Expression Of Recombinant Mutant Fusion Protein Human Serum Albumin-hepatocyte Growth Factor And Its Activity Analysis

Hepatocyte growth factor?HGF?,mainly secreted by hepatic non parenchymal cells,is a multifunctional cytokine which can stimulate the repair of various tissues and organs.HGF has a certain therapeutic potential in many diseases,including liver fibrosis,acute renal failure,chronic leg wounds and amyotrophic lateral sclerosis and so on.In addition,the clinical safety of HGF in treatment of nephritis has been preliminarily evaluated abroad.It has been reported that HGF could promote liver regeneration and anti-liver fibrosis,suggesting that it may be a therapeutic drug molecule suitable for the treatment ofliver fibrosis.Todesign and evaluate the drug properties of HGF,molecular modification was carried out to ensure its biological activity and improve its stability,providing a potential drug candidate for the treatment of liver fibrosis.The main results were shown as followed:?1?Design and modification of HGF drug candidate moleculesHGF was synthesized and secreted in single inactive precursor form in vivo.Active heterodimers need the extracellular enzyme hydrolysis of HGF 494th restriction sites,and the two subunits assembled with disulfide bond.HGF expressed in fermentation engineering is also an inactive single chain molecule,which needs to be cut and reprocessed in vitro to form an active heterodimer molecule.According to the structural characteristics of mature HGF,the enzyme hydrolysis sites in HGF 494th was mutated to develop mutant molecules HGF?R494E?that do not require activation process.Mutant molecule can promote the scattering of MDCK cells and stimulate the phosphorylation of HGF receptor tyrosine residues on hepatocyte,indicating that single chain HGF mutants maintain biological activity.Because the poor stability and short half-life of HGF,albumin HSA was fusion with HGF?R494E?to form fusion protein HSA-HGF?R494E?according to mature albumin fusion technique in our laboratory.HSA-HGF?R494E?still promote the scattering of MDCK cells and stimulate the phosphorylation of HGF receptor tyrosine residues on hepatocyte,indicating that HSA-HGF?R494E?also maintain biological activity.The half-life study in mice showed that the half-life of HSA-HGF?R494E?increased from 3 min to 3 h,which suggested that the HSA-HGF?R494E?stability in vivo was significantly improved.?2?The Biologic preparation of rhHGF and the expression of HGF?R494E?It has been reported that HGF expressed in E.coli,pichia yeast and CHO system.Because the larger molecular weight,numerous disulfide bonds and the complicated spatial structure,E.coli is not suitable for the expression of HGF.Proteins expressed in pichia yeast maybe caused immune toxicology by the problem of glycosylation heterogeneity,and we choose CHO system in this study.To reference antibody expression system of CHO,the signal peptide suitable for HGF expression was screened.The signal peptide Gaussia luciferase was selected which promote HGF secretion in CHO obviously,and the expression of HGF?R494E?was guided by Gaussia luciferase signal peptide.On the other hand,HGF?R494E?molecules does not require subsequent reprocessing step,result in the simple preparation process.HGF?R494E?has the potential to be a drug candidate for acute diseases such as acute renal failure.?3?Expression of fusion protein HSA-HGF?R494E?in CHO cellsBoth of Pichia pastoris and CHO system have been used to express albumin fusion proteins.For example Albuferon,developed by Novartis company,is a fusion product of interferon and albumin produced by yeast.But a case of death caused by immune toxicity occurred when the drug was used in a clinical study in Germany.Therefore,CHO system was selected as the host cells to express HSA-HGF?R494E?.However,in the process of fusion protein expression,once accumulated to a certain extent of fusion protein in the medium,the target protein no longer increased.When 20 mg?L^-1 ubiquitin ligase inhibitor thalidomide was added at intervals of 2 days in the expression process,the target protein HSA-HGF?R494E?accumulated in the whole process with a yield increased from 50 mg?L^-1 to 130 mg?L^-1.The degradation of CHO intracellular fusion protein resulting from extracellular accumulation of HSA-HGF?R494E?is the major limiting factor for the expression of this protein.Compared to natural protein,the activity of the fusion protein was reduced to about 5%,which was no more than 100 times and met the requirements of synthetic properties.?4?Pharmacodynamic study on anti-liver fibrosis of fusion protein HSA-HGF?R494E?HSA-HGF?R494E?promoted the recovery of hepatocyte damaged by CCl₄ and inhibited the expression of hepatic fibrosis proteins?-SMA and COL I in vitro.Moreover,in vivo,after damaged by CCl₄ for a period of time,liver structure was seriously destroyed.A liver protective drug silymarin can improve liver status significantly after intragastric administration every day.The fusion protein HSA-HGF?R494E?even in a low amount and frequency of intravenous injection?three days?could significantly improve liver function and alleviate fibrosis,which suggested HSA-HGF?R494E?had good liver protection and anti-hepatic fibrosis effect.The novel antifibrotic drug precursor molecule HSA-HGF?R494E?was designed in this study.The main restriction reason for the accumulation of fusion protein in medium was analyzed,and the stable and efficient expression of HSA-HGF?R494E?in CHO was realized.It is important that HSA-HGF?R494E?has obvious effect on liver protection and anti-fibrosis.