Production method of escherichia coli expressed human iduronate-2-sulfatase

Abstract

The invention discloses a production method of escherichia coli expressed human iduronate-2-sulfatase. The method employs a 5L fermentation tank of escherichia coli recombinant bacteria for high-density culture and induction expression, the fermentation broth subjected to induction expression is centrifuged to collect thalli, high-pressure homogenization technique is employed to crush the recombinant bacteria to obtain a recombinant protein inclusion body, the recombinant protein inclusion body is dissolved and is then loaded to a Ni column for affinity chromatography purification, and after desalination and freeze-drying, the recombinant human iduronate-2-sulfatase can be obtained. The method provided by the invention simplifies the tedious washing steps of the inclusion body, explores appropriate ultrasonic conditions to transform insoluble inclusion body protein into soluble protein, avoids the denaturation and renaturation operations on the inclusion body brought about by use of 8Murea, 6M guanidine hydrochloride or other strong denaturing agents, and can achieve a protein purity of 95% or more and a recovery efficiency of 70% or above by Ni column affinity chromatography, thus reaching the effect of simple, effective and rapid purification.

Description

technical field [0001] The invention relates to a production method for expressing human iduronate-2-sulfatase in Escherichia coli. Background technique [0002] Mucopolysaccharidosis type II, also known as Hunter syndrome, is an X gene-linked recessive genetic disease. The inability to metabolize leads to a large accumulation of dermatin and heparan sulfate in various organs and tissue lysosomes of the whole body, causing disease. With the accumulation of glycosaminoglycans, mucopolysaccharides gradually accumulated in the body can lead to diseases such as cell congestion, organ enlargement, tissue destruction and system dysfunction. On July 24, 2006, the FDA approved ShireHumanGenetic Therpapies' idose sulfatase injection, whose trade name is Elaprase, as the first drug for the treatment of the rare genetic disease MPSII. In foreign countries, human idose-2-sulfatase is expressed and produced by mammalian CHO cells. The literature "Low-scale expression and purification o...

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Problems solved by technology

In foreign countries, human idose-2-sulfatase is expressed and produced by mammalian CHO cells. The literature "Low-scale expression and purification of an Active putative induronate 2-sulfate sulfatase-Like enzyme from Escherichia coli K12" has expressed similar human idose Duuronate-2-sulfatase, but its gene sequence is not the full-length sequence, and the gene sequence has been artificially modified. At present, there is no expression of human iduronate-2-sulfatase in Escherichia coli in China Full-length gene related literature

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