Study On The Recombinant Expression And Purification Of A Fusion Protein ACTH-TPAST-IGFI In E.coli

Objective: Whenever there is a tension hypothalamus got activated and release CRH(corticotropin releasing hormone)and this works on anterior pituitary gland which is located below hypothalamus to release Adrenocorticotropic Hormone(ACTH),than ACTH works on adrenal cortex to release both glucocorticoid and androgens.Secondry Adrenal Insufficiency is a disease in which there is deficiency of ACTH.the main cause of this disease is pituitary tumor..when there is no ACTH it will not effect on adrenal cortex to release glucocorticoid and androgen.Acth have fast metabolism as compose of just 39 aminoacids.ACTH which is used in clinical medicine is extracted from animal tissues which poses a potential risk for infection from animal diseases and is also expensive.Moreover,their half life is only 15 minutes and so requires prolonged drug infusion time with high costs.Hence,it is crucial and relevant to have a recombinant human ACTH hormone that has increased half life and enhanced activity.Producing large scale biological drugs by gene engineering is a new trend in pharmaceutical industry.This study can help to produce high purity,low cost,large scale ACTH.Method: To express a recombinant fusion protein of ACTH fused to Tissue Plasminogen Activator(TPAST)fused to Insulin Like Growth Factor(IGF1)(His Tag-Linker-ACTH-TPAST-IGFI-PET21)in E.coli and purify using Ni-NTA agarose beads.The reason IGF1 is fused to ACTH through a TPAST substrate linker is because in the blood,there is the Tissue Plasminogen Activator(TPAST)that will act on TPAST substrate linker and cleave the site to release the ACTH and IGF1.IGFI can enhance the effects of ACTH twenty times more.The recombinant plasmids were transformed into E.coli strain BL21-DE3 for protein expression.After analysing if the expression of the ACTH-TPAst-IGF1 fused recombinant protein was in the Supernatant or pellet fraction,it was further purified using Ni-NTA agarose beads.Since the protein was expressed in the pellet fraction of E.coli they are denatured and particulate due to formation of Inclusion Bodies.Hence,they need to be renatured and solubilized to refold them to increase activity.The next steps are to test the functionality of this protein both in-vitro cell-based assays and in-vivo assays.Results: The fused gene product was created by Overlap PCR and then subcloned into p ET21 a vector.Plasmids of wild type(WT)ACTH-TPAST-IGFI,were constructed and thereafter validated by DNA sequencing: All the above were expressed and purified from E.coli strain BL21-DE3.Coomassie brilliant blue staining was done on the SDS-PAGE gel showing the expression of the 14 kd fusion protein in the pellet fraction of the IPTG induced culture and in the elute fraction after protein purification fractions.However,since the protein was insoluble,it was dialysed and refolded and concentrate the protein.(1)Molecular experimental results include :1.Plasmid map,2.Double enzymatic cleavage,3.Cloning of fusion gene PCR product into vector,4.DNA sequencing,5.Recombinant fusion protein expression in E.coli,6.Protein Purification and Renaturation of eluted protein.7.SDS-PAGE.gel stained with comassie blue.8.ELISA to determine the production of c AMP,CREB,Glucocorticoid concentration in mice(waiting to be done due to the current circumstances of Covid-19)Conclusion:We were successful in cloning and expressing the recombinant fusion protein in E.coli in the pellet fraction.The protein purification of this 14 KD protein using Ni-NTA beads resulted in a pure elute fraction and the concentration of the pure protein was 0.03 mg/ ul.We were successful in renaturing and refolding the protein of the 14 kd fusion protein using dialysis.