Recombinant human interleukin-1 receptor antagonist was expressed in E. coli as an insoluble inclusion body. The inclusion body was dissolved in the 8 M urea and then the solution was diluted untill the concentration of urea became 2 M. By ion exchange chromatography the protein in the solution of 2 M urea was refolded and purified. At last the purity of product is more than 95% and its bioactivity is more than 1 x 10(5) IU/mg while it has little endotoxin. Western-Blotting also indicates that recombinant protein can react with antibodies against anti-hIL-1ra.
Escherichia coli ; genetics ; metabolism ; Humans ; Inclusion Bodies ; metabolism ; Interleukin 1 Receptor Antagonist Protein ; biosynthesis ; genetics ; Protein Folding ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification

Interleukin-1 receptor antagonist (IL-1ra), a member of IL-1 family, is a naturally occurring IL-1 inhibitor as "receptor antagonist", which blocks biological responses mediated by IL-1. Recombinant human IL-1ra (rhIL-1ra, Kineret) was introduced in clinical trials involving patients with RA. Between 2001 to approximately 2002, rhIL-1 ra was approved by the US Food and Drug Administration and the European Agency for the Evaluation of Medicine Procedure. Unfortunately, 10,000 to 100,000-fold excess amounts of IL-1ra are needed to relieve disease because minimal IL-1 can induce complete biological responses, and the dosage of 100 to approximately 150mg/day in a RA patient is so big that it greatly influence patients' physical, psychological and economical situation. In this study, IL-1ra mutants were established by site-specific mutagenesis to improve its stability. The sites of mutagenesis included R6 K7-AA,R93 K94-AA and K97 R98-AA. IL-1ra and its mutants were expressed in E. coli BL21 (DE3) using pTIG-Trx expressing system with the induction of IPTG. The recombinant proteins were purified by Ni2+ chelate chromatography and Sephadex G75 gel filtration chromatography. The activity of mutants is as high as IL-1ra. We characterized the pharmacokinetic profile of IL-1ra and its mutants. The third mutant's half life is 2.26 times than wt IL-1ra. The study has provided some approaches and experience for further research to improve the metabolism stability of IL-1ra.
Animals ; Escherichia coli ; genetics ; metabolism ; Female ; Humans ; Interleukin 1 Receptor Antagonist Protein ; biosynthesis ; genetics ; pharmacokinetics ; Mutagenesis, Site-Directed ; methods ; Mutant Proteins ; biosynthesis ; pharmacokinetics ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacokinetics

Both interleukin-1 and IgE are important in the pathogenic mechanism of the allergy asthma. cDNA of interleukin-1receptor antagonist (IL-1ra) and IgE were cloned and a prokaryotic expression vector IL-1ra-Fcepsilon/pBV220 was constructed. The vector was transformed into Escherichia coli BL21(DE3). The fusion protein was expressed successfully in the form of inclusion body. The recombination protein of IL-1ra-Fcepsilon was highly purified by chromatography of gel filtration and ion exchange, which was identifited by Western blotting. The cell assay showed that the activity of IL-1ra-Fcepsilon was as high as IL-1ra in vitro after refolding. The pharmacokenetic profile of IL-1ra-Fcepsilon and L-1ra was analyzed, and the half time of IL-1ra-Fcepsilon is 4.78 times than that of IL-1ra.
Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Gene Fusion ; Immunoglobulin E ; genetics ; metabolism ; Immunoglobulin Fc Fragments ; genetics ; metabolism ; Interleukin 1 Receptor Antagonist Protein ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; metabolism

OBJECTIVETo investigate the causes and influencing factors of heterogeneity of HSA/IL1ra fusion protein expression in Pichia pastoris.

METHODSThe heterogeneity of HSA/IL1ra fusion protein expressed in Pichia pastoris was studied by removing glycosylation and inhibiting glycosylation, as well as by different ways of fusion, different clones, and different expression host.

RESULTGlycosylation caused expression heterogeneity of fusion protein, but in SMD1168 and some GS115 clones there was no this phenomenon.

CONCLUSIONThe expression heterogeneity of HSA/IL1ra fusion protein in Pichia pastoris is due to the glycosylation, and different ways of fusion, different clones, different expression host also have some impact.

Escherichia coli ; genetics ; metabolism ; Genetic Heterogeneity ; Genetic Vectors ; genetics ; Humans ; Interleukin 1 Receptor Antagonist Protein ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Serum Albumin ; biosynthesis ; genetics

The study investigated the effects of adenovirus-mediated gene transfection of basic fibroblast growth factor (bFGF), bFGF combined with interleukin-1 receptor antagonist protein (IL-Ra) and/or insulin-like growth factor-1 (IGF-1) both in human osteoarthritis (OA) chondrocytes and rabbits OA model. Human OA chondrocytes were delivered by adenovirus-mediated bFGF, IL-Ra and IGF-1 vectors, respectively. Chondrocyte proliferation, glycosaminoglycan (GAG) content, expression of type II collagen, ADAMTS-5, MMP-13, MMP-3 and TIMP-1 were determined. Rabbit OA model was induced by anterior cruciate ligament transaction (ACLT) in knees. Adenoviral vectors encoding human bFGF, IL-Ra and IGF-1 were injected intraarticularly into the knee joints after ACLT. The effects of adenovirus- mediated gene transfection on rabbit OA were evaluated. In vitro, the transfected genes were expressed in cell supernatant of human OA chondrocytes. AdbFGF group significantly promoted chondrocyte proliferation, and increased GAG and type II collagen synthesis than in the OA group. As two or three genes were transfected in different combinations, there was significant enhancement on the GAG content, type II collagen synthesis, and TIMP-1 levels, while ADAMTS-5, MMP-13, and MMP-3 levels were reduced. In vivo, the transfected genes were expressed in synovial fluid of rabbits. Intraarticular delivery of bFGF enhanced the expression of type II collagen in cartilage and decreased cartilage Mankin score compared with the OA control group (P = 0.047; P < 0.01, respectively). Multiple-gene transfection in different combinations showed better results than bFGF transfection alone. This study suggests that bFGF gene transfection is effective in treating experimental OA. Multiple gene transfection has better biologic effects on OA.
Adenoviridae/*genetics ; Animals ; Chondrocytes/drug effects/*metabolism ; Collagen Type II/genetics/metabolism ; Fibroblast Growth Factor 2/*genetics ; Gene Therapy/methods ; Genetic Vectors/administration & dosage/*genetics ; Humans ; Insulin-Like Growth Factor I/*genetics/metabolism ; Interleukin 1 Receptor Antagonist Protein/*genetics/metabolism ; Interleukin-1/genetics/metabolism ; Matrix Metalloproteinase 13/genetics/metabolism ; Matrix Metalloproteinase 3/genetics/metabolism ; Osteoarthritis/*therapy ; Rabbits ; Tissue Inhibitor of Metalloproteinase-1/genetics ; Transfection

OBJECTIVETo assess the association of TNF-α and IL-1RA SNPs with the risk of silicosis in Chinese workers exposed to silica particles.

METHODSCase-control study design was used to enroll 68 silicotic patients induced by silica particles and 68 healthy workers matched for length of silica particle exposure as controls. Both cases and controls were from the same company in southwest China, and each of them was requested to complete a questionnaire. Blood samples were drawn for genomic DNA extraction from each participant. The genotyping of TNF-α (-238 and -308) and IL-1RA (+2018) was performed using polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) and SYBR green-based quantitative polymerase chain reaction (qPCR), respectively. Unconditional logistic regression model was used to estimate odds ratios (ORs) and their 95% confidential intervals (CI) for SNPs.

RESULTSNo significant differences were found between cases and controls in particles exposure length, body mass index (BMI), and status of smoking and alcohol consumption except for age (P=0.001) and blood type (P=0.042). The frequencies of TNF-α (-238) and IL-1RA (+2018) genotypes in cases were significantly different from those in controls, (P=0.001 and P=0.002, respectively), while a borderline significant difference was found in the frequencies of TNF-α (-308) between cases and controls (P=0.063). The variants of three SNPs increased the risk of silicosis in the Chinese workers exposed to silica particles. The adjusted ORs of TNF-α (-308), TNF-α (-238) and IL-1RA (+2018) were 2.8 (95% CI: 1.1-7.5), 20.9 (95% CI: 1.8-236.4) and 4.0 (95% CI: 1.6-10.1), respectively.

CONCLUSIONIt is suggested that cytokine polymorphisms of TNF-α (-238, -308) and IL-1RA (+2018) are associated with the risk of silicosis in the Chinese workers exposed to silica particles. Further independent studies on the interaction between SNPs and exposure to silica particles with a larger sample size are therefore warranted.

Adult ; Asian Continental Ancestry Group ; Case-Control Studies ; Genetic Predisposition to Disease ; Genotype ; Humans ; Interleukin 1 Receptor Antagonist Protein ; genetics ; metabolism ; Logistic Models ; Male ; Middle Aged ; Odds Ratio ; Polymorphism, Genetic ; Silicon Dioxide ; toxicity ; Silicosis ; genetics ; Tumor Necrosis Factor-alpha ; genetics ; metabolism

OBJECTIVETo purify the recombinant human serum albumin fusion protein with interleukin 1 (HAS/IL1ra) and to detect the bioactivity of the fusion protein.

METHODSThe recombinant HAS/IL1ra protein was purified by affinity chromatography and ion exchange chromatography, the bioactivity of the fusion protein was detected by IL1-induced A375 S2 cell killing.

RESULTThe purity of the fusion protein was at least 98 % as assessed by HPLC and the protective effect from the IL1-induced A375 S2 cell killing was similar to natural IL1ra.

CONCLUSIONThe purified recombinant HAS/IL1ra protein in this study has a satisfactory bioactivity.

Apoptosis ; drug effects ; Cell Line, Tumor ; Humans ; Interleukin 1 Receptor Antagonist Protein ; biosynthesis ; genetics ; Melanoma ; pathology ; Pichia ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification ; pharmacology ; Serum Albumin ; biosynthesis ; genetics

In order to increase the plasma half-life and tissue specificity of IL-1 receptor antagonist, a recombinant fusion protein IL-1Ra-HSA, linked by a rigid peptide linker PAPAP, was engineered and expressed by the Pichia pastoris host cells. The fusion protein was secreted to the host cells culture, identified by Western blot, and purified by affinity chromatography. This was followed by a further examination of its bioactivity and pharmacokinetics. Our results demonstrated that the fusion protein retained the antagonist activity of IL-1Ra, capable of binding specifically to the IL-1 receptor on human melanoma A375.S2 cells, and inhibits the cytolytic effect of IL-1beta to A375.S2 cells. Albumin fusion dramatically extended the half-life of IL-1Ra and resulted in a specific accumulation of IL-1Ra in the arthritic paws and a lower distribution of IL-1Ra in other organs such as liver, kidney, spleen and lung in mice with collagen-induced arthritis. The findings reported herein indicate that the fusion protein is likely to have greater clinical applications in areas such as the treatment of rheumatoid arthritis.
Animals ; Apoptosis ; drug effects ; Arthritis, Experimental ; metabolism ; Cell Line, Tumor ; Forelimb ; metabolism ; Half-Life ; Humans ; Interleukin 1 Receptor Antagonist Protein ; genetics ; metabolism ; pharmacokinetics ; pharmacology ; Interleukin-1beta ; toxicity ; Male ; Melanoma ; pathology ; Mice ; Mice, Inbred DBA ; Pichia ; genetics ; metabolism ; Plasmids ; Recombinant Fusion Proteins ; genetics ; metabolism ; pharmacokinetics ; pharmacology ; Serum Albumin ; genetics ; metabolism ; pharmacokinetics ; pharmacology ; Tissue Distribution

BACKGROUNDOsteoarthritis (OA) is a chronic and incurable disease, lacking effective treatment. Gene therapy offers a radical different approach to the treatment of arthritis. Even though the etiology of OA remains unclear, there is now considerable evidence to suggest that interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) are the main mediators in the pathogenesis of OA. The goal of this study was to determine the efficacy of local expression of interleukin-1 receptor antagonist (IL-1Ra) and soluble tumor necrosis factor-alpha receptor type I (sTNF-RI) by direct adenoviral-mediated intra-articular gene delivery in the rabbit model of osteoarthritis.

METHODSAdenoviral vectors containing IL-1Ra or sTNF-RI genes were constructed. OA was induced in both hind knees of 12 New Zealand white rabbits by the excision of the medial collateral ligament plus medial meniscectomy. Five days after surgery, approximately 1 x 10(8) plaque-forming units (pfu) of adenovirus were injected into the joint space of the knee through the patellar tendon. A total of 12 operated rabbits were divided into four groups. Three experimental rabbit groups received 1 x 10(8) pfu of adenovirus encoding either IL-1Ra (3 rabbits), sTNF-RI (3 rabbits) or IL-1Ra and sTNF-RI in combination (3 rabbits), into both knee joints respectively. An inflamed control group of 3 rabbits received approximately 1 x 10(8) pfu of Ad-GFP into both joints. Three days after injection of the adenovirus, both knees of each rabbit were lavaged with 1 ml of saline solution through the patellar tendon. At day 7, the rabbits were sacrificed, and the knees were lavaged, dissected and analyzed for effects of transgene expression. Levels of IL-1Ra and sTNF-RI expression in recovered lavage fluids were measured using a cytokine ELISA kit. Cartilage from the lesion areas of medial femoral condyle and synovium were fixed, embedded, sectioned and stained with hematoxylin and eosin (cartilage and synovium) and toluidine blue (cartilage). The samples were examined by light microscopy and quantitatively evaluated.

RESULTSIntra-articular delivery of IL-1Ra resulted in a significant inhibition of cartilage degradation, but did not affect synovial changes. In contrast, rabbit knee joints receiving sTNF-RI alone showed no detectable reduction in cartilage degradation. However, double gene transfer of IL-1Ra and sTNF-RI resulted in a higher suppression of the cartilage degradation and an observable reduction in synovitis. These data add to and confirm that IL-1Ra has good chondroprotective properties, but TNF-alpha blockade has little effect on joint destruction.

CONCLUSIONThe enhanced therapeutic effects of both antagonists in combination suggest inhibition of multiple inflammatory cytokines may be more efficacious than blockade of either cytokine alone in treating OA.

Adenoviridae ; genetics ; Animals ; Arthritis, Experimental ; genetics ; therapy ; Cartilage ; metabolism ; pathology ; Cartilage, Articular ; metabolism ; pathology ; Cell Line ; Cells, Cultured ; Genetic Therapy ; methods ; Genetic Vectors ; genetics ; Humans ; Interleukin 1 Receptor Antagonist Protein ; Osteoarthritis ; genetics ; therapy ; Rabbits ; Receptors, Tumor Necrosis Factor, Type I ; genetics ; physiology ; Sialoglycoproteins ; genetics ; physiology ; Synovial Fluid ; metabolism ; Synovial Membrane ; cytology ; metabolism ; Transfection ; methods