Studies On The Production Techniques Of Recombinant Salmon Calcitonin

In this study we primarily established a production seed bank of gene engineering E. Coli which efficiently expressed the rsCT fusion protein from the initial strain. We also finished a series study on fermentation, purification and in vitro modification of expressed product,and set up a manufacturing technique of rsCT suitable for pilot production.First, production seed bank was prepared from initial seed stock. The genotype of the seed bank is verified to be gal-, met-, mal+ andλS, which are identical to the initial strain. The kanamycin resistance in the strain was also detected.In order to perform a high density fermentation of E. coli cell and high expression, we selected a suitable fermentation medium, and established the optimize fermentation parameters including inoculant quantity, pH, dissolved oxygen, the concentration of IPTG for induction, induction time, post-induction fermentation time. In a 40 L ferment tank (the fermentation volume is 18L), the optimize parameters of fermentation and induction including: inoculant quantity is 5～10%, dissolved oxygen is not under 40%, pH is 7.0, and the concentration of IPTG is 0.15 mmol/L, post-induction fermentation time is 4 hours. In the conditions, the quantity of the expressed fusion protein (GST-rsCT-Gly)is up to 4.87g/l.An affinity chromatography is used in the initial purification. The cell homogenate containing the fusion protein is mixed with the affinity resin, which consists of agarose beads with immobilised glutathione, to purify and concentrate the interest product simply.To avoid fracture of the side radical of the peptide chain during the process of cleavage, the initial purified fusion protein was S-sulphonated and concentrated before chemical cleavage. Then the fusion protein cleavage was performed following CNBr treatment, and the peptide SO3-rsCT-Gly is gotten.A key part of the production of biologically active rsCT is the in vitro C-terminal amidation stage. In this studyα-Amidating Enzyme(α-AE) was utilized to amidate the C-terminal glycin of cleavage product(convert the C-terminal Pro-Gly to prolinamide). Before this step, the residual SO3-GST and CNBr following cleavage were removed by CEX chromatography and desalted by RP chromatography. SO3-rsCT-Gly was converted withα-AE to rsCT with prolinamide in C-terminal, which is fully identical to natural salmon calcitonin. After renaturation the disulphide bridge was formed between the native Cysl- Cys7 and rsCT has the full biological activity.In order to further remove the impurities and potential contaminants in the product, a series of steps including CEX, RP were performed to purify rsCT to achieve a final purity＞98%. Finally the product was lyophilised to produce rsCT bulk powder.In conclusion, in this study we established the technique of fermentation, purification and chemical modification. The total yield of rsCT achieved 30-50 mg/L. It laid solid foundation for commercial production and clinical application.