Study On High Density Fermentation, Purification And Engineering Research Of Recombinant Human Keratinocyte Growth Factor-2(rhkgf-2)

With the advance of biotechnology and the use of gene technology, genomics,proteomics, bioinformatics and other related technologies, it has become possible that clone proteins known or unknown then make the functional and characteristic research.But amplification and industrialization of the basic research need solving many technical difficulties.Because it not only associated with the upstream construction and expression, but also with downstream fermentation, purification, and other aspects, and at the same time,because the special character of biological products,including the stability, biological activity and safety etc,need establishing the appropriate test methods and quality standards.This makes a number of scientific researches restrict at basic research, stay in the laboratory level,then can not meet the needs of production and commercialization.Keratinocyte growth factor-2(Keratinocyte growth factor-2,KGF-2),also known as fibroblast growth factor-10,is secreted mainly by fibroblasts and other mesenchymal derived cells,and plays a specific role for epithelial cell proliferation, differentiation and migration by stromal-epithelial interactions in paracrine manner.It has a broad clinical application of repair of traumatic,inflammatory mucosa after chemotherapy,treatment of venous ulcers,diabetic ulcers,corneal,radiation damage and other effects,Current common problem of research and development of keratinocyte growth factor-2 is that the soluble expression amount of proteins is little and the product is low,the production process is complicated,poor recovery rate and protein stability has become a serious bottleneck in engineering.Therefore the project KGF-2 research is of great significance.At present,foreign countries for venous ulcers,chemotherapy-induced mucositis and treatment of ulcerative colitis has conducted Phase II clinical trials,the China's external KGF-2 formulations in lyophilized powder and biological fluid membrane used for anti-ulcer and promoting wound healing,has entered the clinical phase I.We ourselves,located in the Bioreactor and Pharmaceutical Development Research Center of the Ministry of Education are also using KGF-2 to treat corneal damage,new drug development of KGF-2 eye drops.1.This study has constructed a highly expressing pET3c-rhKGF-2/E coli BL21 (DE3) plysy engineering strain, and after continuous 50 generations, plasmid loss of only 2%,the morphological characteristics of engineering bacteria was no significant difference;The DNA of different generations times obtained by the size of restriction fragments is consistent with expectations,demonstrating that the inserted gene is not lost because of cell division;the expression strain between the primary passage and 50 times was no significant difference, indicating that strains can be stably expressed,and Western blot analysis proved that the genetic engineering bacteria had good stability.The amount of protein expression can be more than 30% of total protein, and after the cells broken, most of the target protein present is in the supernatant indicating that the recombinant protein expression is soluble.2.This study were optimized by orthogonal experiments to determine the appropriate medium composition by the shaking conditions,the influence of factors fermentation temperature,pH,dissolved oxygen.acetic acid concentration and induction conditions.On this basis,establish the high-density fermentation and cell culture phase technology;Adjust the carbon origin acceleration rate according to the dissolved oxygen change in circumstances;in the product expression phase,according to pH changes,adjust accelerating the rate of carbon origin of fed-batch production process access to higher product volume and expression.The average of cell wet weight of continuous fermentation three batches is 75.7g/L by IPTG induction and the average expression rate is 33.66%.3.We used lactose as an alternative to IPTG for the inducible expression of exogenous protein Different from IPTG, it requires the help of lactose permease into the cell,and transformed byβ-galactosidase enzyme into isomeric lactose then it can work.At the same time, lactose itself as a carbon source,and is constantly used by bacterial metabolism.Therefore,the induction process of lactose as an inducer, the inducer concentration is always in the process of dynamic change,and induction process is more complex, the induction conditions required more detailed study and optimization. In this paper,based on IPTG induction, lactose induction process was optimized, the average of wet weight of three batches of bacterial fermentation is 105.5g/L by lactose induction, the average expression rate is 32.44%, and this has met the requirement of engineering production.4.This research has established purification process for production.According to nature and characteristics of the target protein,we use cation exchange chromatography and heparin affinity chromatography.Using AKTAPuriferlO protein purification system, in salt concentration of 0.1-2.0mol/L gradient,find out the target protein elution peak concentration and electrical conductivity,and as a basis explore more optimized purification and amplification carried out in pilot scale,and determine the composition of purification buffer. Select acetate buffer (pH4.0,5.0),citric acid buffer (pH5.0,6.0),phosphate buffer (pH6.0,6.5,7.0) and Tris-HCl buffer (pH7.5,8.0),examines the different buffer system,pH value and storage temperature on protein stability,and further optimize the purification of the production process.After the optimized purification of three continuous batches.the average protein yield is 745mg/200g wet cell,purity can reach to 99.0%. 5.We established quality inspection methods and standards of rhKGF-2 stock solution, measured the production process rhKGF-2's molecular weight19446D by UItraflexⅢTOF/TOF Mass Spectrum Report MS,consistent with the theoretical molecular weight; N-terminal sequencing results,consistent with the report;established high performance liquid detection method, the purity greater than 98.0%;West-Blotting analysis with its antibody; part results of the safety consistent with the 2005 version of Part III of the relevant requirements of Chinese Pharmacopoeia.Used in the biological activity of KGF-2 by high-affinity binding of the FGFR2-Ⅲb genes through lentivirus-mediated BaF3 cells (mouse primary B cells). The results showed a typical S-curve,and in a certain range in a dose-pendent,not only between batch differences but also in batch differences were within less than 10%,showing good reproducibility and reliability.In short,we had a more detailed study of recombinant human keratinocyte growth factor-2(rhKGF-2) on high density fermentation,purification and engineering to establish a high-density fermentation technology and production technology suitable for purification,and establish quality standards to meet engineering requirements,lay a good foundation for the further research and development of rhKGF-2.