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Preparation method of recombinant uricase

A uricase and recombinant protein technology, applied in the fields of genetic engineering and protein biochemistry, can solve the problem of not solving the method of efficient fusion expression of uricase, etc.

Inactive Publication Date: 2015-02-11
吉林金梓源生物科技股份有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0009] According to the knowledge of the inventors, the prior art has not yet solved the method of efficiently expressing uricase in Escherichia coli, and at the same time provides a method for preparing purified recombinant uricase soluble uricase with high purity, high activity and high recovery rate

Method used

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  • Preparation method of recombinant uricase
  • Preparation method of recombinant uricase
  • Preparation method of recombinant uricase

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0119] Cloning and expression of embodiment 1 Candida utilis uricase

[0120] 1. Extraction of Candida utilis genomic DNA: After resuscitating the freeze-dried Candida utilis (2.0120) strain according to the instruction manual, inoculate it into YPD culture (yeast extract 5g, peptone 10g, glucose 10g, add water to 500ml, Adjust the pH to 6.0) and cultivate in shake flasks at 30°C. After two consecutive generations of culture, Candida utilis can be seen to grow normally. The cells were collected by centrifugation, and the genomic DNA was extracted with a yeast genomic DNA extraction kit.

[0121] 2. Amplification of the target gene: According to the gene sequence of Candida utilis uricase in GenBank, a pair of primers were designed and synthesized (synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.), and NdeI and HindIII restriction sites were introduced into the primers.

[0122] P1: 5'-ggA ATT CCA TAT gTC AAC AAC gCT CTC ATC-3'

[0123] P2: 5'-CCC AA...

Embodiment 2

[0128] Purification and identification of embodiment 2 recombinant uricase

[0129] Purification of the target protein: centrifuge the induced bacterial solution at 5000r / min and 4°C for 20min, collect the bacterial cells, wash them twice with PBS (pH7.4), resuspend the bacterial cells with PBS, and ultrasonically disrupt the bacterial cells ( The whole process was carried out in an ice bath), centrifuged at 12000r / min, 4°C for 45min, and the supernatant was collected; the supernatant was subjected to ammonium sulfate fractional precipitation, and the recombinant uricase was mainly contained in the 40%-60% ammonium sulfate saturation fraction Protein, the precipitate of this fraction was collected, dissolved in 50mmol / L glycine buffer (pH10.0), desalted with Sephadex G25, and then purified with Resource Q anion exchange column ( Figure 10 ), collected the eluate containing recombinant uricase, and carried out molecular sieve purification with a Superdex 75 column ( Figure 1...

Embodiment 3

[0132] The activity measurement of embodiment 3 recombinant uricase

[0133] Activity analysis: The method reported by Legoux et al. (1992) was used. The amount of enzyme required to oxidize 1 μmol of uric acid to allantoin per minute is 1 International Unit (IU) of enzyme activity. In a 5ml reaction system, add 3ml triethanolamine buffer solution (pH8.9), 1.5ml 0.2μmol / L uric acid stock solution, 1μg (10μl) purified uricase, react at 30°C for 5min, then add 0.5ml 20% KOH was used to terminate the reaction, and the concentration of uric acid was measured with a detection wavelength of 292nm.

[0134] The activity of the enzyme was calculated according to the standard curve, and there was no difference in the activity of the recombinant uricase expressed from the wild-type Candida utilis uricase gene and the optimized sequence, and the specific activities were 36.1U / mg and 37.9U / mg, respectively.

[0135] The above results are summarized in Table 1.

[0136] Table 1 Activity...

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Abstract

The invention provides a nucleotide sequence suitable for secretory expression of recombinant candida utilis uricase (uricase, EC1.7.3.3) by virtue of escherichia coli, a carrier containing the nucleotide sequence, an engineering cell containing the carrier, and a method for purifying the recombinant candida utilis uricase from the engineering cell. On the basis of a natural sequence of a uricase gene, a codon of the uricase gene is optimized, the transformed uricase gene is cloned to a prokaryotic expression vector pET42a(+); an escherichia coli Rossetta strain is transformed; the expression level can reach 50% of soluble protein of the escherichia coli by IPTG induction; the expressed protein is soluble protein; and the biological activity of the expressed protein can be up to that of 37.89U / mg recombinant protein. The recombinant candida utilis uricase product can be efficiently and simply obtained at low cost. The invention also comprises an application of the recombinant candida utilis uricase prepared by the method in preparation of medicines, foods, health care products and cosmetics for treating hyperuricemia-related diseases or pathological symptoms of a human body.

Description

technical field [0001] The present invention relates to a method for efficiently expressing Candida utilis uricase recombinant protein in Escherichia coli using DNA recombination technology and using related protein purification technology to separate and purify recombinant uricase, including the uricase recombinant protein prepared by the method and its use, It belongs to the field of genetic engineering and protein biochemistry. Background technique [0002] With the improvement of people's living standards, the intake of high-protein foods has increased year by year, and the incidence of hyperuricemia and gout has gradually increased. Hyperuricemia and gout are related to obesity, hyperlipidemia, and hypertension. , diabetes, atherosclerosis and other diseases are significantly positively correlated, while gout can not only invade bones and joints, but also easily involve the kidneys and cardiovascular system, which is extremely harmful. [0003] Uricase is an enzyme in ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/06C12N15/53C12N15/70C12N1/21C12R1/72
CPCC12N9/0048C12Y107/03003
Inventor 李慎涛庞海刘爽
Owner 吉林金梓源生物科技股份有限公司
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