SUMO蛋白酶Ulp1的固定化及其应用研究
发布时间：2018-08-05 19:32
【摘要】：SUMO融合表达系统广泛应用于实验室表达那些易于错误折叠、难溶或有毒性的蛋白,可以得到很好的表达效果。6*His是目前实验室最为广泛使用的融合标签之一,但是由于其分子量很小,容易被目的蛋白包裹,若将SUMO标签置于6*His与目的蛋白之间,可以很好的排除这种情况,使得挂柱效率大幅度提升。但是,某些分子量较小的蛋白由于大标签SUMO的引入可能导致其功能丧失。SUMO蛋白酶Ulp1可以很好的解决这个问题,通过识别SUMO的三级结构,切割SUMO羧基端的GG-,从而得到目的蛋白,因此不会留下任何氨基酸残基,也不会影响对目的蛋白进一步的研究。但是商业化的Ulp1价格昂贵,也有的实验室自己表达纯化Ulp1,我们发现Ulp1在经过反复冻融之后很容易失活,即使长期保存在-80℃冰箱中,也存在失活的情况,从而大大增加了纯化的人力与物力。本研究拟通过共价结合固定的方法,提高Ulp1的稳定性,扩大它的应用范围。本文主要从以下几个方面展开研究工作:(1)在前人的研究中我们了解到Ulp1的活性位点是580位的半胱氨酸和514位的组氨酸,而赖氨酸残基并不在它的活性位点上,因为我们考虑通过赖氨酸残基或者是氨基末端将其固定在活性载体上。为此,我们制备了可靠的溴化氰活化的琼脂糖,醛基化的琼脂糖和N-羟基琥珀酰亚胺活化的琼脂糖,并检验了三种beads可以很好的用来交联蛋白。(2)在溴化氰活化的琼脂糖,醛基化的琼脂糖和N-羟基琥珀酰亚胺活化的琼脂糖均可以有效的用来固定化蛋白前提下,我们对这三种beads固定化Ulp1蛋白做了研究,发现只有N-羟基琥珀酰亚胺活化的琼脂糖可以在有效地共价结合Ulp1的同时不影响其活性,通过用BSA做标样,BCA定量法和考马斯亮蓝法测定了固定化效率为1.68 mg/mL。(3)对固定化Ulp1的酶学性质进行研究,结果表明:固定化Ulp1的pH适用范围广泛,可以从4.5-11.0,在11.0的时候,酶活略有减弱,和游离Ulp1基本相似。由于酶切体系是液体,固定化Ulp1是固态,因此不利于混匀,为此在做切割速率试验时,加入了游离酶3倍酶量的固定化酶,结果表明它们的切割速率一致,所以在使用的时候为了提高切割效率,可以通过增加固定化酶的用量。在对于小分子耐受试验中,发现固定化和游离的Ulp1均可以耐受800 m M的氯化钠、1 M的尿素、100 mM的二硫苏糖醇、400 m M的咪唑,对于变性剂十二烷基硫酸钠均不耐受。此外检测了Ulp1对有机试剂的耐受,发现20%的乙醇均不影响固定化和游离Ulp1的活性,而15%DMSO会导致游离Ulp1失活,但不影响固定化Ulp1的活性。(4)对固定化Ulp1的稳定性研究,结果表明:固定化Ulp1在热稳定性上得到了显著的提高,在37℃处理24小时后,游离Ulp1已经完全失活,但是固定化Ulp1仍有97%的活性;而30℃处理72小时后,游离Ulp1已经完全失活,但并没有降低固定化Ulp1的活性;同样的25℃处理14天,固定化Ulp1仍有93%的活性,在4℃条件下保存50天,仍有活性。当用极端pH去处理Ulp1后,发现固定化Ulp1可以耐受4.5-10.5的极端pH处理,而Ulp1在pH 10.5处理12个小时后已表现出活性丧失。通过对其重复使用次数探究,发现固定化Ulp1在重复使用15次后仍保持90%的活性。(5)为了进一步扩大固定化Ulp1的使用范围,为此构建了快速纯化系统,可以在1个小时之内获得高纯度且没有标签的目的蛋白,若配合使用96孔板,可以同时纯化多种蛋白,为工业化奠定了基础。
[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.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q814
，
本文编号：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.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q814
，
本文编号：2166783
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