Development And Research Of SH2 Superparent Based On Phage Display Technology

The systematic analysis of the phosphorylation signaling network,comprising numerous kinases and phosphorylation sites,provides abundant information for measuring the pathological state of cells,identifying cancer biomarkers,and discovering drug targets.The developed SH2 superbinder that is more effective than commercial anti-p Tyr antibodies in enriching phosphotyrosine(p Tyr)is easy to manufacture and has a small molecular weight(13KDa).The enrichment experiment,based on SH2 superbinder,was able to quantitatively determine the highly active kinases,as a drug targets,in tumor cells.Therefore,SH2 superbinder has broad application prospects in the diagnosis and treatment of tumor diseases.However,recent studies have shown that SH2 superbinder also binds to sulfotyrosine(s Tyr).While s Tyr and p Tyr have similar molecular weights and physical and chemical properties,which makes it difficult to distinguish.Therefore,it is required to develop a new type of SH2 superbinder that binds to p Tyr with high specificity.In this study,phage display technology was used to construct a Fyn-SH2 phage display library(>10⁹unique SH2 variants),and the binders of p Tyr-containing peptides(p Y)were enriched after multiple rounds of affinity panning with p Y.This study involved that p Y-binding positive clones,of which the DNA sequence was confirmed by Sanger sequencing,were identified by phage-ELISA;the binding affinity and binding specificity between the mutants and the p Y were determined with the purified variant proteins.Finally,we obtained the SH2 superbinder with high affinity and high specificity,named variant 3(V3).The first part is the construction and quality assessment of the Fyn-SH2 phage display library.We first construct the phage display vector of Fyn-SH2 wild type for phage generation,and the phage-ELISA experiment shows that the SH2 domain displays well on the phage display system.Fyn SH2 wild type as the template,Fyn SH2 variant library was generated by randomizing 8 variable residues(K15,E37,T38,T39,A42,S44,K59,K62).The library was evaluated by about 5 million sequences by next-generation sequence.The result shows that a difference existed between the construction library and the designed library,but no extreme case in the mutation distribution was observed.The second part is panning of the Fyn SH2 variant library and identifying positive single clones.A solid-phase selection method was applied for library panning with target peptide p Y(EPQp YEEIPIYL).Phage pool ELISA suggests that positive clones were enriched at round 3 and round 4 after 4 rounds of panning.Twenty-five unique positive clones,isolated from round 3 and round 4,were identified by phage.The third part is the identification and kinetic analysis of SH2 superbinders.EC50 experiment was performed to distinguish the binding strength between SH2variant proteins and p Y,then the binding affinities of those mutants(V3,V10,V13,V17,V24),which of EC₅₀values were lower than 200n M,were measured by fluorescence polarization(FP).The KD values were 38n M,32n M,12n M,84n M,and23n M,respectively.To determine whether those mutants have increased affinity for single p Tyr,the KD values between mutants and the peptide GGp YGG were determined.The KD values between V3,V13,V24,and GGp YGG were 1.627?M,0.62?M and 2.57?M respectively.The experiment results of biolayer interferometry(BLI)show that mutants,performing high affinity with p Y,have slower dissociation rates.V3 was a novel SH2 superbinder with high specificity for p Y confirmed by protein ELISA.