Expression, Purification And Biological Activity Of Human Galectin-1

Galectin-1 is a phylogenetically conserved member of galectins, as a shared consensus of amino-acidsequences of 135 amino acids and the carbohydrate recognition domain (CRD) responsible forβ- galactoside binding. Galectin-1 occurs as a monomer as well as a non-covalent homodimer, and is expressed in many types of cells in the thymus, muscle, kidney, heart, skeletal muscle, with important biological functions. In the nervous system, Galectin-1 involved in regμlating nervous system development and cell proliferation. In the immune system, Galectin-1 promotes the apoptosis of activated T cells and thymocytes ,and suppresses the secretion of inflammatory factors by regμlating activity of T cells. Galectin-1 overexpression in some tumors generally involved in oncogenesis, migration, adhesion and tumor angiogenesis.A number of studies show that Galectin-1 can prevent and treat various autoimmune diseases, and possess a wide prospect of clinical application. However, the relevant animal model experiments must be given a relatively high concentration of Galectin-1 to get the desired resμlts. If applied to humans, correspond to a man of 60kg bodyweight taking 240mg galectin-1 every day .So a large dose of Galectin-1 no doubt lead to difficμlties in clinical. Therefore, how to get high activity of Galectin-1 became the subject of an urgent need to solve the problem.The data demonstrat the higher activity of the galectin-1 dimer in immune homeostasis. Patrick B?ttig show that they designed a covalently linked for dimeric form of galectin-1is a potent inducer of apoptosis in murine thymocytes as well as murine mature T cells at concentrations 10-fold lower than wild-type galectin-1. Therefore, we will adopt a similar design to express the monomeric Galectin-1 and the Gal-1②concatemer and screen high activity protein.First of all, we get the genes of Galectin-1 and Galectin-1②by PCR , plasmid pACT-Gal-1 as a template. The product is cloned into the pQE-30 and pET-22b(+) vector , and then transformed into E. coli M15 and E. coli BL21(DE3). Human Galectin-1 expression is induced by IPTG and analyzed by SDS-PAGE. The expressed protein can be purified by Ni affinity chromatography, anion exchange and cation exchange chromatography. Finally, the biological activity of recombinant human Galectin-1 was evaluated by hemagglutination test and inhibition of cell proliferation test.In the study ,the following three results were obtained:1) M15/pQE-30-Gal-1, BL21/pET-22b (+)-Gal-1, BL21/pET-22b(+)- Gal-1②engineering bacteria were successfully constructed. The human Galectin-1 cDNA fragment was successfully inserted into the vector, which was confirmed by DNA sequencing and double restriction enzyme digestion.2) The His-Gal-1 protein was purified by Ni affinity chromatography and reached a purity of more than 95%; the Gal-1 protein and the Gal-1②protein were purified by Q anion-exchange chromatography and SP ion exchange chromatography and the purity was about 90%. Western blot showed that they are expected protein.3) Three recombinant proteins have shown the erythrocyte agglutination activity and inhibition of Jurkat cells proliferation .Moreover, Gal-1②concatemer has higher biological activity than Gal-1 and His-Gal-1 protein.In summary, we got a high activity of Gal-1②recombinant protein, may become candidates for treatment of autoimmune diseases.