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题名	乳腺生物反应器表达的重组人乳清蛋白质的纯化策略与应用
作者	张焱
学位类别	博士
答辩日期	2010-07-28
授予单位	中国科学院研究生院
导师	苏志国
关键词	乳清白蛋白 乳铁蛋白 同源蛋白质 分离纯化 牛乳预处理 乳腺生物反应器
其他题名	Purification Strategies and Applications for Recombinant Human Whey Proteins Expressed by Mammary Gland Bioteactor
学位专业	生物化工
中文摘要	具有营养和医药价值的重组人乳清白蛋白（rHLA）和人乳铁蛋白（rHLF）已经分别在牛乳腺生物反应器中得到了高效的表达，但是这些重组蛋白质的纯化存在特殊的困难。牛乳中含有大量的牛源蛋白质，包括酪蛋白胶体颗粒和牛乳清蛋白质。由于目标蛋白质与牛自身表达的牛乳清白蛋白（BLA）和乳铁蛋白（BLF）蛋白质之间存在高度的同源性，分离难度很大。为了有效地从重组牛乳中纯化目标蛋白，我们进行了如下的研究工作： 1）牛乳预处理方法的研究。本文系统地考察了三种常用牛乳预处理方法的应用范围以及在这些方法中不同乳清蛋白质的回收率。结果表明，凝乳酶法和钙盐沉淀法的应用范围较宽，可以沉淀初始蛋白浓度为10-80g/L脱脂乳中的酪蛋白；酸沉淀法的应用范围略窄，无法沉淀初始蛋白浓度高于60g/L脱脂乳中的酪蛋白。凝乳酶法中乳清蛋白质的回收率最高，可以达到90%以上；钙盐沉淀法中的大部分乳清蛋白质的回收率较高，但是乳清白蛋白（α-LA）的回收率较低（30%-80%）；酸沉淀过程中由于存在包裹效应和共沉淀效应，rHLA的回收率略低（76.1%）。 2）同源蛋白质性质的研究。本研究通过对同源乳清蛋白质氨基酸序列的分析和计算比较了了它们在疏水性、静电电势和分子量等理化性质方面的异同。结果表明，同源蛋白质的分子量非常接近，而疏水性和静电电势有一定的区别。在此基础上建立了鉴定两对同源蛋白质的高效反相检测方法，并定量检测了重组牛乳中两对同源蛋白质的含量，结果显示rHLA浓度1.2~1.5g/L，BLA浓度约1.1g/L；rHLF浓度2.5~3.4g/L，BLF浓度约0.1g/L。 3）重组人乳清白蛋白的纯化研究。本研究比较了离子交换层析和疏水层析分离同源乳清白蛋白的效果。结果表明，同源乳清白蛋白在离子交换层析中的分离度不高。通过层析条件的优化，我们通过非钙离子依赖型的丁基疏水层析有效地分离了rHLA和BLA，并以疏水层析为核心建立了两条rHLA的纯化工艺，目标蛋白回收率分别为67.1和48.6%，所得产品中BLA含量<5%，产品纯度>90%。对纯化所得转基因蛋白性质鉴定的结果表明，rHLA与天然蛋白分子量相同（14,078 Dalton），具备天然的二级结构，无糖基化，具备调节β-1，4-半乳糖苷转移酶促乳糖合成的生物活性。 4）重组人乳铁蛋白的纯化工艺及中试放大。使用SP阳离子交换层析可以从重组牛乳中高效地纯化rHLF，并有效地去除rHLF产品中痕量的BLF蛋白。在此基础上我们完成了5批次200L转基因牛乳的纯化中试，得到了3.11Kg的rHLF纯品。目标蛋白回收率约72%，产品纯度>95%，所得产品中牛乳铁蛋白含量<5%。对纯化所得rHLF进行了性质鉴定，结果表明，rHLF分子量为79,481 Dalton，具备正常铁离子吸附解吸活性和抗菌活性。
英文摘要	Recombinant human α-lactalbumin (rHLA) and lactoferrin (rHLF) with physiological and nutritional value have been expressed successfully in bovine mammary bioreactor. However, purification processed for them could be big challenges because the milks contain lots of animal proteins, including casein micelle and bovine whey proteins. Because of high homologies between recombinant proteins and their bovine courtparts, the separations are very difficult. To purify recombinant proteins efficiently from transgenic bovine milks, we did following works. 1) Study of milk pretreating methods. The applied ranges and efficiencies of three common methods were studied here. Calcium precipitation and rennetcoagulation could give wide application ranges. They could effectively remove casein from milk with protein concentration from 10 to 80g/L. By contrast, applied range of acid precipitation is limited. If protein concentration in milk is above 60g/L, the casein would not be precipitated with acid. On the other hand, the recoveries of all whey proteins in rennet coagulation are very high, above 90%. The calcium salt precipitation also could give satisfactory recovery for most whey protein except α-lactalbumin. Only 30-80% rHLA was retained in the whey. Because of package and co-precipitation effect in acid precipitation, the rHLA recovery was lower, about 76.1%. 2) Study of homologous protein properties. The properties such as hydrophobicity, electric potential and molecular weight between homologous whey proteins were compared through analysis of amino acids sequence. The results showed that the molecular weights between homologous proteins were very close. By contrast, the surface hydrophobicities and electric potentials of rHLA and BLA exhibit some difference, based on which a detect method using high performance reverse phase chromatography was established and the contents of homologous whey proteins in transgenic milk were quantitively monitored. The results showed 1.2~1.5g/L for rHLA, about 1.1g/L for BLA, 2.5~3.4g/L for rHLF and about 0.1g/L for BLF. 3) rHLA purification. The separation efficiencies of rHLA and BLA in hydrophobic interaction chromatography (HIC) and ion exchange chromatography (IEC) were compared. It is shown that the resolution of homologous α-lactalbumin is not high enough in IEC. By contrast, through optimization of solution conditions, rHLA and BLA had been separated successfully in calcium independent butyl HIC column. Then two purification processes were established resulting an rHLA purity > 90% with recovery 67.1 or 48.6%. The BLA content in the final product is lower than 5%. The properties and activities of rHLA were also monitored. The molecular weight of rHLA is equal to that of natural protein (14,078 Dalton). Furthermore, native state, non-glycosylation and regulatory activity for β-1, 4-galactosyltransferase of the purified rHLA were confirmed. 4) rHLF Purification at laboratorial and pilot scale. The rHLF could be purified effectively from transgenic milk using cation exchange chromatography and trace BLF could be removed. Then the purification was processed at pilot scale, in which 200L transgenic milk was treated, for five times and 3.11 Kg pure rHLF product were obtained. The recovery of rHLF is about 72% and the purity >95%, while the BLF content in product <5%. Detection results showed that the molecular weight of rHLF was 79,481 Dalton. The purified rHLF exhibited close iron absorption / desorption properties and antibiotic activities to the natural HLF.
语种	中文
公开日期	2013-09-17
页码	173
内容类型	学位论文
源URL	[http://ir.ipe.ac.cn/handle/122111/1487]
专题	过程工程研究所_研究所（批量导入）
推荐引用方式 GB/T 7714	张焱. 乳腺生物反应器表达的重组人乳清蛋白质的纯化策略与应用[D]. 中国科学院研究生院. 2010.

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