Study On The Scale Preparation And Freeze-dried Preparation Of Tat-PTD-Endostatin-RGD

Endostatin is a 20 kDa protein,which is used as an effective angiogenic inhibitor.Angiogenesis is the formation of new blood vessels from the existing vessels,which is involved in a variety of physiological and pathological processes,such as tumor progression and diabetic retinopathy(DR),etc.The development of angiogenesis inhibitors provides new strategy for the treatment of many angiogenesis related diseases.In the previous studies of our lab,blood ocular barrier membrane penetrating peptide Tat-PTD and targeting peptide RGD were designed to be attached at the terminals of endostatin,and after expression and purification,the bioactivity of Tat-PTD-Endostatin-RGD were also studied.Excellent anti-proliferative activity,anti-migration activity and blood ocular barrier penetrating activity were also found.Blood ocular barrier penetrated Tat-PTD-Endostatin-RGD via eye drops can combine with fundus vascular cell surface receptors specificly to inhibit retinal angiogenesis.Tat-PTD-Endostatin-RGD prepared in the previous experiment is expressed in the form of inclusion bodies,and the yield is not high;In addition,Tat-PTD-Endostatin-RGD is unstable and easy to precipitate during placement,which is not conducive to future development and application.To solve these problems,our study mainly focuses on the large-scale preparation and freeze-dried preparation of Tat-PTD-Endostatin-RGD,which lays the foundation for the development of new drugs.The research contents and results are as follows:1 Soluble expression and purification of Tat-PTD-Endostatin-RGDTarget gene plasmid was respectively introduced into E.coli BL21?E.coli Rosetta and E.coli Origami2(DE3),and then was respectively induced by lactose and IPTG.,then observed The expression of soluble protein was observed to obtain appropriate expression vector and inducer.For the primary purification of target protein,the proper imidazole concentration of wash buffer through Ni2+-Sepharose was studied.For further purification,Sephdex-G50 was used.The results showed that when E.coli Origami2(DE3)was used as the target protein expression vector,and lactose was used as the inducer,1.3 mg of target protein can be purified from 1L of bacteria when the imidazole concentration of flushing buffer was 45 mmol/L.2 Large-scale preparation of soluble expression ofTat-PTD-Endostatin-RGDLarge-scale fermentation of Escherichia coli were carried out in 30 L f fermentor.The inducer do sage,the induction time and the separation and purification conditions of target protein were all optimized.After that,CCK-8 assay was carried out to study the anti-proliferation activity.The results showed that when 360g/tan of inducer lactose was added,and the induction time was 22-25h,76 mg of target protein was purified from 15L of culture medium.The results of CCK-8 assay proved that Tat-PTD-Endostatin-RGD displays good anti-proliferative activity.3 Study on proper lyoprotectant for Tat-PTD-Endostatin-RGDOrthogonal experiment of both single-factor and multi-factors were all carried out.For single factor orthogonal experiment,human serum albumin(HSA),trehalose,mannitol,sucrose,lactose,fructose,sorbitol,arginine,glycine,histidine hydrochloride was respectively freeze-dried together with target protein.For Orthogonal experiment of three factors and three levels,certain lyoprotectants was chosen to obtain appropriate formula of lyoprotectants.The single factor experiment results showed that 3%HSA(w/v)can provide the best protection.Results of three factors and three levels of orthogonal experiment showed that,5%trehalose,1%mannitol and 1%arginine,3%HSA(w/v)can provide the best protection.4 The stability and acute toxicity test of Tat-PTD-Endostatin-RGDThe stability and acute toxicity of the target protein were investigated.Samples were respectively placed at the temperature of-20?,4? and 25?.Samples were taken out weekly,and the moisture content,appearance,redissolution time and residual protein concentration were all determined,and the decomposition polymerization was observed by SDS-PAGE.Sixty-four times the dose of eye drops was used to inject the mice though tail vein and the acute toxicity was observed.The results show that the target protein can be preserved for a long time after freeze drying,and it will be slow to inactivate,and the speed is 25?>4?>-20 ?.No acute toxicity in mice after the injection through tail vein was observed.The results and conclusions obtained in this research:(1)The suitable soluble expression conditions for Tat-PTD-Endostatin-RGD were screened and the expressed protein can be successfully purified.(2)Soluble Tat-PTD-Endostatin-RGD can be scale-expressed in fermentor.The fermentation conditions were optimized.(3)A suitable Tat-PTD-Endostatin-RGD's lyoprotectants was screened,and the target protein could be preserved for a long time.(4)The stability and acute toxicity of Tat-PTD-Endostatin-RGD were preliminarily investigated.It was found that the target protein freeze-dried samples were slow to inactivate and had no acute toxicity to mice.