Studies On Preparation,Stability And Pharmacological Effects Of Recombinant Long-lasting Human Adrenocorticotropic Hormone

Objective Adrenocorticotropic hormone (ACTH) is a peptide hormone and it is widely used in clinical practice, especially as a specific medicine for the treatment of infantile spasms, however the metabolic rate of wide type ACTH is relatively high in human body; the half-life of which in vivo is about 15 min. In the present study, we explored the preparation of genetically engineered long-lasting human ACTH.Method First, the proteolytic restriction sites in ACTH that susceptible to proteases present in human blood were analyzed utilizing protease database and mutated by overlapping PCR method. Second, Constructed fusion expression plasmid (HisTag-Linker-EKst-ACTH-pET30a) containing His tag and enterokinase cleavage site (EKst). The third,the recombinant plasmids were transformed into E. coli strain BL21-DE3 for protein expression. The fourth, the fusion protein was purified using Ni columns, then the proteins were cleaved with enterokinase and purified with a CM- cellulose column. After obtaining purified mutant ACTHs, qPCR and ELISA methods were used to compare the stability and pharmacological effects of mutant ACTHs and wild type ACTH in vivo and in vitro.Results (1) Plasmids of wild typy (WT) ACTH, ACTH24 and site-mutant ACTHs(E5D, F7L, G10A, Y2S) were constructed and thereafter validated by DNA sequencing; (2)All the above ACTHs were expressed and purified from E. coli strain BL21-DE3. Coomassie brilliant blue staining and Western Blotting results showed that high purity and yields of purified ACTH; (3) In vivo stability experiments showed that the blood concentration of E5D mutant ACTH is higher than the wild type at 45 min after injection (P<0.01); (4) In vivo experiments showed that the effects of E5D and Y2S mutant ACTH on the levels of glucocorticoids (GC) in the blood were equivalent to that of WT ACTH. In vitro pharmacological studies showed that the effects of E5D and Y2S mutant ACTH on the level of GC secreted from Y1 cells, intracellular levels of cAMP and phosphorylated CREB, and the transcription levels of critical genes involved in GC synthesis including SF-1, STAR,P450-scc, 3?-HSD were equivalent to those of WT ACTH.Conclusion (1) The methods used in the present study to prepare and purify wild type and mutant ACTH are practical. (2) The stability of recombinant E5D mutant human ACTH in the blood was higher than that of the wild type ACTH, and the pharmacological effects of E5D muntant ACTH showed no significant difference compared to those of the wild type ACTH.