Purification of inclusion bodies using PEG precipitation under denaturing conditions to produce recombinant therapeutic proteins from Escherichia coli
Purification of inclusion bodies using PEG precipitation under denaturing conditions to produce recombinant therapeutic proteins from Escherichia coli
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在变性条件下使用 PEG 沉淀纯化包涵体，以从大肠杆菌中产生重组治疗蛋白
DOI：
10.1007/s00253-017-8265-x
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发表时间：
2017-04
影响因子：
5
通讯作者：
Zhu JW
中科院分区：
文献类型：
--
作者：
Chen Huanhuan;Li Ninghuan;Shi Siwei;Zhu Chencen;Luo Han;Chen Junsheng;Zhang Lei;Zhao Menglin;Lu Huili;Zhu Jianwei;Xie Yueqing;Jiang Hua;Cagliero Cedric;Zhu Jianwei;Yang Xiaoyi;Feng Lei;Lu HL;Zhu JW
已经有文献证明，通过尺寸排阻色谱法（SEC）从大肠杆菌中纯化包涵体可能有利于随后的复性和重组蛋白的回收。然而，柱的装载体积和高成本限制了其在生物制药蛋白质的大规模生产中的应用。我们报告了一种新的方法，使用聚乙二醇（PEG）沉淀在变性条件下，以取代SEC的快速纯化包涵体含有重组治疗蛋白。以重组人白细胞介素15（rhIL-15）为例，将rhIL-15的包涵体溶于7 M盐酸胍中，并通过加入PEG 6000沉淀rhIL-15。发现5%（w/v）PEG 6000的最终浓度对于沉淀靶蛋白并提高回收率和纯度是最佳的。与先前报道的S-200尺寸排阻纯化方法相比，PEG沉淀更容易放大，并且获得相同的蛋白质产量和产品质量。PEG沉淀也减少了约50和95%的材料成本的制造时间。重组人IL-15经复性和纯化后，纯度较高，生物活性与标准品相当。我们的研究表明，在变性条件下的包涵体的PEG沉淀作为从E. coli蛋白表达系统。
It has been documented that the purification of inclusion bodies from Escherichia coli by size exclusion chromatography (SEC) may benefit subsequent refolding and recovery of recombinant proteins. However, loading volume and the high cost of the column limits its application in large-scale manufacturing of biopharmaceutical proteins. We report a novel process using polyethylene glycol (PEG) precipitation under denaturing conditions to replace SEC for rapid purification of inclusion bodies containing recombinant therapeutic proteins. Using recombinant human interleukin 15 (rhIL-15) as an example, inclusion bodies of rhIL-15 were solubilized in 7 M guanidine hydrochloride, and rhIL-15 was precipitated by the addition of PEG 6000. A final concentration of 5% (w/v) PEG 6000 was found to be optimal to precipitate target proteins and enhance recovery and purity. Compared to the previously reported S-200 size exclusion purification method, PEG precipitation was easier to scale up and achieved the same protein yields and quality of the product. PEG precipitation also reduced manufacturing time by about 50 and 95% of material costs. After refolding and further purification, the rhIL-15 product was highly pure and demonstrated a comparable bioactivity with a rhIL-15 reference standard. Our studies demonstrated that PEG precipitation of inclusion bodies under denaturing conditions holds significant potential as a manufacturing process for biopharmaceuticals from E. coli protein expression systems.
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影响因子：
2.9
作者：
Vyas VV;Esposito D;Sumpter TL;Broadt TL;Hartley J;Knapp GC 4th;Cheng W;Jiang MS;Roach JM;Yang X;Giardina SL;Mitra G;Yovandich JL;Creekmore SP;Waldmann TA;Zhu J
通讯作者：
Zhu J
影响因子：
13.5
作者：
J. Van den Bergh;E. Lion;V. V. Van Tendeloo-V.;E. Smits
通讯作者：
J. Van den Bergh;E. Lion;V. V. Van Tendeloo-V.;E. Smits
影响因子：
1.6
作者：
Lan Qian;Shunying Zhu;Jiaqing Shen;Xiao-dong Han;Jin Gao;Mingyuan Wu;Yan Yu;Huili Lu;W. Han
通讯作者：
Lan Qian;Shunying Zhu;Jiaqing Shen;Xiao-dong Han;Jin Gao;Mingyuan Wu;Yan Yu;Huili Lu;W. Han
影响因子：
4.7
作者：
Etsushi Yamamoto;S. Yamaguchi;Teruyuki Nagamune
通讯作者：
Etsushi Yamamoto;S. Yamaguchi;Teruyuki Nagamune
影响因子：
6.4
作者：
Rahmen N;Fulton A;Ihling N;Magni M;Jaeger KE;Büchs J
通讯作者：
Büchs J