Secretion Of Active Peptides Expression

The active peptides have very important physiological functions in the bodies and are being studied and developed as new peptide drugs. There are two major approaches to obtain the active peptides; one is extraction from the tissues of animal and plant, the other is chemical synthesis. For most of active peptides, it is impossible to obtain enough amount for clinical application by means of extraction from the tissues due to their contents are too low in the tissues. Although many peptides have been synthesized, it is still very difficult to synthesize those peptides with 30 or more amino acids. The recombinant DNA technology has been used successful to prepare a lot of proteins in a large scale. However, it is very difficult to obtain active peptides by genetic engineering method because of low molecular weight and unstability, i.e. the expressed peptides are easy to be degraded by host cells. Therefore, there are few reports that the active peptides could be expressed in high level and purified by simple methods. Based on comparing the advantages and disadvantages of the existing expression systems, we suggest that the secretory expression system may be the best approach to express active peptides. Glucagon containing 29 amino acids and without disulfide bond is a kind of widely used peptide drug. In my thesis, glucagon was used as target active peptide model to study the secretory expression of recombinant active peptides. Firstly, our study showed that phoA (alkaline phosphatase) secretory expressionsystem could be used to express glucagon at high level. When fermentation was carried out in 15L fermentor, the expression yield of recombinant glucagon was found to be 80 mg/L, approximately 30% of the total proteins in supernatant. The purified recombinant glucagon was obtained by precipitation with TCA, gel filtration and HPLC. The biological activities and the physicochemical properties of the purified recombinant human glucagon were found to be the same as that of native glucagon. Secondly, strong PL promoter and phoA signal peptide were used to construct secretory expression system. Our result showed that strong PL promoter could be used to increase the expression level of glucagon and phoA signal peptide could direct recombinant glucagon into culture medium. In shaking flask, the expression level of glucagon reached 3.46 mg/ L/ A600 at 40℃. But, the majority of recombinant glucagon precursor with signal peptide could not be processed and blocked into the cytoplasm of E. coli. Thirdly, the effect of phoA signal peptide and the N-terminal histidine of glucagon on expression was studied. It was found that the phoA signal peptide is necessary for the stability of glucagon in the cytoplasm of E. coli and that the stability of the glucagon precursor was decreased when the hydrophobic core of phoA signal peptide was mutated by polylucine. This result is much different from other reports. In addition, it was found that the N-terminal Histidine of glucagon influenced the secretory efficiency of glucagon. When this histidine was deleted, the expression level of desHis1-glucagon could increse about 40% compared with native glucagon. Fourthly, the expression system containing PL promoter and phoA signalpeptide was used to express superactive glucagon, [Lys17, 18, Glu21]-glucagon (SA-glucagon). Expression studies were carried out in E. coli BL21 transformed by pBLSG7. It was found that the expression yield of SA-glucagon reached 3.65 mg/liter/A600, about 19.5% of total proteins in the supernatant under shake flask conditions.