Construction Expression And Activity Identification Of Recombinant HSA/IL 1ra Fusion Protein

IL1ra which is the first described naturally occurring specific receptor antagonist of any cytokine or hormone is a member of IL1 gene family. The binding of IL1ra to the IL1 receptor on the cell surface is almost irreversible and shows no agonist activity in the body. By occupying the IL1 receptor IL1ra effectively stops the IL1 signal transduction. There is increasing evidence that IL1 is a key mediator in the autoimmune disease rheumatoid arthritis (RA). Because of effectively stops the IL1 signal transduction IL1ra became a potential drug to RA.In 2001 recombinant human Interleukin-1 receptor antagonist (rhIL1ra) has been approved by FDA and EMEA as the first of a new class of drugs to treat RA. But IL1ra's half-life is short because of its small molecular weight. So rhIL1ra must be given daily by subcutaneous injection produced a sustained clinical response at 24 weeks. Therefore it's necessary to develop some kind of long-acting rhIL1ra for optimizing treatment effect.Human serum albumin (HSA) is the major protein component of human plasma. Due to its remarkably prolonging half-life together with its wide in vivo distribution and its lack of enzymatic and immunological functions, HSA represents an optimal natural carrier for therapeutic peptides aimed at interacting with cellular or molecular components in vivo. The development of genetic engineering has opened up the possibility to obtain recombinant proteins without the danger of contamination of human pathogens. HSA/IL1ra fusion protein was produced as a recombinant protein composed of human serum albumin genetically fused at its C-terminus to the N-terminus of IL1ra in Pichia pastoris. The HSA/IL1ra fusion protein could be given less frequently than IL1ra to achieve similar therapeutic effects. It would have great potential in research and development of long-acting preparation on RA treatment.1. Construction of pPIC9-HSA/ILlra recombinant expression plasmidHSA gene was amplified from cDNA library of human liver by PCR. The PCR product was isolated and ligated with pGEM-T vector. Construct HSA/IL1ra fusion genes and subclon them into expression vector pPIC9. The recombinants were identified by endonuclease digestion (with PstI,XhoI and EcoRI) and the result indicated the target gene had been successfully cloned into pPIC9 vector. The pPIC9-HSA/ILlra recombinant plasmid had been finally identified by sequencing.2. Transformation of Pichia and PCR identification of Pichia transformantThe linearized pPIC9-HSA/ILlra recombinant plasmids were digested by SaiI and transformed GS115 (SMD1168) competent cells by electroporation. Transformants were growed on RDB without histidine after 72h at 30℃. Using genomic DNA isolated from Pichia clones as templates. Amplified the interert gene by primering withα-factor-up and AOX1-down. The PCR results showed that the expected size of our interest gene was about 2.2kb. This provided information that the gene of fution protein had been integrated into Pichia genome.3. Expression of recombinant fution proteinPicking up several identified recombinant colonies, cultivated in BMGY medium in shake flasks (250rpm/min) at 30℃. After one day of cultivation, the cells were transferred to BMMY medium and 100% methanol was added to final concentration of 1% for induction of the recombinant proteins every 24h. After additional 72h of cultivation, the cells were harvested and the supernatants were collected for expression analysis. SDS-PAGE results indicated the recombinant protein with 84kD as expected had been expressed through secretion way and the production of recombinant protein increased with induced time extended. The yield of HSA-G-IL1ra at 72h was about 200mg/L4. Purification of HSA-G-ILlra fusion proteinThe fermentation supernatant was concentrated through ammnonium sulfate precipitation then was purified through affinity column,ion exchange column and desalting column with AKTA Exerplorer. The purity of fusion protein exceeded 90% by HPLC analysis.5. Western Blotting analysis of HSA-G-ILlra fusion proteinWestern Blotting result demonstrated that fusion protein could be recognized by rat-anti-IL1ra antibodies and rabbit-anti-HSA antibodies. This result proved that the recombinant expression product was included two domains of HSA and IL1ra.6. Activity of fusion proteinThe Activity of fusion protein was determined by inhibiting the lethal effect of IL1 to A375.S2 cells. The results suggest that the fusion protein could effectively inhibit the lethal effect of IL1 to A375.S2 cells with dose-dependent trend.7. Pharmacokinetics of fusion proteinHSA-G-IL1ra fusion protein was administered subcutaneously (SC) to mice and the serum samples were collected at different times. The drug concentration in serum was analyzed by ELISA. Pharmacokinetic parameters were calculated with SARS software. The t1/2 CL of the fusion protein was 8.125h,0.182L/h/kg, respectively. A seventeenfold increase in plasma half-life and a sixfold decrease in clearance were seen with HSA-G-IL1ra fusion protein compared to IL1ra protein alone. These results suggested HSA-G-IL1ra fusion protein had a better long-acting effect compared to IL1ra.8. Structure identification of fusion proteinThe structure of fusion protein was identified by Western Blotting aminoacid-sequencing and circular dichroism spectra. The result indicated that the fusion protein simultaneously possess the correct amino-acid sequences and structure of HSA and IL1ra. Concluding as following:HSA/IL1ra fusion proteins were successfully expressed and purifide from the Pichia. pastoris system. Using SDS-PAGE Western Blotting amino-acid sequencing and circular dichroism spectra assay, HSA-G-ILlra fusion protein had been identified. The bio-activity of fusion protein was determined by inhibiting the lethal effect of IL1 to A375.S2 cells. The results suggest that the fusion protein had a similar bioactivity with ILlra and could effectively inhibit the lethal effect of IL1 to A375.S2 cells with dose-dependent trend. The pharmacokinetic studies of HSA-G-IL1ra fusion protein proved that the fusion protein had a better long-acting effect than ILlra. These studies suggested that the fusion protein could be given less frequently than ILlra to achieve similar therapeutic effects in patients.