Expression, Purification And Identification Of Structure Modified Human Plasminogen Kringle 5 Genes In Escherichia Coli

Background & Objective Pathological angiogenesis is one of the key stages of tumor growth and metastasis, thus blocking or inhibiting angiogenesis is an effective strategy of anti-tumor therapy. Meanwhile pathological angiogenesis is found in many diseases such as diabetic retinopathy, atherosclerosis and rheumatoid arthritis. In recent years, extensive and in-depth researches have performed by researches on the formation of vascular, has proved that angiogenesis is affected by a variety of factors and found a variety of promoting angiogenesis factor and angiogenesis inhibitors. Anti-angiogenesis has become a hot topic for cancer therapy. A number of angiogenesis inhibitors have entered clinical trials.Human Plasminogen Kringle 5 (K5) is a newly discovered angiogenesis inhibitors. It is the fifth kringle loop of human plasminogen. It has some more than 80 amino acid residues. Its molecular weight is about 14 kDa. Kringle loop contains three strictly conservative disulfide bonds. K5 can make effect on the proliferating endothelial cells, inhibit cell proliferation, migration, and induce apoptosis. It is one of the strongest endogenous angiogenesis inhibitors, but the mechanism is still not clear. At the same time with exploration the mechanisms, modifying K5 structure to enhance its activity has become another hot topic. Recently, some scholars won a mutation of K5 (liteK5) through genetic engineering methods to remove part of K5 on both sides of the amino acid arm, retaining the integrity of the three disulfide bonds. This mutant K5 show higher activity in experimental animals, the ability of inhibit vascular endothelial cell proliferation is twice of wild-type K5.Integrin is cell membrane receptor, it mediate the adhesion between cells and basement membrane through binding with its corresponding ligands. RGD (Arg-Gly-Asp) sequence as binding sites of integrin with its ligands, widely exist in the body. Adhesion protein in extracellular matrix is the most common protein containing RGD sequence in human. The expression of integrinα_vβ₃ was up-regulated in the surface of activating tumor cells and tumor endothelial cells. Peptides containing RGD sequence can not only specifically bind tumor endothelial cells, but also could bind tumor cells. It provides a good delivery way for anti-tumor drugs. In recent years, scholars have explored effects of many short peptides containing RGD. The RGDS short peptide shows a good capacity of binding with integrinα_vβ₃, and can significantly inhibit tumor growth. Another study found that ploy(RGD) will have priority for adhering RGD-dependent integrin. Therefore, fusing K5 and peptides containing RGD can collaborate their anti-tumor effects. Methods The genes of RGDRGD-liteK5 and RGDS-liteK5 were amplified from the K5 cDNA by PCR and inserted into plasmids pGEXl-λT and pGEX-4T-1 respectively to construct prokaryotic fusion expression vectors. Expression of fusion proteins in Escherichia coli Rosetta was detected after induction by IPTG. The expression products were collected by affinity chromatography with GSTrap FF column and analyzed by Western blot.Result Acquired two genes RGDRGD-liteK5 RGDS-liteK5 through PCR mutation, their length are 274bp and 268bp respectively. Confirmed by enzyme digestion and sequence analysis, the genes RGDRGD-liteK5 and RGDS-liteK5 were correctly cloned into the prokaryotic expression plasmids pGEX-1λT and pGEX-4T-1. The recombinant plasmids pGEX-RGDRGD -liteK5 and pGEX-RGDS-liteK5 successfully expressed fusion proteins GST/RGDRGD-liteK5 and GST/RGDS-liteK5 in E. coli Rosetta. The fusion proteins were confirmed by Western blot analysis using anti-GST tag antibody. Their molecular weights are about 36 kDa. Eventually we got proteins RGDRGD-liteK5 and RGDS-liteK5 with the molecular weight about 10 kDa through digested the fusion proteins by thrombin.Conclusion The experiment succeeded in modifying the human plasminogen Kringle5 gene, constructing of recombinant plasmids pGEX-RGDRGD-liteK5 and pGEX-RGDS-liteK5, and acquire recombinant proteins RGDRGD-liteK5 and RGDS-liteK5 by using genetic engineering methods. It pays the way for further research on their biological function and has made a positive test to explore and establish multi-target genetic engineering anti-tumor drugs.