Solubilization and refolding of bacterial inclusion body proteins
Solubilization and refolding of bacterial inclusion body proteins
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DOI：
10.1263/jbb.99.303
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发表时间：
2005-04-01
影响因子：
2.8
通讯作者：
Panda, AK
中科院分区：
文献类型：
--
作者：
Singh, SM;Panda, AK
大肠杆菌中产生的包涵体由密集包装的变性蛋白质分子以颗粒形式组成。包涵体蛋白的重折叠过程繁琐，回收率低，是从大肠杆菌中生产重组蛋白的主要成本。杆菌随着细菌包涵体中蛋白质聚集体结构和功能的新信息，已经有可能开发出更高回收率的生物活性蛋白质的改进的增溶和重折叠程序。包涵体由部分折叠的蛋白质中间体形成，并且由大多数单一类型多肽的聚集体组成。这有助于在溶解和重折叠之前将蛋白质聚集体分离和纯化至均匀。包涵体聚集体内的蛋白质具有天然的二级结构。据推测，与使用高浓度离液剂的增溶相比，使用温和的增溶条件恢复这种天然样二级结构将有助于改善生物活性蛋白的回收。在包涵体形成过程中导致聚集的主导力量的分析提供了信息，以开发合适的温和的溶解程序的包涵体蛋白。由于天然样二级结构的恢复，从这种溶解的蛋白质的重折叠将非常高。从大肠杆菌中纯化人生长激素包涵体。在溶解和重折叠之前的大肠杆菌细胞中。纯包涵体在2M尿素溶液存在下在碱性pH下溶解。溶解的蛋白质使用脉冲复性过程进行重折叠，随后使用色谱程序进行纯化。超过40%的包涵体蛋白可以复性为具有生物活性的天然构象。因此，温和增溶是从包涵体中高回收生物活性蛋白的关键。
Inclusion bodies produced in Escherichia coli are composed of densely packed denatured protein molecules in the form of particles. Refolding of inclusion body proteins into bioactive forms is cumbersome, results in poor recovery and accounts for the major cost in production of recombinant proteins from E. coli. With new information available on the structure and function of protein aggregates in bacterial inclusion bodies, it has been possible to develop improved solubilization and refolding procedures for higher recovery of bioactive protein. Inclusion bodies are formed from partially folded protein intermediates and are composed of aggregates of mostly single types of polypeptide. This helps to isolate and purify the protein aggregates to homogeneity before solubilization and refolding. Proteins inside inclusion body aggregates have native-like secondary structures. It is assumed that restoration of this native-like secondary structure using mild solubilization conditions will help in improved recovery of bioactive protein in comparison to solubilization using a high concentration of chaotropic agent. Analysis of the dominant forces causing aggregation during inclusion body formation provides information to develop suitable mild solubilization procedures for inclusion body proteins. Refolding from such solubilized protein will be very high due to restoration of native-like secondary structure. Human growth hormone inclusion bodies were purified to homogeneity from E. coli cells before solubilization and refolding. Pure inclusion bodies were solubilized at alkaline pH in the presence of 2 M urea solution. The solubilized proteins were refolded using a pulsatile renaturation process and subsequently purified using chromatographic procedures. More than 40% of the inclusion body proteins could be refolded back to the bioactive native conformation. Mild solubilization is thus the key for high recovery of bioactive protein from inclusion bodies.