Purification of recombinant protein expressed in E. Coli

The aim of molecular cloning vector construction is to obtain target proteins. E. coli, yeast, insect, plant and mammalian cell expression systems are common expression systems. E. Coli expression system is the most popular one with many advantages such as the identified genetic background, simple operation, high yield, low price and so on. Separation and purification of recombinant protein is the base of developing and producing proteins, antibodies and Elisa kits.Common purification methods include ion exchange chromatography (IEX),Hydrophobic interaction chromatography (HIC), size exclusion chromatography (SEC) and electrophoresis, etc. The purity, reproducibility, stability and cost should be considered during purification. Now we take several recombinant protein expressed in E. coli as examples to introduce the purification process to provide references for researchers.

Under certain conditions protein expressed will form inclusion bodies that are not insoluble in water and accumulates intracellularly in E. coli. Inclusion bodies can be coated by membrane or formed in uncovered structure. In this case, it is more difficult for purification. Sometimes soluble proteins can be obtained during expression. The purification methods for both soluble proteins and proteins expressed in inclusion bodies will be introduced below.

1. Soluble protein. If the protein is soluble, cells can be disrupted in lysis buffer with protease inhibitors. In this condition, the protein is fully released without degradation. Target protein will be purified by immobilized metal affinity chromatography (IMAC). Below is the electrophoresis result of human AQO10 purified by IDA nickel column.

 Fig 1 SDS-PAGE (human AQO10)

 Lane 1: E. coli extract

 Lane 2: IMAC flow-through

Lane 3: IMAC eluted pool

As showed in Fig 1, in lane 1 there are a lot of bands, corresponding to various proteins in the sample; In lane 2, the target band is more obvious compared to lane 1, because the concentration of target protein is higher so that it can be detected in the flow-through; In lane 3, there is only one target band, showing the high purity of the protein.

2. In order to purify recombinant protein contained in inclusion bodies, extraction requires solubilization of the inclusion bodies. Meanwhile ,other components such as protein, nucleic acid, cellular debris and lipoid should be removed followed the procedure below:

1) Resuspend and disrupt cells. Resuspend the cell paste in resuspension buffer added Triton X-100 and EDTA, then disrupt cells with ultrasonication on ice. Triton is used as a detergent which can remove lipid component and increase the permeability of cell membrane, improving the effect of disruption. EDTA is used as a chelating agent for metalloproteinase. 

2) Washed by detergent. To dissolve inclusion bodies, pellet is resuspended in lysis buffer added Triton X-100 which is used to remove impurity. Collect the paste after centrifugation.

3) Washed by high-salt solutions. Resupend the pellet by high-salt buffer to exclude interference of residues on previous step. Meanwhile, it can prevent the interaction of charge between proteins. Also it can promote dissolution of released nucleic acid so that it can be removed for next step.

4) Washed by denaturing agents. Tris, Urea or β-ME are added in the suspension, then collect the pellet by centrifugation. Low concentrations of urea can be used to remove other protein. EDTA is a chelating agent for metalloproteinase. And β-ME is a reducing agent which can unfolds the mismatched protein.

5) Solubilization using denaturing agents. Resuspend the cell paste in 10 ml resuspension buffer（20mM Tris,6M GdnHCl,2mM β-ME,PH8.0) and incubate overnight in room temperature. Centrifuge at high speed and collect the supernatant. High concentrations of denaturing agents is used to dissolve insoluble protein, while β-ME is used to open the mismatched disulfide bond and promote dissolution.

 6) Purify the denatured recombinant protein using immobilized metal affinity chromatography (IMAC) as soluble protein.

Fig. 2 SDS-PAGE (AKA4 ag7854 rat APPBP2(28a))

Lane 2: IMAC flow-through

Lane 3: IMAC eluted pool

The quality of purified protein will be verified after elution. As showed in Fig 2, the purify of protein expressed in inclusion bodies is as high as soluble protein. SDS-PAGE will be used to identify the molecular weight and purity of purified protein at first. Subsequent verification of protein identity could be obtained by HPLC and the purity should be over 95%. The purification method of protein is based on the similarity and difference among various proteins. Nonprotein substance can be removed according to the similarity of proteins, while target protein can be separated according to the difference. Protein purification is a complex job and we offer the common method of purifying protein expressed in E.coli for reference.

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