Expression Of Recombinant Insulin Degludec Precursor Protein,Optimization And Scale-up Of Its Fermentation Process

The clinical application of basic insulin and similar drugs,The whole blood sugar of diabetic patients has effectively controlled.However,due to its short acting time,poor stability and poor compliance,researchers are urged to develop a new insulin with stable and lasting effects,little variability of pharmacodynamics in body and less side effects.Insulin degludec is one of these new types of insulin.This paper aims to construct the expression system of recombinant insulin degludec precursor protein,and focus on enlarging its fermentation process from laboratory scale to pilot scale,laying a foundation for industrial production of insulin degludec.The artificial human insulin precursor gene INS and HS631 plasmid were ligated into the recombinant plasmid HS631-INS,and transferred into E.coli BL21(DE3)to construct a recombinant engineering strain HS631-INS/BL2 1.which stably expressed the propeptide insulin precursor protein.The restriction enzyme map and sequencing results showed that the size and sequence of the expression vector and the precursor gene were consistent with the theory.The precursor protein was purified in multiple steps to obtain a recombinant insulin sample.Mass spectrometry indicated that the molecular weight was consistent with the theoretical value of insulin degludec,both of which were 5826 Da.The product has a potency of 26.38 IU/mg as determined by insulin bioassay and has good biological activity.In shaking flask experiment,the effects of different media,carbon source,nitrogen source,carbon-nitrogen ratio and inorganic salt on the density of recombinant bacteria and the expression of insulin degludec precursor protein were preliminarily compared and analyzed.The optimum medium formulations were selected as follows:glucose 20g/L,yeast exact 5g/L,ammonium sulfate 1.Og/L,dipotassium hydrogen phosphate trihydrate 16.42g/L,potassium dihydrogen phosphate 3.81 g/L,magnesium sulfate heptahydrate 4.93g/L and defoamer 0.2ml/L.The effects of culture temperature,pH,inducer concentration,feeding medium,feeding rate and inoculation amount on cell density and precursor protein expression were studied in a 19L fermentor.The optimum operating parameters were obtained as follows:inoculation amount 1.0%,incubation temperature 37.0 C,pH 6.7 and dissolved oxygen concentration 30%+15%.The feeding rate was 0.5 ml/min.L in 60%glucose solution and 0.25 ml/min.L after induction.When the cell density OD600 reached 75,IPTG with the final concentration of 0.5 mol/L was added,and the induction time was 10 hours.The cell density OD600 reached 184.0.and the expression of insulin degludec precursor protein was 10.45 g/L,which was much higher than the reported level of insulin degludec precursor protein expressed by Pichia pastoris(4.5 g/L).The fermentation process was further scaled up to a 500L scale by geometric similarity method.The results of three batches of 500L fermentation showed that the OD600 value of bacterial density reached 163.3,and the expression of insulin precursor protein reached lOg/L.Stability experiments showed that the cell density and precursor protein expression of 500L fermentation were stable,reproducible and easy to operate.At the same time,it realizes the localization of raw materials and reduces the production cost.