Pig-human Uric Acid Purification Of Enzyme Modification Research And PEG

Uric acid is the human body, the final product of purine metabolism in, Uric acid into most of purine raw liver enzymes and oxidative metabolism in the liver by, Uric acid70%is excreted in the urine,30%are excluded from the gut。Trigger or exacerbate many diseases due to uric acid and its salts of low solubility in blood and ease of deposition makes hyperuricemia, Hyperuricemia, uric acid crystals deposited acute inflammation and pain caused by peripheral joints is a major cause of gout, severe joint dysfunction can cause gout。The uric acid can stimulate the proliferation of vascular smooth muscle cells, leading to endothelial cell dysfunction。Plasma uric acid is an important risk factor of atherosclerosis and other cardiovascular diseases。 Hyperuricemia, uric acid deposition in the renal tubules can cause kidney dysfunction, high levels of plasma uric acid is acute renal failure, renal tubular injury, a major risk factor for IgA nephropathy inflammation。 Large number of tumor cell lysis in leukemia, lymphoma, myeloma and other blood cancer chemotherapy, a large number of degradation of intracellular nucleic acid, a sharp increase in plasma uric acid, caused by the the hyperuricemia main metabolic disorders caused by patients with kidney failure and death, thisphenomenon called tumor lysis syndrome。Urate oxidase (urate oxidase, uricase) is one kind of enzyme in the process of purine metabolism of uric acid is decomposed into allantoin, carbon dioxide and hydrogen peroxide, and uric acid using molecular oxygen oxidized hydrogen peroxide and uric acid solubility than5times more allantoin. Key enzyme in the metabolism of uric acid。Study abroad uricase quite rapidly in Europe, from aflatoxin prepared uricase treatment with to leukemia chemotherapy-related severe hyperuricemia report; a recombinant aflatoxin uric acid enzyme by Europe and the United States approved for clinical useprevention and treatment of tumor lysis syndrome,But the uricase is a foreign protein, directly or reused easily lead antibodies reduce the efficacy of, and allergies, so for long-term treatment, obviously requires long-acting and non-immunogenicity of uricase preparations PEGylation of uricase can be solvedproblems encountered in the application of natural enzymes,Polyethylene glycol (polyethylene glycol. PEG) is a CH2CH20a basic structure of macromolecules of linear or branched polymers. Protein immunogenicity by the molecular surface of the antigenic determinant the decision. PEG is a linear, a hydrophilic, flexible exclude charged molecules can be used as a barrier, when the combination of it with the protein non-essential group covalently.blocking the protein antigenic determinants.The low molecular weight PEG (less than400D) after in vivo by the action of alcohol dehydrogenase to produce toxic metabolites, shows toxicity: However, when the PEG molecular weight is greater than the IOOOD lose toxicity. Polyethylene glycol having immunological inert, even if the molecular weight of up to5.9×106Da, itself also low immunogenicityo The activated polyethylene glycol coupling protein molecules, the spatial structure of the protein, eventually leading to protein various biochemical changes in the nature:increased chemical stability, resistance to protease hydrolysis ability immunogenicity and reduced toxicity ordisappeared in vivo half-life, plasma clearance was reduced。But currently the treatment of hyperuricemia caused disease or to promote the excretion of uric acid and control acute onset of drug colchicine, non-steroidal anti-inflammatory drugs, using drugs such as the classic allopurinol or probenecid, Medication during most of the make uric acid decreased, but the withdrawal rebound, need long-term medication, many patients can not tolerate the toxicity of the drugs leaving sicker; allopurinol treatment strategies efficacy is not ideal。 Therefore, there is a huge demand for drugs on the market to reduce plasma uric acid; the Allantoin the excellent water-soluble and kidney uricase into allantoin efficient excretion ideal drug candidate for the treatment of hyperuricemia。 High levels of xanthine, should be accompanied by the plasma from the push segment of uric acid metabolism, hyperuricemia experiments also confirmed the hyperuricemia associated with high concentrations of serum xanthine。 Uricase preparations than the higher activity of the treatment required dose of the smaller, lower treatment costs and security anti protection. Therefore, the effectiveness of the anti-xanthine and catalytic uricase is the ideal medicinal uricase treatment of hyperuricemia。Research objective:The engineering bacteria BL21/pET28a completed the induced expression and expression of the target protein after purification through the purity reached to96%and above;Then the high purity uric acid enzyme PEG modification and to explore the optimal modification conditions;Finally the modified uric acid enzyme preparation into freeze-dried powder injection.experimental methods:The BL21/pET28a gene was induced by lactose,Induction of5h at37℃High expression of the product of the enzyme activity and protein content were measured by enzymatic method and Brodford method,Then through the SDS-PAGE protein electrophoresis the protein bands;According to the factors of target protein isoelectric point, ionic strength, the first choice of anion exchange chromatography,Anion exchange chromatography for protein volume is larger, higher protein concentrations, and the separation effect is good, easy to operate and has the characteristics of concentrated action to remove a portion of protein, and the recovery of added sodium sulfate the protein solution,By increasing the concentration effect of hydrophobic interaction protein and hydrophobic interaction chromatography media, so as to improve the hydrophobic protein purified by hydrophobic interaction chromatography, again,In order to further separate the different polymer form and separation of different molecular weight and improve the purity of protein, the molecular sieve purification,At each step of purification are respectively by enzymatic method and Brodford method for the determination of the enzyme activity and protein content, and by SDS-PAGE protein electrophoresis and its purification effect;Then mPEG-SC-lOKD modification of the purification of uricase,The influences of uricase and different PEG molar ratio on the modification reaction,Different uricase protein concentration and modification of PEG results,Different carbonate buffer pH modification effect on PEG results,Different carbonate buffer concentration modification effect on PEG results were studied in these four aspects;Finally the modified uricase adding excipients powder injection, prepared by freeze drying method.experimental results:Induced expression product by protein electrophoresis in0.1M PH6.8phosphate buffer is not soluble protein,After10.14carbonic acid buffer complex precipitation is the size of33KD protein,Breaking all bacteria live detection by enzyme, and uric acid reaction, determination of its high activity of11.7U/ml.Use the Bradford method to determine the content of enzyme protein uric acid is4.9ug/ul, the specific activity was2.4U/mg, enzyme activity of11U/ml complex solution precipitation.Protein content was3.4ug/ul, the specific activity was3.4U/mg,Although the two more than activity increased, but the result is lower, and this uricase protein purity is not high,By protein electrophoresis map can clearly see that contains a lot of Escherichia coli endogenous protein solution, need through the late purified to high purity, high uric acid enzyme specific activity.The insoluble carbonate buffer to phosphoric acid containing the target protein precipitate dissolved in0.02M pH10.14,In the three step chromatography,Protein specific activity by the sample of untreated4.6U/mg increased to15.3U/mg,Purity of sample increased nearly four times, the purity is still low, protein loss of more than20%;In the improved21times by hydrophobic chromatography purification purity, specific activity reached50U/mg, remove most of impurity protein;Finally, through the molecular sieve purification, will eventually target protein uric acid enzyme was increased by26times, the specific activity was62.2U/mg,Than that reported in the literature48U/mg higher than10U.By SDS-PAGE electrophoresis and gel system strips to determine the purity of more than96%.By uricase PEG modified,The best modification results for pH9.16, carbonate buffer solution of0.02mol/l, the enzyme concentration is8mg/ml, modifier in a1:13.5molar ratio for the optimal modification conditions.The experiment with mPEG-SC-lOKD modification agent was modified according to its modifier molecular weight greater shielding effect to the antigen is more ideal, modified with less toxicity and side effect, immunogenic itself is very low;Research on animal experiment of mPEG-SC-5KD modifier due to the shielding effect to the antigen is not ideal, the modified side effects are more, so its preliminary experiment;While the mPEG-SC-lOKD modification agent because of its ideal effect for formal experiment.Finally by freeze drying method for preparation of modified uric acid into powder for injection of2.0ml/bottles.Conclusion:The success of this experiment expresses uricase protein,And the various methods of isolation and purification to the purity reached more than96%,To explore the best conditions of PEG modification and the modification was prepared by powder injection,To lay the foundation for later research in animal experiment.