Mouse Endostatin Gene And Mutation In E. COLI Expression Of Recombinant Protein Refolding And Activities

Endostatin, is a strong angiogenesis inhibitor which is a 20-kDa C-terminal cleavage product of collagen XVIII. It has been shown to regress tumors in mice, with no toxic side effects observed. Endostatin was originally isolated from the supernatant of a cultured murine hemangioendothelioma cell line in which the yield was less than 2%. The endostatin gene has been cloned and expressed as a recombinant protein in Escherichia coli and yeast expression systems. Bacteria could produce large quantities of recombinant protein in rapid, often inexpensive, stable and high purity. However, recombinant endostatin prepared from Escherichia coli was deposited in insoluble and inactive aggregates or inclusion bodies, and more than 99% protein lost during the refolding procedure. A soluble form of endostatin was available from the yeast system with relatively low yield (about 1/3 of bacteria system) and high cost, which has made it difficult to produce endostatin in quantities sufficient for extensive clinical evaluation.To improve the yield of active and soluble endostatin, in the present paper mouse endostatin was cloned into PBAD vector and expressed in Escherichia coli Top10. The inclusion bodies were purified and the factors affected the refolding productivity were optimized including low molecular additives (L-arginine, sucrose, glycerol, PEG4000), detergent, pH, reduced/oxidized reagents, and the initial concentration of denaturing recombinant protein. At last a simple and effective protocol was generated with the final refolding yield of 60% at the concentration of 1mg/ml. Precipitated protein was recovered and redenatured for the next refolding cycle. At last we got more than 120mg of soluble protein from 1L culture medium. To improve the activity and the specificity of endostatin, two short peptides screened by phage display technology that had been shown to home specifically to tumor blood vessels were recombined to the N-terminal of the endostatin, formed two mutants ME1 and ME2. In vivo and in vitro effect of endostatin and its two mutants were evaluated after the refolding procedure. The in vitro assay indicated that when immobilized on the plate both endostatin and its mutants had the ability to promote the attachment and spreading of HUVEC. Soluble recombinant endostatin and two mutants inhibited the proliferation and migration of endothelial cells in response to stimulation by basic fibroblast growth factor. With the presence of 10ug/ml recombinant protein there was 70% survival for endostatin and 20% for ME1 or ME2 after 24 hours incubation. In vivo antiangiogenic effect was tested with chorioallantoic membrane assays (CAM)and the result provided a formal proof that endostatin and the two mutants acts as an antiangiogenic agent. More than 60% or 90% of angiogenesis induced by bFGF was inhibited when 20ug/embryo of recombinant ME lor ME2 was added and we failed to see such a dramatic response for endostatin.This study presents a simple and effective protocol for the generation of large quantity of soluble and biologically active form of mouse endostatin expressed as the inclusion bodies produced by Escherichia coli, providing enough protein for fundamental and applied studies on the mechanisms of tumor growth suppression by angiogenesis inhibitors. And the effect of two mutants showed striking improvement might offer a new method to find more effective and special drug for tumor therapy.