Verso la caratterizzazione molecolare dei domini extracellulari del recettore tyrosine kinase orphan receptor 1 (ROR1).

Receptor tyrosine kinase orphan receptor 1 (ROR1) is a 130 kDa transmembrane enzyme. Its ectodomain organization is similar to that of different members of the receptor tyrosine kinase (RTK) superfamily, and although ROR1 has been considered a pseudokinase due to its reduced enzymatic activity, it has been classified within the VIII RTK subfamily. This transmembrane receptor enzyme is widely expressed during embryonic development and it is also involved in skeletal muscle regeneration in adults. High ROR1 expression levels are also associated with the onset of several types of cancer, including aggressive breast cancers and hematopoietic malignancies such as chronic lymphocytic leukemia (CLL). ROR1 has also been reported as involved in the Wnt pathway, a crucial development signalling mechanism fundamental for many cellular processes. For these reasons, ROR1 is currently considered a very promising target for cancer-specific therapeutic development, in particular through immunotherapy. In addition, recent reports identify ROR1 as a putative interactor of the muscle-specific kinase (MuSK), another RTK essential for neuromuscular junction (NMJ) development, formation and function. Despite these numerous important features, at present the molecular knowledge about human ROR1 is very limited, and in particular no three-dimensional structure information are available about the extracellular receptor domains of this molecule. My research work focused on the development of efficient strategies to express in recombinant form and purify various fragments corresponding to the ROR1 ectodomain. In particular, I designed optimized recombinant production and purification methods for the immunoglobulin (Ig) and the Kringle (KR) domains of ROR1, in order to obtain milligram amounts of pure protein sample suitable for crystallization trials and structural biology investigations. Moreover, I performed initial biochemical attempts to investigate whether the isolated recombinant ROR1 domains could suffice to reconstitute stable complexes with MuSK ectodomain. To reach my goals, I used multiple biochemical methods that include: molecular biology methodologies to verify the expression plasmids for recombinant protein production; small- and large-scale recombinant protein production of different domains of ROR1 using prokaryotic (E. coli) and also eukaryotic (HEK293 cells cultivated in suspension) expression systems. The obtained protein samples were purified using chromatographic techniques such as immobilized metal ion affinity chromatography (IMAC) and size-exclusion chromatography (SEC); the latter was also used to perform co-elution trials with available MuSK ectodomain constructs. Numerous crystallization trials using various preparations of the purified proteins were performed to obtain structural information about the domains using X-ray crystallography. Collectively, these results pave the way to a molecular characterization of ROR1, and provide recombinant reagents highly suitable to characterize the implications of this orphan receptor in organism development, tissue regeneration, and cancer.