4) the stability of the immobilized Ulp1 is studied. The results show that the immobilized Ulp1 has been remarkably improved in the thermal stability. After 24 hours of treatment at 37 C, the free Ulp1 has been completely inactivated, but the immobilized Ulp1 still has the activity of 97%, while 30 C for 72 hours. After that, the free Ulp1 has been completely inactivated, but does not reduce the activity of immobilized Ulp1; the immobilized Ulp1 still has 93% activity for 14 days at the same 25 C treatment, and remains active at 4 C for 50 days. After the treatment of Ulp1 with extreme pH, it is found that the immobilized Ulp1 can tolerate the extreme pH treatment of 4.5-10.5, and Ulp1 is treated for 12 hours after pH 10.5. In order to further expand the use of immobilized Ulp1, a rapid purification system was constructed to obtain a high purity and unlabeled target protein within 1 hours, if combined with the use of the immobilized Ulp1 for 15 times. 96 orifice plates can simultaneously purify many kinds of proteins, which laid the foundation for industrialization.
【學位授予單位】：西南大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：Q814
，
本文編號：2166783
[Abstract]:SUMO fusion expression system is widely used in laboratory to express those proteins that are prone to error folding, insoluble or toxic..6*His is one of the most widely used fusion labels in the laboratory. But because of its small molecular weight, it is easy to be encapsulated by the eye protein. If the SUMO label is placed in 6*His and its purpose is placed. Between the proteins, it can be well eliminated and the efficiency of the column is greatly improved. However, some of the smaller molecular weight proteins, due to the introduction of the large label SUMO, may lead to the loss of the function of the.SUMO protease Ulp1, which can be a good solution to this problem. By identifying the three stage structure of the SUMO, the GG- of the SUMO carboxyl terminus is cut. The protein, therefore, does not leave any amino acid residues and does not affect further research on the target protein. But the commercialized Ulp1 is expensive and some laboratories themselves express the purified Ulp1. We found that Ulp1 is easily inactivated after repeated freezing and thawing. Even if the long term is at -80 centigrade refrigerators, there is also an inactivation situation. The purpose of this study is to improve the stability of Ulp1 and expand its application range by covalent and immobilized method. This paper mainly studies the following aspects: (1) in previous studies, we have learned that the active sites of Ulp1 are the cysteine and 514 - bit histidine, which are 580 bit and 514. The amino acid residue is not on its active site, because we consider immobilized on the active carrier through the lysine residue or the amino terminal. To this end, we have prepared a reliable cyanogen activated agarose, aldehyde agarose and N- hydroxy succinimide activated agarose, and tested that three kinds of beads can be good. (2) (2) in the presence of cyanogen activated agarose, aldehyde based agarose and N- hydroxy succinimide activated agarose can be used to immobilize protein effectively, we studied the three beads immobilized Ulp1 proteins, and found that only N- hydroxysuccinimide activated agarose could be effectively covalently covalent. Combining with Ulp1 without affecting its activity, the enzymology properties of immobilized Ulp1 were studied by BCA quantitative method and Coomassie brilliant blue method by using BSA as standard sample, BCA quantitative method and Coomassie brilliant blue method. The results showed that the pH of immobilized Ulp1 was widely used, and the enzyme activity was slightly weakened and free Ulp1 base could be reduced from 4.5-11.0, at 11. This is similar. As the enzyme cutting system is a liquid, immobilized Ulp1 is solid, so it is not conducive to mixing. Therefore, when the cutting rate test is done, the immobilized enzyme with 3 times the amount of free enzyme is added. The results show that the cutting rate is consistent, so in order to improve the cutting efficiency, the dosage of immobilized enzyme can be increased. In the small molecular tolerance test, it was found that both the immobilized and free Ulp1 can tolerate 800 m M of sodium chloride, 1 M urea, 100 mM two sulphose alcohol, 400 m M imidazole, and the tolerance to the denaturant twelve alkyl sulfate. Furthermore, the tolerance of Ulp1 to organic reagents and the occurrence of the present 20% ethanol do not affect the immobilization and free Ulp1 activity. 15%DMSO can cause the inactivation of free Ulp1, but does not affect the activity of the immobilized Ulp1. (4) the stability of the immobilized Ulp1 is studied. The results show that the immobilized Ulp1 has been remarkably improved in the thermal stability. After 24 hours of treatment at 37 C, the free Ulp1 has been completely inactivated, but the immobilized Ulp1 still has the activity of 97%, while 30 C for 72 hours. After that, the free Ulp1 has been completely inactivated, but does not reduce the activity of immobilized Ulp1; the immobilized Ulp1 still has 93% activity for 14 days at the same 25 C treatment, and remains active at 4 C for 50 days. After the treatment of Ulp1 with extreme pH, it is found that the immobilized Ulp1 can tolerate the extreme pH treatment of 4.5-10.5, and Ulp1 is treated for 12 hours after pH 10.5. In order to further expand the use of immobilized Ulp1, a rapid purification system was constructed to obtain a high purity and unlabeled target protein within 1 hours, if combined with the use of the immobilized Ulp1 for 15 times. 96 orifice plates can simultaneously purify many kinds of proteins, which laid the foundation for industrialization.
【學位授予單位】：西南大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：Q814
，
本文編號：2166783
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