Screening The Binding Protein Of Angiogenesis Inhibitor Kringle 5 And Interaction Between Kringle 5 And The Binding Protein

Kringle 5 which was got with Cao in 1977 by protease digestion and molecular cloning method, the fifth fragment of plasminogen, is about 8 KD. Kringle 5 is known to be important for inhibiting the proliferation and migration of vascular endothelial cell （VEC） and leading the apoptosis of vascular endothelial cell by causing a stagnation of the VEC growth cycle, while not having any effects on normal endothelial cells, such as fibroblast and human stem cells. Although kringle 5 can inhibit the growth of endothelial cells and is very important for cancer treatment, what are its targets and how kringle 5 lead to the apoptosis of vascular endothelial cell? We all do not know why. So the binding protein of kringle 5 was screen and validated with the expressing recombinant kringle 5 in a prokaryotic system as the target protein using Ph.D.-7 phage display peptide library with molecular docking, along with surface plasma resonance （SPR）. This study also found a rapid screening system to obtain ligand of protein using Ph.D.-7 phage display peptide library with molecular docking, along with SPR. These data lay a foundation for understanding the potential of screening receptor or ligand of protein using phage display peptide library and molecular docking technology. This research mainly obtains the following results:（1） The primers were designed to clone the sequence of kringle 5 according to the sequence of kringle 5 from NCBI, and the sequence of kringle 5 was cloned into the prokaryotic expression vector pET28b and the protein of kringle 5 was expressed in BL21. The SDS-PAGE showed that the kringle 5 with HIS tags appeared in the surpernate of broken BL21. The compound kringle was got and the purity was 86%after purifying using Ni2+-chelated Sepharose 6 Fast Flow column and Sephadex G-25 pre-packed column （10×100nm）.（2） The possible binding protein of Kringle 5 in vitro was screened and validated using Ph.D.-7 phage display peptide library. The yield coefficient was increased from 6.25×10^-9 in the second screening round to 1.57×10^-7 in the fourth screening round, which indicated that the pool of phage became enriched in favor of the sequences that bind to the Kringle 5. After four rounds of panning, fifty well-separated individual phage clones were then randomly selected from the sample group LB culture plates, on which the total number of phage clones were no more than 100. The sequencing data revealed that 50 phage clones selected from the sample group LB culture plates had 13 different amino acid sequences. The affinity of these short peptides for Kringle 5 was tested using ELISA and the results showed that the IGNSNTL sequence was selected as the high affinity peptide of the Kringle 5. By comparing the seven peptides （IGNSNTL） in the NCBI anthropogenic protein database,103 proteins were revealed. These proteins were divided into twenty-eight types according to the structure and function of them. Considering the acting function of these proteins in the human body, we found that there were nine kinds of proteins which were more likely to the binding protein of plasminogen Kringle 5. Furthermore, through considering and assessing the acting function and pathway of the plasminogen Kringle 5 in the human body, we speculated that the most likely binding protein of plasminogen Kringle 5 were most likely Laminin subunit alpha-3 isoform 2 precursor, ABCA12 and voltage-dependent anion channel-1 （VDAC-1）.（3） The crystal structures of nine screening protein were built using homology modeling and direct modeling. The interactions between Kringle 5 and these nine proteins were analyzed by HEX software and the results showed that Laminin subunit alpha-3 isoform 2 precursor, VDAC-1 and Protein C-ets-2 could bind with Kringle 5 and the binding energy were -837.55 J/mol,-822.65 J/mol and -832.6 J/mol, respectively. Analizing the protein active sites of these nine proteins, Kringle 5 could bind with protein C-ets-2 and did not enter into the active sites of it. But Kringle 5 entered into the active sites of Laminin subunit alpha-3 isoform 2 precursor, VDAC-1, ABCA12 and Endothelin B receptor isoform X2 when Kringle 5 bound with them. Analyzing the amino acids of active sites of these four proteins which took part in binding with Kringle we found that the amino acids of active sites of Laminin subunit alpha-3 isoform 2 precursor, VDAC-1 and ABCA12 apeared in the sequence of IGNSNTL. These results showed that the short peptide IGNSNTL simulated the sequence of active sites of these three proteins. According to the principle of lowest energy of chemical reaction and the principle of space matching of chemical reaction, Laminin subunit alpha-3 isoform 2 precursor and VDAC-1 might be the binding protein of Kringle 5.（4） The nine binding protein of Kringle 5 had been found using phage display technology. Prokaryotic expression vectors of these nine screened protein were built and nine proteins were induced expression in BL（21） for proving wether these nine screened protein could bind with Kringle 5. The interaction between Kringle 5 and these nine proteins were analyzed by SPR and results showed that eight proteins could not bind with Kringle 5 except VDAC-1.（5） The prokaryotic expression vector of Kringle 5 was built for proving the interaction between Kringle 5 and VDAC-1. The prokaryotic expression vector pET28b-VDAC-1 was built successfully and the VDAC-1 was induced expression in BL（21）. The protein was expressed in the form of inclusion body. The method of separation after refolding and the method of on-column refolding were used to separating VDAC-1 and the results showed that protein yield using the method of separation after refolding was higher than that of using the method of on-column refolding. The protein yield was highest when the condition of refolding were pH8.0,0.2 mmol/L oxidized glutathione and 1 mmol/L reduced glutathione, and the protein yield was 55.6%.（6） After optimizing the immobilizing conditions of Kringle 5, Kringle 5 was immobilized on the chip of CM5 and the thermodynamic parameter of Kringle 5 and VDAC-1 was analyzed. The results showed that the association rate constant and dissociation rate constant were respectively 2.43×e3 L/mol and 4.12×e-4 L/mol. Frontal chromatography was also used to study the interaction between Kringle 5 and VDAC-1, and the results showed that the association rate constant and dissociation rate constant were respectively 23.54 L/mol and 0.097 L/mol.The possible ligand of Kringle 5 in vitro was screened and validated using Ph.D.-7 phage display peptide library with molecular docking, along with surface plasma resonance （SPR）. SPR showed that this method could effectively reduce the scope of searching for the ligand of Kringle 5. VDAC-1 which was one of the screened three proteins was the right ligand of Kringle 5 proving by SPR. So phage display peptide library with molecular docking, along with surface plasma resonance （SPR） was a usefull method to screen the ligands of protein.