Low temperature alkaline phosphatase and preparation method thereof

A phosphatase and alkaline technology, applied in the field of genetic engineering, can solve problems such as high acquisition cost and inactivation, and achieve the effects of low production conditions and equipment requirements, easy operation and increased yield

Inactive Publication Date: 2013-01-02
FUDAN UNIV
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

However, the acquisition cost of the enzyme is relatively high, and it is easy to be gradually inactivated in the reaction

Method used

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  • Low temperature alkaline phosphatase and preparation method thereof

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Embodiment Construction

[0027] Use polymerase chain reaction (PCR) method to amplify the TAB5-AP sequence, digest the fragment and vector plasmid with restriction enzymes, and then insert the fragment into the vector to construct a TAB5- Plasmid of AP fragment. Finally, it was transferred to E. coli competent for positive screening.

[0028] Pick a positive single clone and activate it overnight in 5ml LB culture medium containing 100ug / ml ampicillin, then transfer it to 1L auto-induction medium, culture it at 37℃ for 4 to 5 hours, and place it in 20℃ low temperature induce expression for 15 hours. After induction of expression, the bacteria were collected by centrifugation at 5,000 g for 20 minutes.

[0029] Resuspend the bacteria with Ni-NTA-eluted Buffer A (20mM Tris-HCl, pH 8.0, 150mM NaCl, 10% glycerol and 10mM imidazole), add 0.2% Triton-X100 and sonicate the bacteria at a speed of 12,000g Centrifuge at 4°C for 20 minutes to take the supernatant. First use Ni-NTA affinity column for rough separa...

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Abstract

The invention belongs to the field of gene engineering, and relates to novel alkaline phosphatase and a preparation method thereof. The cDNA sequence of low temperature alkaline phosphatase shown as SEQ ID NO 1 in a sequence table is cloned to a vector for genetic engineering, and expressed to obtain the low temperature alkaline phosphatase. According to codon bias of Escherichia coli, the DNA sequence of the TAB5 low temperature alkaline phosphatase is transformed, so that the DNA sequence is highly expressed in the Escherichia coli; the alkaline phosphatase is purified by nickel affinity chromatography and ion exchange chromatography; and more than 90mg of TAB5 alkaline phosphatase with the purity of over 99 percent is obtained from each liter of Escherichia coli liquid finally. The invention can be applied to research in the field of molecular biology of the field of life science, particularly multi-step reaction for heat inactivation of alkaline phosphatase, such as sample treatment before DNA sequencing and the like.

Description

Technical field [0001] The invention belongs to the field of genetic engineering and relates to a DNA sequence. Specifically, the present invention relates to an alkaline phosphatase Alkaline Phosphatase (TAB5-AP) existing in the Antarctic strain TAB5, and also relates to a method for expressing and purifying the protein in an E. coli expression system. Background technique [0002] Alkaline phosphatase is a non-specific enzyme widely present in various organisms and plays an important role in organisms. Most alkaline phosphatases are homodimers composed of two identical subunits, and there are 10 groups of conservative β-sheet sequences in the middle of the enzyme. As an enzyme commonly used in laboratories, alkaline phosphatase is often used to non-specifically dephosphorylate groups in molecular cloning and weaken the environment from empty vectors or recombinant vectors. The phosphatase-treated fragment lacks the 5'phosphoryl tag, so it can avoid self-cyclization and reduce...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/16C12N15/55C12N15/70
Inventor 丁澦陆致晟
Owner FUDAN UNIV
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