Purification Pilot Study Of Non-purification Tag RLj-RGD3(H~-) Which Come From Lamprey

Lj-RGD3is a secretion protein which is extracted from the Japanese lamprey oralglands, it consists of118amino acid residues. Because it contains three RGD（Arg-Gly-Asp）motif named. In addition,it also includes a pair of cysteines and17histidines. rLj-RGD3isOur previous obtained by cloning means that Lj-RGD3recombinant protein whichhashistidine (His) purification tag, it is a plasmid with6His fusion protein. Experimentalresults show that rLj-RGD3protein with anti-angiogenic, anti-tumor cell migration, invasionand other functions, it expected to become anti-thrombotic and anti-tumor drugs, our group isresearching its anti-tumor and anti-thrombotic before clinical.The purpose of this study: rLj-RGD3(H-) is the recombinant protein in rLj-RGD3basisremoval of purified His tagged proteins, Which contains sequence identical with the deducedgene of Lj-RGD3sequence. According to "People’s Republic of China Pharmacopoeia" onrecombinant protein drug requirements, recombinant proteins used shall not contain fusedpurification tag, the purpose of this study is removing rLj-RGD3,s His label by molecularcloning and obtaining without any purification tag rLj-RGD3(H-), pilot study itsfermentation and purification. Research Methods: Molecular cloning methods was used toadd the termination codon behend Lj-RGD3gene, and then construct back to the originalpET23b vector, a His-tag that does not express on the recombinant plasmid. Using thecalcium chloride transformation method for the recombinant plasmids were transformed intoE. coli BL21and induced with a final concentration of1mM IPTG overnight at a lowtemperature, and the protein named rLj-RGD3(H-). This topic use20L,90L differentspecifications fermenter for high-cell-density fermentation conditions fumble and useBIO-RAD and AKTA protein purification system. We use GE’s Chromatography to studyprotein purification,eg: Affinity chromatography HisTrap HP (Ni2+column), Talon (Co2+column), HiTrap IMAC (Cu2+column); cation exchange chromatography SP FF column, SPHP column; hydrophobic interaction chromatography HiTrap Phenyl HP, Phenyl FF LS,HitrapButyl FF column; heparin affinity chromatography. In the preparation of the study, weconduct a preliminary study to optimize the formulation of pharmaceutical preparations byadding different stabilizers or antioxidants drug dressing such as sorbitol, sodium chloride,glycine, vitamin C. We verified the biological function preliminary optimization rLj-RGD3(H-). Results: the subject using molecular biology methods successfully constructedpET23b-RGD3(H-) recombinant vector, and obtained the expression of protein Soluble. Theresults of the high-density fermentation experimental show that20L fermenter can produce2.76kg cells. Purification process using histidine affinity chromatography, SP column ion exchange chromatography, hydrophobic interaction chromatography, three steps to complete.We added vitamin C, sorbitol, glycine, sodium chloride and other protective agents in thepurified protein and detected by SDS-PAGE, these protective agents have good stability andprotection for the purified proteins. It functions experiments showed that it equally hasanti-angiogenic and antithrombotic function. Conclusion: Subject completion of thenon-purification tag rLj-RGD3(H-) protein,s cloning,expression,fermentation and purificationprocess optimization, and conducted a preliminary study of its pharmaceutical formulations.We demonstrate that their have anti-thrombotic and angiogenic activity.With the subject indepth, will be completed rLj-RGD3(H-) pharmaceutical research and carried out for itsanti-thrombotic, anti-tumor preclinical studies and IND reporting experimental data and testdrugs.