Hieh-level Expression Of IL-1ra-G-HSA Fusion Protein And Its Preliminary Pharmacodynamic Study On Animals

Cytokines are classified as proteins or peptides, which function as a category of signaling molecules in intercellular communication and immuno modulating agents. The levels of cytokines are controlled by a complex regulatory system in the body. When agonists are overexpressed, receptor antagonists can inhibit the function of agonists by bind their receptor but without efficacy for their cognate receptors. Interleukin-1receptor antagonist (IL-1ra) was first discovered in1984, belongs to the interlukin-1cytokine family. It binds to the interlukin-1receptor and inhibits of the pro-inflammatory effect of interlukin-1, alpha and interlukin-1, beta. Many reports demonstrated that interlukin-1, alpha and interlukin-1, beta was associated with increased risk of autoimmune disease (i.e., rheumatoid arthritis). Kineret is an recombinant form of IL-1ra produced by the pharmaceutical company Amgen, and have approved for treatment of rheumatoid arthritis by FDA and EMEA. Since2009, several animal and human experiments revealed that IL-1ra could also reduce hyperglycemia in the type2diabetes.However the molecular weight of kineret is only17.3kDa, which is susceptible to excretion in the glomerular and has a half-life of4-6hours. In order to achieve effective plasma drug concentration for patients, subcutaneous injection twice per day is required. Frequent administration increases the burden of financial, physiological and psychological for patients, therefore it is necessary to develop the long-acting IL-1ra analog in clinical. Human serum albumin (HSA) is the most abundant protein in circulatory system, because of its widely distribution in the body, long half-life and low immunogenicity, it is an ideal candidate for producing long-acting protein drug. In the present study, the IL-1ra cDNA was fused to the amino terminus of the cDNA encoding HSA, and the fusion gene was expression in Pichia pastoris. The results of the present study may provide theoretical and experimental basis for development of long-action IL-1ra analog.Construction of fusion protein expression vector and transformation of P.pastorisIL-1ra gene with flanking Xho I and BamH I restriction sites was amplified from pET41a-IL-1ra, while HSA gene with flanking BamH I and EcoR I restriction sites was amplified from pGEM-T-HSA. The PCR products of IL-1ra and HSA were digested with Xho Ⅰ/BamH Ⅰ and BamH Ⅰ/EcoR I, respectively. Then the above two digested fragments were ligated into the vector pPIC9cut with Xho1/EcoR I, to create pPIC9-IL-1ra-G-HSA. pPIC9-IL-1ra-G-HSA validated by DNA sequencing for correct sequence was introduced in to P. pastoris strain GS115. The positive transformants were confirm by PCR analysis with primers a factor and3’-AOX.Expression of IL-1ra-G-HSA, establishing quality control standards and determination its half-life bioactivityIL-1ra-G-HSA was expressed in19L fermenter, and then purified by ultrafiltration and Chroma to graphy. Quality control standards were established for the purified fusion protein, immunoassay showed that it could react with both IL-1ra antibody and HSA antibody. Bioactivity assay of the purified IL-1ra-G-HSA in vitro revealed that still, it had the ability to bind with interleukin-1receptor. Pharmacokinetic analysis showed that the biological terminal half-life of IL-1ra-G-HSA was about20-fold longer than that of IL-1ra.Study of the degraded fragment of IL-1ra-G-HSA expressed in P. pastoris Expression of IL-lra-G-HSA in P. pastoris strain GS115resulted in aberrant proteolysis of the corresponding protein. The results of Western blotting, N-terminal sequence and MALDI-TOF showed that the degraded product of fusion protein contained complete sequence of IL-lra but C-terminal sequence of HSA was missing. The YPS is a family of aspartic proteases that have common substrate specificity to cleave at basic amino acid residues. The proteinase A and B are key proteases in vacuolar activation cascade, which may also be involved in the degradation process of HSA. In the present study, the SMD1168strain, SMD1163strain and seven yps-deficient mutant strains were compared with GS115strain for expression of cleaved IL-1ra-G-HSA fusion protein. It was confirmed that deficient of proteinase A in GS115strain could had the ability to reduce the degradation of fusion protein but not single yps protease-deficient strainsThe effect of gene copy number and co-expression of chaperone on production of IL-1ra-G-HSATo improve the expression level of IL-1ra-G-HSA, we examined the effect of gene copy number or co-expression of two chaperones such as PDI and BiP with high gene copy number on IL-1ra-G-HSA expression in P. pastoris. The IL-1ra-G-HSA fusion gene was ligated into the vector pPICZaB to give pPICZaB-IL-1ra-G-HSA, and then the pPICZaB-IL-1ra-G-HSA was introduced into P. pastoris strain GS115for construction multi-copy strain. It was observed that the levels of secretory IL-1ra-G-HSA produced in strain with higher IL-1ra-G-HSA gene copy number increased about1.6fold. Base on the multi-copy strain, the expression level of IL-1ra-G-HSA was further improved about2.4fold after being co-expressed with protein disulfide isomerase (PDI)IL-lra-G-HSA reduces hyperglycemia in GK ratRecently, many experiment and clinical studies revealed that type2diabetes was associated with cytokine imbalance, therefore some scientists advocated that inflammatory mediators trigger islet P-cell apoptosis and islet dysfunction. Treatment of type2diabetes animal and human with IL-1ra were found reduced hyperglycemia and increased β-cell function. Goto-Kakizaki (GK) rat is a well known genetic model of type2diabetes, it is reported that IL-1ra treatment reduced GK hyperglycemia. Therefore, in this study, GK rat was used as the model for investigation of the bioactivity of IL-1ra-G-HSA. Taking into account the half-life of IL-1ra-G-HSA, the administration frequency of IL-1ra-G-HSA was reduced to once daily. The overall results showed that IL-1ra-G-HSA could reduce hyperglycemia in GK rat.The most important discoveries in this paper1. Pharmacokinetic analysis showed that the biological terminal half-life of IL-1ra-G-HSA was about20-fold longer than that of IL-1ra.2. The expression level of IL-1ra-G-HSA was improved about4fold after increasing the gene copy number and being co-expressed with protein disulfide isomerase.3. IL-1ra-G-HSA could reduce hyperglycemia in GK rat.