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Fusion collagenase to which affinity tag is attached, and method for producing same

A technology of collagenase and marker, applied in the field of collagenase

Active Publication Date: 2011-10-12
MEIJI SEIKA KAISHA LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Furthermore, there is no report on the decomposition of collagenase when the collagenase derived from Clostridium histolyticum is expressed as a recombinant protein in the case of Escherichia coli or the like as a host

Method used

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  • Fusion collagenase to which affinity tag is attached, and method for producing same
  • Fusion collagenase to which affinity tag is attached, and method for producing same
  • Fusion collagenase to which affinity tag is attached, and method for producing same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] [ Example 1 ] Expression of a fusion collagenase with a histidine tag attached to the carboxyl terminus of collagenase G derived from Clostridium histolytica (fusion collagenase G)

[0084] (1-1) Preparation of collagenase G gene fragment

[0085] The 5' side end to the 3' side end of the Clostridium histolyticum-derived collagenase G gene were amplified by PCR using the Clostridium histolyticum-derived genomic DNA as a template. At this time, it is designed to form at the 3' side end of the amplified collagenase G gene Xba I recognize the sequence, and add the stop codon linked to the gene Bam Primers for the HI recognition sequence. As a result, mutations were introduced at two positions (amino acids at positions 1007 and 1008 in SEQ ID NO: 1) of the carboxy-terminal amino acid sequence of native collagenase G described in Non-Patent Document 1, and the sequence numbers were changed to: The amino acid sequence described in 2.

[0086] Primers used in this PCR ...

Embodiment 2

[0112] [ Example 2 ]Cultivation of Fusion Collagenase G Expressing Escherichia coli and Selective Recovery of Collagenase G with CBD

[0113] (2-1) Culture of Escherichia coli expressing fusion collagenase G

[0114] The fused collagenase G-expressing Escherichia coli obtained in Example 1 was inoculated into a 250 ml Erlenmeyer flask supplemented with 100 ml of medium, and cultured with stirring at 200 rpm at 28° C. for 16 hours. The culture medium used in this cultivation is TB culture medium (1.2% tryptone, 2.4 % yeast extract, 0.94% dipotassium hydrogen phosphate, 0.22% potassium dihydrogen phosphate, 0.8% glycerin).

[0115] (2-2) Preparation of Extracts of Collagenase G Expressing Escherichia coli

[0116]The culture-terminated solution obtained in (2-1) was centrifuged to recover bacterial cells, and the recovered bacterial cells were lysed with 10 ml of POPculture Regent (manufactured by Merck Corporation) to extract proteins in the bacterial cells. In order to re...

Embodiment 3

[0121] [ Example 3 ] Expression of a fusion collagenase with a histidine tag linked to the carboxyl terminus of collagenase H derived from Clostridium histolyticum

[0122] (3-1) Preparation of Collagenase H Gene Fragment

[0123] The 5' side end to the 3' side end of the Clostridium histolyticum-derived collagenase H gene were amplified by PCR using the Clostridium histolyticum-derived genomic DNA as a template. At this time, it is designed to form at the 3' side end of the amplified collagenase H gene Xba I recognize the sequence, and add the stop codon linked to the gene Bam Primers for the HI recognition sequence. As a result, a mutation was introduced at one position (amino acid at position 980 of SEQ ID NO: 5) in the carboxy-terminal amino acid sequence of the amino acid sequence of native collagenase H described in Non-Patent Document 2, and changed to SEQ ID NO: 6 The amino acid sequence described in.

[0124] Primers used in this PCR are as follows.

[0125] ...

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Abstract

A fusion collagenase obtained by attaching an affinity tag to a carboxyl terminal of a collagenase is expressed as a recombinant protein. By purifying the thus-obtained fusion collagenase by affinity chromatography, a collagenase having a collagen-binding site can be selectively collected.

Description

technical field [0001] The present invention relates to a collagenase used for isolating cells (blocks) such as islets from internal organs such as pancreas. Background technique [0002] As a radical treatment for diabetes, islet transplantation of islets (insulin-producing cells) isolated from protease-treated pancreas by dripping into the portal vein of diabetic patients is known. This transplantation method has attracted attention in recent years as a safe and simple radical treatment of diabetes because it does not require an open surgery on the patient. [0003] The isolation of islets from the pancreas is carried out by treating the pancreas with collagenase and neutral metalloprotease. As the collagenase used for this purpose, Clostridium histolyticm-derived collagenase is particularly effective (non-patented Literature 1, 2). [0004] Collagenase derived from Clostridium histolyticum is known to consist of collagenase G and collagenase H, two enzymes having differ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/00C07K19/00C12N1/21C12N5/071C12N9/52C12N15/09
CPCC07K14/78C07K2319/21C12N9/52C07K19/00C12N15/62C12N15/70
Inventor 福岛崇惠横山健吾村岛弘一郎后藤昌史山形洋平
Owner MEIJI SEIKA KAISHA LTD
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