Purification and renaturation of the hottest prote

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Protein purification and renaturation

when recombinant proteins are highly expressed in E. coli, they often exist in cells in the form of insoluble and inactive protein aggregates, namely inclusion bodies. The inclusion body must be separated from the cell, and dissolved with a high concentration of denaturant (such as 7.0mol/l guanidine hydrochloride, 8.0mol/l urea), then the denaturant is removed or the concentration of denaturant is reduced to renature the inclusion body protein, and finally the target protein is purified by chromatography, with a compression force of 20 to 30 kg. The refolding and purification of inclusion body protein is the core of the whole process

at present, the common problems in the production of recombinant protein are: (1) low refolding efficiency. The traditional renaturation methods are dilution and dialysis. Dilution renaturation method dilutes the sample dozens or even hundreds of times, which will greatly increase the volume of the sample, and bring great difficulties to the subsequent separation and purification, and a large renaturation container is required in the renaturation process. Dialysis takes a long time, and the dialysis solution needs to be changed many times. The common disadvantage of these two methods is that egg white matter will aggregate and produce a large amount of precipitation during the renaturation process, and the renaturation efficiency is low. Generally, the activity recovery of protein is only 5~20%, and the renatured protein solution contains a large number of miscellaneous proteins, which need to be further separated and purified. (2) The process route is cumbersome and the production cycle is long. In the traditional process of recombinant protein separation and purification, the classical soft gel separation medium is mostly used. Because the particles of this medium are large and the separation efficiency is poor, it is often necessary to use a variety of different modes of chromatographic operations to purify the target protein in order to obtain the target protein with a certain purity standard. In addition, the pressure resistance of this chromatographic medium is very poor, so it can only be operated under the condition of low flow rate, and the separation and purification time is long. Many separation and purification steps and long separation time make the quality recovery and activity recovery of protein very low. Moreover, in the traditional recombinant protein production process, protein renaturation and purification are two independent unit operations in the production process, which also restricts the production efficiency to a great extent. (3) High production cost and large equipment investment. Because renaturation and separation and purification are carried out separately, and there are many separation and purification steps, each step requires supporting equipment, resulting in large equipment investment and high production cost. With the increase of production scale, this drawback will become more and more serious

In 1991, Professor Geng Xindu first used high performance hydrophobic interaction chromatography (hphic) for the renaturation of denatured proteins, which has solved the above problems. Now it has been successfully used in recombinant human interferon-g (rhifn-g), recombinant human interferon-a (rhIFN-a), human granulocyte colony stimulating factor (rhG CSF), recombinant human proinsulin Renaturation and simultaneous purification of recombinant proteins such as recombinant bovine prion and standard model proteins such as lysozyme and nuclease. At present, exclusion chromatography, ion exchange chromatography and affinity chromatography have also been used in protein renaturation and simultaneous purification. Compared with the traditional dilution method and dialysis method, the advantages of protein renaturation by chromatography are: ① denaturant can be removed quickly after injection; ② Due to the adsorption of relatively denatured proteins fixed by chromatography, the generation of protein aggregates and precipitates in the renaturation process can be significantly reduced or even completely eliminated, so as to improve the quality and activity recovery of protein renaturation; ③ At the same time of protein renaturation, the target protein can be separated from the miscellaneous protein to achieve the purpose of purification, so that renaturation and purification can be carried out at the same time; ④ It is convenient to recover the denaturant to reduce the cost of wastewater treatment. In short, chromatographic renaturation can improve the activity and quality recovery of protein, integrate protein renaturation and purification in one step, shorten the operation steps and production time, reduce equipment investment, and greatly reduce the production cost. It has attracted the attention of many biochemical researchers and recombinant protein drug manufacturers all over the world. Due to the high separation efficiency of high performance liquid chromatography (HPLC), proteins with satisfactory purity can be obtained in one step, and the separation speed is fast, which has greater advantages in application

refined 2

refined 1


finished product

renaturation (dilution or dialysis)



crude purification

Figure 1 Schematic diagram of the downstream process of shortening recombinant protein with preparative usrpp

the following is a summary of some relevant literature. The full text can be requested, Shaanxi Xida Colin gene Pharmaceutical Co., Ltd.

purification of recombinant bovine normal prion protein PrP (104 – 242) by hphic

chaozhan Wang, Xindu Geng, Dawei Wang, Bo Tian, Journal of chromatography B, 806 (2004) 185–190

Purification of the prion protein (PrP) is a major concern for biological or biophysical analysis as are the structural specificities of this protein in relation to infectivity. A simple and efficient method for purification of recombinant bovine normal prion protein containing residues 104–242, PrP(104–242) expressed in Escherichia coli by high performance hydrophobic interaction chromatography (HPHIC) was presented in this work. The solution containing denatured and reduced protein in 8.0 mol/L urea extracted from the inclusion body was directly injected into the HPHIC column, aggregates were prevented by the interaction between the denatured PrP(104–242) molecules and the stationary phase during the chromatographic process, the soluble form of PrP(104–242) in aqueous solution was obtained after desorbed from the column. Several factors, including pH value, types of stationary phase and salt, and gradient mode, influencing the purification results were investigated. Optimal conditions were obtained for the purification of PrP(104–242) by HPHIC. This procedure yield PrP (104 – 242) of a purity of 96% with a recovery of 87%, carefully, for a single step purification of 40 min.

reforming of denatured/reduced lysozyme using weak category exchange chromatography

Yan Wang, Bo Lin Gong, Xin this series of electronic universal experimental machines adopts highly integrated DSP central processor and exchange servo electromechanical control system Du Geng, Chinese chemical letters, 14 (2003) 828 – 831

Oxidative refolding of the denatured/reduced lysozyme was investigated by using

weak-cation exchange chromatography (WCX). The stationary phase of WCX binds to the reduced lysozyme and prevented it from forming intermolecular aggregates. At the same time urea and ammonium sulfate were added to the mobile phase to increase the elution strength for lysozyme. Ammonium sulfate can more stabilize the native protein than a common eluting agent, sodium chloride. Refolding of lysozyme by using this WCX is successfully. It was simply carried out to obtain a completely and correctly refolding of the denatured lysozyme at high concentration of 20.0 mg/mL.

High-performance hydrophobic interaction chromatography as a tool for protein refolding

Xindu Geng and Xiaoqing Chang, J. Chromatogr., 599 (1992)

A method for the refolding of previously unfolded proteins with a concentrated solution of denaturing agent is presented, involving the use of high-performance hydrophobic interaction chromatography (HPHIC) to separate the denaturing agent completely from the unfolded protein and to provide a suitable environment for its refolding. The retention, peak shape and peak height in HPHIC and size-exclusion chromatography, UV spectra, circular dichroic spectra and bioactivity were used to test the possibility and the completeness of the protein refolding. The proposed method permits the extracted solution from Escherichia coli cells to be injected directly into the HPHIC column and, at the same time, the refolding and purification of the proteins to be effected. The renaturation and purification of recombinant human interferon g form E. coli cells is one example of the application of the method in biotechnology.

progress of hydrophobic chromatography and its application in biochemical research

Liu Tong, Geng Xindu, chromatography, 1998, 16 (1):

this paper focuses on the theoretical research of hydrophobic chromatography and the new development of inorganic fillers, organic fillers, non porous fillers and macroporous membranes in hydrophobic stationary phases, and the application of hydrophobic chromatography in the separation, purification and biochemical research of biological macromolecules, including protein renaturation The applications of folding and molecular conformation change are introduced. The full text includes 62 literatures and a table

multifunctional protein to avoid deviation in experimental results development and performance study of activator

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