title: Development of novel gelatin-binding proteins for targeting therapeutics to cartilage lesions in the osteoarthritic joint
Creators: name: Jackson, Gemma
Publisher: name: University of Liverpool Repository
Year: 2023

description: Osteoarthritis (OA) is the most common chronic joint disease appearing to be increasing in prevalence amongst a population that is now on average living significantly longer. Age is a well-established risk factor for OA. Type II (TII ) gelatin, derived from TII collagen is found abundantly in damaged regions of the OA joint, making it an ideal target for binding to target a therapeutic. Retention and integration of mesenchymal stem cells (MSCs) within the damaged regions of the OA joint could facilitate not only joint repair, a prolonged improvement in pain and mobility for OA patients, but also could be defined as the first disease modifying OA therapeutic. The collagen binding domain (CBD) of Matrix-metalloproteinase-2 (MMP-2) binds to TII gelatin and has previously been used by the group as a starting point for designing mutants with increased affinity for TII gelatin. 222 is a chimeric protein previously developed by the Hollander group and proven to bind with an affinity fourteen times greater than CBD to TII gelatin. However, it was concluded that 222 would be challenging to exploit therapeutically because of poor solubility and variable efficacy, therefore the aim of this thesis was to further enhance binding efficacy and/or to improve solubility of 222. Subsequent designed mutant proteins here were intended to be used to coat mesenchymal stem cells (MSCs), to promote potent adherence to TII gelatin in the OA joint. In this work a combination of in silico and in vitro experiments were conducted with the aim of developing proteins that bind with a greater affinity to TII gelatin than 222. Firstly, binding residues were assessed in silico for surface exposure and stability, to impact upon binding and be recoverable in vitro. Four mutants were selected to take to in vitro experiments. Asn (N) 11, 69 and 127 (equivalents in the three modules of 222) were, identified as the most important binding residues. As their mutation to Ala (A) caused the greatest decrease in binding affinity, when assessed in vitro using a TII gelatin binding assay. All three important residues were then mutated to every alternate in silico. Docking predicted 222W, with Trp (W) (substituted at position 11, 69 and 127) as the only such mutant predicted in silico to have stronger binding affinity than 222. Use of a Maltose-Binding Protein (MBP) tag was successful in aiding soluble expression of this mutant 222W. However, this mutation seemed also to alter the characteristics of the protein, preventing 3C protease cleavage, meaning protein of interest (POI) alone could not be isolated. Alongside, the CamSol webserver was utilised to design six mutants with increasing solubility. The CamSol tool predicted mutant protein CamSol6 (CS6) to be the most soluble with three substitutions Val (V) 4 to Glu (E); Phe (F) 6 to E; Tyr (Y) 9 to E and an insertion of EEE between Gly (G) 97 and Y98. CS6 was expressed, purified, and characterised for solubility and binding to TII gelatin. An amorphous precipitation assay with polyethylene glycol (PEG) and ammonium sulfate was used to give a measure of apparent solubility. CS6 was more soluble than both CBD and 222, however binding to TII gelatin was reduced compared to 222. The work presented in this thesis has identified key residues important for the binding of 222 to TII gelatin, as well as those important for enhancing solubility. To be used as a therapeutic a protein must be shown to be stable, soluble, demonstrate minimal heterogeneity, minimal contamination as well as being suitable to scale, with consistent and reproducible expression, purification and physiochemistry. Further work is warranted to achieve these properties and develop a strongly binding and more soluble mutant of 222, with a balance of both characteristics for optimal therapeutic potential.