流通色谱介质的研制和生物大分子色谱分离
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摘要
本文主要研究了以双乳化法制备新型流通色谱介质及其在生物大分子的分离过程中的应用。
以甲基丙烯酸缩水甘油酯为单体,二甲基丙烯酸乙二醇酯为交联剂,甘油溶液为超孔致孔剂,甲苯与正庚烷为微孔致孔剂,安息香乙醚为引发剂,采用双乳化法制备乳化体系,紫外光引发悬浮聚合制备出刚性双孔介质。介质明显地含有超孔与微孔。经二乙胺修饰后,获得的阴离子交换介质,对BSA的静态吸附容量与不含超孔的微孔介质接近,但在高的流速下它有比微孔介质大大高出的动态吸附容量,并用混合模型蛋白溶液来考查介质的分离效果,发现增加流动相流速对双孔介质的分离效果影响不大。
再采用碳酸钙悬浮液为超孔致孔剂,环己醇和十二醇为微孔致孔剂,紫外光悬浮聚合制备出双孔介质,比使用甘油溶液为超孔致孔剂,甲苯与正庚烷为微孔致孔剂制备双孔介质,并使制备过程中的乳化体系更加稳定。经乙二胺修饰制得阴离子交换介质,实验证明流速对双孔介质的动态吸附容量影响更小。为了进一步考察自制双孔介质性能,它被用来分离纯化蛋白(分子伴侣GroEL)和核酸(质粒DNA)。
先发酵带有分子伴侣GroEL基因质粒的大肠杆菌,然后利用超声法对菌体进行破碎并制得澄清的待分离液,经过小量进样与梯度洗脱确定了对GroEL的洗脱条件,然后在不同的流速下(150和1500 cm/h)对分离液进行穿透吸附,并使用阶跃洗脱得到了电泳纯的GroEL产品。
对于质粒DNA的分离也是先发酵带有pcDNA3质粒(5.4 kb)的大肠杆菌,利用碱裂解法制得粗提液,使用梯度洗脱的方法可以得到电泳纯的质粒DNA,并考察了流速、样品盐浓度对分离结果的影响,最后在不同的流速下(150和1500 cm/h)对粗提液进行穿透吸附,并使用阶跃洗脱法得到了电泳纯的质粒DNA产品(0.014和0.113 mg plasmid DNA/min·mL bed)。从而证明了自制双孔介质能够对生物大分子进行很好的分离纯化。
The thesis focuses on the fabrication of bipores beads for high-speed flow- through chromatography and its application in the purification of biomacromolecules.
A novel rigid biporous bead (BiPB) had been fabricated by double emulsification to prepare a (w/o)/w emulsion and a subsequent polymerization. The polymerization of monomers, glycidyl methacrylate and ethylene glycol dimethacrylate, was initiated with benzoin ethyl ether by ultraviolet irradiation. The BiPB with an average diameter of 42.8μm was characterized to possess two types of pores, i.e., micropores (20-100 nm) and superpores (300-4000 nm). Comparing with the traditional beads (micropores beads, MiPB), BiPB have the similar static adsorption capacity. However, frontal analysis demonstrated that the dynamic binding capacity of the BiPB column was 1.6-2.4 times higher than that of the MiPB at high flow rates ranging from 1200 to 2400 cm/h. Moreover, separation of a model protein mixture (myoglobin and BSA) was conducted at mobile phase velocities up to 3000 cm/h to compare the performance of the two stationary phases.
To obtain more stable emulsion, we choose the calcium carbonate suspension and cyclohexanol / dodecanol to create superpores and micropores. Derivatized with diethyl amine (DEA), the biporous beads were changed into a kind of anion-exchange matrix (denoted as DEA-B). The static adsorption capacity of the DEA-B was close to that of the DEA-M for BSA (bovine serum albumin). However, frontal analysis demonstrated that the dynamic binding capacity of the DEA-B column was two times higher than that of the DEA-M at a flow rate of 1800 cm/h. The results showed that the velocity of mobile phase have a little effect on the dynamic adsorption capacity of DEA-B.
To test the property of our customized bipores beads, we use it to purify the biomacromolecules from the E. coli broth. After fermentation, ultrasonication and centrifugation of E. coli, the supernatant containing the GroEL was obtained. After some experiment for purification of GroEL, electrophoresis purity of GroEL was got at mobile phase velocities of 150 cm/h and 1500 cm/h. To obtain the pcDNA3 plasmid DNA (5.4 kb) solution, the E. coli was lysed by alkalinelysate method. Purified by customized biporous beads (DEA-B) column with gradient elution, the electrophoresis purity of plasmid DNA was obtained. And the effects of velocity of mobile phase and salt concentration of sample on the purification of plasmid DNA were studied. Finally, we breakthrough the biporous beads column using alkaline lysate at 150 cm/h and 1500 cm/h, and got electrophoresis purity 0.014 and 0.113 mg plasmid DNA/min·mL bed after step elution at 63% buffer B, respectively.
All the results indicate that the bipores bead contains interconnected flow-through pores is promising for high-speed flow-through chromatography.
以甲基丙烯酸缩水甘油酯为单体,二甲基丙烯酸乙二醇酯为交联剂,甘油溶液为超孔致孔剂,甲苯与正庚烷为微孔致孔剂,安息香乙醚为引发剂,采用双乳化法制备乳化体系,紫外光引发悬浮聚合制备出刚性双孔介质。介质明显地含有超孔与微孔。经二乙胺修饰后,获得的阴离子交换介质,对BSA的静态吸附容量与不含超孔的微孔介质接近,但在高的流速下它有比微孔介质大大高出的动态吸附容量,并用混合模型蛋白溶液来考查介质的分离效果,发现增加流动相流速对双孔介质的分离效果影响不大。
再采用碳酸钙悬浮液为超孔致孔剂,环己醇和十二醇为微孔致孔剂,紫外光悬浮聚合制备出双孔介质,比使用甘油溶液为超孔致孔剂,甲苯与正庚烷为微孔致孔剂制备双孔介质,并使制备过程中的乳化体系更加稳定。经乙二胺修饰制得阴离子交换介质,实验证明流速对双孔介质的动态吸附容量影响更小。为了进一步考察自制双孔介质性能,它被用来分离纯化蛋白(分子伴侣GroEL)和核酸(质粒DNA)。
先发酵带有分子伴侣GroEL基因质粒的大肠杆菌,然后利用超声法对菌体进行破碎并制得澄清的待分离液,经过小量进样与梯度洗脱确定了对GroEL的洗脱条件,然后在不同的流速下(150和1500 cm/h)对分离液进行穿透吸附,并使用阶跃洗脱得到了电泳纯的GroEL产品。
对于质粒DNA的分离也是先发酵带有pcDNA3质粒(5.4 kb)的大肠杆菌,利用碱裂解法制得粗提液,使用梯度洗脱的方法可以得到电泳纯的质粒DNA,并考察了流速、样品盐浓度对分离结果的影响,最后在不同的流速下(150和1500 cm/h)对粗提液进行穿透吸附,并使用阶跃洗脱法得到了电泳纯的质粒DNA产品(0.014和0.113 mg plasmid DNA/min·mL bed)。从而证明了自制双孔介质能够对生物大分子进行很好的分离纯化。
The thesis focuses on the fabrication of bipores beads for high-speed flow- through chromatography and its application in the purification of biomacromolecules.
A novel rigid biporous bead (BiPB) had been fabricated by double emulsification to prepare a (w/o)/w emulsion and a subsequent polymerization. The polymerization of monomers, glycidyl methacrylate and ethylene glycol dimethacrylate, was initiated with benzoin ethyl ether by ultraviolet irradiation. The BiPB with an average diameter of 42.8μm was characterized to possess two types of pores, i.e., micropores (20-100 nm) and superpores (300-4000 nm). Comparing with the traditional beads (micropores beads, MiPB), BiPB have the similar static adsorption capacity. However, frontal analysis demonstrated that the dynamic binding capacity of the BiPB column was 1.6-2.4 times higher than that of the MiPB at high flow rates ranging from 1200 to 2400 cm/h. Moreover, separation of a model protein mixture (myoglobin and BSA) was conducted at mobile phase velocities up to 3000 cm/h to compare the performance of the two stationary phases.
To obtain more stable emulsion, we choose the calcium carbonate suspension and cyclohexanol / dodecanol to create superpores and micropores. Derivatized with diethyl amine (DEA), the biporous beads were changed into a kind of anion-exchange matrix (denoted as DEA-B). The static adsorption capacity of the DEA-B was close to that of the DEA-M for BSA (bovine serum albumin). However, frontal analysis demonstrated that the dynamic binding capacity of the DEA-B column was two times higher than that of the DEA-M at a flow rate of 1800 cm/h. The results showed that the velocity of mobile phase have a little effect on the dynamic adsorption capacity of DEA-B.
To test the property of our customized bipores beads, we use it to purify the biomacromolecules from the E. coli broth. After fermentation, ultrasonication and centrifugation of E. coli, the supernatant containing the GroEL was obtained. After some experiment for purification of GroEL, electrophoresis purity of GroEL was got at mobile phase velocities of 150 cm/h and 1500 cm/h. To obtain the pcDNA3 plasmid DNA (5.4 kb) solution, the E. coli was lysed by alkalinelysate method. Purified by customized biporous beads (DEA-B) column with gradient elution, the electrophoresis purity of plasmid DNA was obtained. And the effects of velocity of mobile phase and salt concentration of sample on the purification of plasmid DNA were studied. Finally, we breakthrough the biporous beads column using alkaline lysate at 150 cm/h and 1500 cm/h, and got electrophoresis purity 0.014 and 0.113 mg plasmid DNA/min·mL bed after step elution at 63% buffer B, respectively.
All the results indicate that the bipores bead contains interconnected flow-through pores is promising for high-speed flow-through chromatography.
引文
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