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Creative BioMart's insight and experience in protein refolding allows us to provide protein refolding service to obtain highly pure and stable recombinant proteins for you to accelerate your research projects.
During protein expression, the target protein usually aggregates in inclusion bodies. The complete mechanism of inclusion body formation is still unclear. It is generally believed that the expression speed is too fast, resulting in the inability to fold in time after the protein is translated, thereby mistakenly forming the inclusion body protein. Protein refolding is a critical step in the large-scale production of recombinant proteins. With rich experience in protein expression, Creative BioMart achieves refolding in vitro through procedures such as dilution, dialysis, and column chromatography to restore the biological activity of the target protein.
Fig 1. The flowchart of two-step refolding of inclusion body proteins. (Seyed B. M, et al., 2018)
Creative BioMart can help researchers obtain correctly folded target proteins and correct inclusion bodies generated during expression of heterologous genes. We use the concentration change of denaturant to refold the inclusion body protein to obtain our target protein. Our protein refolding services include but are not limited to the following items.
The customer needs to provide the target gene sequence, and we will synthesize and optimize the target gene according to the actual situation of the experiment, then construct the expression vector, and finally synthesize the target recombinant protein.
We use washing solutions such as urea to wash away as much of the membrane components, DNA and other proteins as possible, leaving only inclusion bodies.
Generally, inclusion bodies are dissolved in high concentrations of denaturing agents such as urea, guanidine hydrochloride, etc. We will use a denaturing agent and add a certain amount of reducing agent (such as β-mercaptoethanol, dithiothreitol, etc.) to dissolve the inclusion bodies.
The purpose of refolding is to "activate" the inactive recombinant protein. We have a variety of biotechnologies for obtaining correctly folded proteins from inclusion bodies, including dilution, dialysis, and ultrafiltration. For high-concentration proteins, gel filtration chromatography can be used to separate proteins and small molecule denaturants to achieve solution exchange and protein renaturation. For the refolding of high-concentration proteins with high yield requirements, it can be achieved by reverse micelle refolding technology or aqueous two-phase extraction refolding technology.
To test whether a protein is properly folded, monodisperse, and fully biologically active, we need to identify the refolded protein. Detection from multiple aspects (biochemical, immunological, physical) is required, such as functional determination of the biological activity and basic functions of proteins; detection of protein determinants by immunoblotting or ELISA; detection of the presence of target proteins in liquid samples by SDS-PAGE; size exclusion chromatography method to verify whether there is an error protein, etc.
Please feel free to contact us if you have any needs for our protein refolding service. We look forward to working with you on attractive projects.
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