Optimization Of Fermentation And Purification For ACE-C Terminal Domain From Pichia Pastoris

The angiotensin I-converting enzyme (ACE, EC 3.4.15.1) plays a key role in the cardiovascular homeostasis and regulation of blood pressure, by generating the potent vasoconstrictor angiotensin II (AngII) after cleavage of the C-terminaldip eptide from angiotensin I (AngI); In addition, the ACE inactivates the bradykinin, blocking its hypotensive effects. ACE is a zinc-dependent dipeptidyl carboxypeptidase with diverse physiological functions, which composed of two separate and independent catalytic domains (N and C domains), arising from a gene duplication event. This study shows that the C-domain of ACE is the predominant site of angiotensin I cleavage in vivo,while the N-domain mainly refers to bleeding regulation. ACE-C domain will provided a specific enzyme for screening specific ACE inhibitor. Currently,the target enzymes used in ACEI screening mainly are ACE which has two domains and there hasn't been any commercial ACE-C domain product. Based on ACE-C domain of target products, this study aimed at the establishment of efficient fermentation and purification process of protein which produced by Pichia pastoris.In this study, we constructed the high expression recombinant Pichia pastoris and performed the screening tests in shake flasks to obtain the optimal values of several key fermentation parameters. Glycerine concentration, temperature and pH of three levels were investigated to determine their influence on cell growth. Methanol concentration, temperature and pH of three levels were investigated to determine their influence on ACE-C domain production. The optimal parameters were successfully scaled up to 5 L bioreactors, and then we optimize several other parameters of fed-batch condition. The best condition was determined by screening different fed-batch strategies of methanol, polypeptone and proportion of bacterial suspension. Establish and optimization of separation and purification condition including equilibration, elution and so on were compared and screened. Lastly, Comparison of the inhibition efficiency the ACE-C domain and ACE By captopril (ACEI) identify that if ACE-C domain has the capability for drug screening.Based on effects on the growth and expression level , the optimal values for the temperature, the concentrations of glycerol and methanol and the pH were 28℃, 2% (V/V), 6.5; and 26℃, 1.5%(V/V), 5.5, respectively. The optimal parameters were successfully scaled up to 5 L bioreactors to achieve high-throughput production: the cell density (OD₆₀₀) of recombinant P. pastoris and the yield of crude target protein were respectively 500 and 446 mg/ L, Compared with the shake flask cell density increased 10 times, ACE-C domain expression increased 16 times. The appropriate chromato- graphy conditions were established, after the purification with Ni+-NTA columns, ACE-C domain was collected with a recovery of 53% , purity of 94%, and the specific activity of it was reached 85.47 U/mg, which is double fold than ACE purchased from Sigma. The target protein has ACE-C domain structural features and a good target protein for selection ACEI drugs. The results provided a zymolytic condition for ACE-C domain industrialscale production, and provided a high specific activity enzyme for screening specific inhibitor to ACE-C domain. The processes developed for ACE-C domain have been shown to be scalable further.This provided a zymolytic condition to be used for ACE-C domain in industrial scale production, and provided a high specific activity enzyme for screening specific inhibitor to ACE-C domain.