Reference : Development of an in vivo phosphorylation system in Escherichia coli
Scientific congresses and symposiums : Poster
Life sciences : Biochemistry, biophysics & molecular biology
Development of an in vivo phosphorylation system in Escherichia coli
Goux, Marine mailto [Université de Liège - ULiège > > > Doct. sc. bioméd. & pharma. (Bologne)]
NanoFar Autumn School 2013
du 21 octobre 2013 au 25 octobre 2013
Saint-Jaques de Compostelle
[en] Nanofitin ; Phosphorylation
[en] Recently, new strategies emerged in the field of monoclonal antibodies radiolabeling for PET imaging with the use of positron emitter such as zirconium-89 (89Zr, t½=78.4h) or gallium-68 (68Ga, t½=1.13h)[a]. Despite their important role in the therapeutic world, antibodies have many disadvantages related to their structure. In addition to their limited stability, the presence of disulfide bridges and their post-translational modifications make their production difficult and expensive. Moreover, conjugation of chelating agent often occurs on lysines, which is non-regioselective and leads to a heterogeneous mixture of products. In addition, the long clearance of antibodies can be a problem to obtain a good contrast when they are used in imaging. To address these different limitations, we developed a chemistry-free chelating system consisting of a highly phosphorylatable peptide tag. A specific phosphorylation step, with the alpha subunit of the casein kinase II (CKIIα), generates a nanocluster of 4 phosphates that can interact strongly with metal ion like zirconium. This strategy has already demonstrated its powerfulness for the stable and specific anchoring of protein on zirconium phosphonate-based microarray[b]. We are now adapting the use of this labeling tag to the stereoselective chelation of radionuclides for PET imaging. To ensure an efficient targeting of the radionuclide, the tag was genetically fused to a Nanofitin®, a protein scaffold developed as an alternative to antibodies. Nanofitins share the advantages of being small (7KDa), cystein-free, easy to engineer, highly stable both to temperature and pH and expressed with high yield in Escherichia coli.

As described previously, functionalizing the tag with a phosphate nanocluster is a key step to provide it with the capacity to specifically chelate metal ions such as zirconium or gallium. In early development the phosphorylation was realized in vitro. In order to save purification steps and decrease production costs, we developed an in vivo phosphorylation system consisting in the co-expression of a Nanofitin® fused to the phosphorylatable tag and the CKIIα. Effectiveness of the in vivo system was demonstrated together by electrophoretic mobility shift assay and staining with a specific phosphoprotein staining gel: ProQDiamond®. To further optimize and validate the system, the rate of phosphorylation is evaluated by an isoelectric focusing method. This technique is based on the fact that the pHi of the Nanofitin® decreases as the protein is phosphorylated. Such a technique allowed us to isolate the different phosphorylation states of the phospho-Nanofitin® (from 0 to 4 phospho-serine). In parallel to the development of the in vivo system, we are developing a phosphoprotein purification protocol by immobilized metal affinity chromatography, relying on the specific interaction of phosphate moeity with gallium(III) or iron(III). The use of a single tag for both the purification and the labeling will provide a much simpler and straightforward system. Finally this fusion system of phosphate nanocluster and a Nanofitin® will allows us to combine the advantages of an original single step regioselective labeling for both zirconium-89 and gallium-68, together with a highly stable targeting moiety.

[a] Heuveling D. A. et al. (2011), Oral Oncology, 47, pp. 2–7
[b] Cinier M. et al. (2012), Journal of Biological Inorganic Chemistry, 17, pp. 399–407
Unité de Fonctionnalité et Ingénierie des Protéines (UMR CNRS 6289)
Région Pays de la Loire

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