Expression Of TAT-HSA-α-MSH Fusion Protein In Pichia Pastoris And Its Anti-neuroinflammation Activity In CNS

Neuroinflammation involves in the progression of many neurodegenerative disorders,and plays an important role in the development of the related diseases.Currently,most of anti-neuroinflammation drug candidates have been screened by researchers based on a single mediator,a signaling pathway or response of one kind of immune cells during the progression of neuroinflammation,rather than the complex and whole profile of neuroinflammation.In addition,the safety and efficacy of these potential neuroprotective therapeutic agents remain to be further evaluated for their controversiy and limition in clinical trial.Therefore,effective neuroprotective agents are still urgently needed to be investigated for battling against neuroinflammation in CNS disorders.α-MSH is an endogenous immunomodulatory peptide,and has been shown to exert anti-inflammatory and protective effects in neurodegenerative diseases and brain damage,which are mediated by MCR broadly distributed on the surface of neuron and immune cells.It has been considered to be a promising drug candidate for anti-CNS disorders.However,α-MSH is composed of 13 amino acid residuals,sensitive to protease and its half-life is only a few minutes.Besides,it is difficult to be delivered effectively into brain tissues via nondirect application.In an attempt to improve the circulate half-life of α-MSH and its ability of crossing the BBB,α-MSH was genetically fused to HSA,and protein transduction domain TAT.The TAT-HSA-α-MSH fusion protein was produced in Pichia pastoris and further purified.The ability of TAT-HSA-α-MSH to cross the BBB was detected.The in vitro and in vivo activity,as well as the half-life of the TAT-HSA-α-MSH fusion protein were examined.Our result will also open a new window to the development of long acting drug candidates for attacking against neuroninflammation and its related diseases.1.A linker containing endogenous protease recognizing site L1 or a flexible linker L2 between HSA and α-MSH was introduced to construct the fusion protein of TAT-HSA-L1-α-MSH or TAT-HSA-L2-α-MSH.The high expression recombinant strain of the fusion protein was screened in shake flask,and the purified TAT-HSA-L1-α-MSH can not be digested by ADAMTS-4 under an optimum condition.As a result,the fusion protein TAT-HSA-L2-α-MSH was selected to be studied for its in vitro and in vivo activity and named as TAT-HSA-α-MSH.2.The high cell density fed-batch fermentation of the high expression recombinant strain of TAT-HSA-α-MSH was conducted in a 20 L bioreactor.The purification strategy of fermental supernatant has been successfully established.The purity of fusion protein TAT-HSA-α-MSH after purification is higher than 98% detected by non-reducing SDS-PAGE.The detected molecular weight is 70692.08 Da by MALDI-TOF.The isoelectric point of TAT-HSA-α-MSH is 5.3 by isoelectric focusing electrophoresis analysis.The antigenicity of TAT-HSA-α-MSH was detected by WB using anti-HSA antibody or anti-α-MSH antibody.The N-terminal and C-terminal are identified with the theoretical sequence of TAT-HSA-α-MSH.3.A dual luciferase reporter assay system was constructed and used to detect the bioactivity of TAT-HSA-α-MSH by analyzing the NF-κB mediated transcription activity in A172 cells.It has confirmed that TAT-HSA-α-MSH significantly inhibited NF-κB mediated transcription induced by TNF-α in vitro.4.TAT-HSA-α-MSH can effectively cross the BBB evidenced by immunohistochemistry and ELISA.The half-life of TAT-HSA-α-MSH is 15.2 h in healthy mouse.5.TAT-HSA-α-MSH significantly inhibited TNF-α production in experimental brain inflammation of mice induced by LPS through ELISA analysis.