Dr Jiří Libus, Charles University
Centre for Knowledge and Technology Transfer of Charles University
Proteins are basic components of living systems. Big investments in research are spent on discovering their numerous properties. Many proteins are produced on industrial scale to be used in medicine and biotechnology (such as insulin, growth factors, and many enzymes). For production purposes as well as for science, it is necessary to isolate efficiently a particular protein from a mixture of molecules. There are a multitude of protein purification methods (most based on affinity chromatography) but all of them suffer from various problems; lack of specificity, inefficient affinity tag removal and compromised structure of the resulting protein are among the common ones.
We introduce an efficient and straightforward purification method that allows the preparation of pure proteins without any tag or carrying a chemical modification or fusion partner. The method consists of 2 fusion proteins that are produced in 2 independent production systems, and it is based on the use of split inteins to purify and/or modify the protein of interest (POI). The first fusion protein (called Base) contains the POI and a tag for affinity chromatography. The second fusion protein (called Modifier) contains a peptide modification, a domain that allows tag-removal from the Base, and another affinity tag. The tagged Base is produced and bound to the affinity matrix. After washing away other proteins, the Modifier is added, resulting in the elution of the POI. It is possible to separate the production of Modifier and Base. If a chemical modification is desired it should be performed on the purified Modifier. Every kind of Modifier results in elution of a different variant of POI. To purify a POI - either tag-less or modified in many various ways, one only needs to produce a single fusion polypeptide. That is a substantial simplification compared to each variant being produced separately. It is then transformed into the desired form by applying the corresponding Modifier. The reaction is more specific then elution of e. g. His tag. There is no need to add and later remove proteases to cut off affinity tags. Chemical modification of proteins becomes available to a wider spectrum of users if Modifiers of the appropriate kind are available. Efficiency of the purification procedure is improved by the possibility of leaving production of Modifiers to an external supplier. However, some Modifiers can be produced in any laboratory equipped for standard protein purification if low budget is the priority.
In comparison to other methods, this purification method is more efficient and specific, all components but the purified (modified) protein of interest remain bound to the matrix. The following protein purification is straightforward, easier, cleaner and performed within fewer steps, which translates to cheaper and faster production in comparison to alternative methods. The method can be provided in a kit format. As possible clients, we see the pharmaceutical industry, medical and biotechnology companies, as well as academic laboratories of small and medium size. Furthermore, we see a commercial opportunity to produce and supply the Modifiers.
Validation of results
Czech patent application: PV 2018-650