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High temp. resistant guanylate kinase gene, its coded polypeptide and preparing method thereof

A high temperature resistant, guanylic acid technology, applied in the field of mutation or genetic engineering, can solve problems affecting binding objects, etc.

Inactive Publication Date: 2003-05-28
HUADA GENE RES & DEV CENT HANGZHOU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This supramolecular regulation may affect binding in the GK domain

Method used

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  • High temp. resistant guanylate kinase gene, its coded polypeptide and preparing method thereof
  • High temp. resistant guanylate kinase gene, its coded polypeptide and preparing method thereof
  • High temp. resistant guanylate kinase gene, its coded polypeptide and preparing method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Construction of a sequencing library

[0039] The sequencing library was constructed using the genome-wide shotgun method. Firstly, Tengchong thermophilic anaerobic bacteria were cultured according to (Yanfen Xue, 2000) modified MB medium (Balch et al., 1979), bacteria were collected according to Marmur (1961) method, and total DNA was extracted. In order to ensure the randomness of sequencing library construction and avoid the problem of breaking hot spots to the greatest extent, the Dolly method and the principle of library construction under different conditions were adopted. First, physical shearing methods (including ultrasonic method and HydroshearMachine shearing) were used, and then AluI was selected for random partial enzyme digestion according to the genome characteristics of the bacteria. Different intensities are used to treat rods during physical shearing, and samples are treated by setting enzyme gradients during enzyme digestion. After the pr...

Embodiment 2

[0040] Example 2: Genome Sequencing

[0041] When sequencing the genome of thermophilic anaerobic bacteria in Tengchong, two automatic sequencers were mainly used: ABI377 and MegaBACE 1000. Both sequencers use the principle of electrophoresis for sequencing (see figure 2 ), 96 samples can be completed each time. ABI377 is a product of PE Company, which is a kind of ABI series. It belongs to the slab gel electrophoresis sequencer. MegaBACE 1000 is a product of Pharmacia, which belongs to capillary gel electrophoresis sequencer.

Embodiment 3

[0042] Example 3: Basecalling and sequencing quality monitoring

[0043] The so-called basecalling refers to the process of obtaining the correct base sequence from the raw data file obtained on the sequencer. Since the sequencer obtains the intensity change traces (traces) of light of different wavelengths corresponding to the four bases A, T, G, and C, it is necessary to use a computer to adopt a certain algorithm to correctly identify the bases corresponding to the different traces. . We used Phred software (Ewing B, Hillier L, 1998) because its results are more reliable and its output is more convenient for further analysis by other programs in the same software package.

[0044] The algorithm principle of Phred's basecalling is to judge the base type based on the shape, distance, and signal-to-noise ratio of each peak in the trajectory, and at the same time give the credibility information for this base, that is, the sequencing quality of the base. In large-scale sequen...

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Abstract

A process for coding the separated DNA with activity or its function equivalent variant and preparing the polypeptide or its function equivalent variant with high-temp resistant guanylate kinase activity from said DNA by recombination is disclosed. The high-temp. resistant guanylate kinase gene is cloned and separated, which can be used to prepare the transgenic microbe, animal, or plant and recover the enzyme coded by said gene. The amino acid sequence of said polypeptide and the processes for preparing, separating and purifying said polypeptide are disclosed.

Description

technical field [0001] The present invention relates to mutation or genetic engineering, in particular to a high temperature resistant guanylate kinase gene sequence, encoded polypeptide and preparation method. Background technique [0002] Guanylate kinase (GUK) is a critical enzyme in the process of nucleoside synthesis. It catalyzes the following reactions: [0003] (d) GMP+ATP => (d) GDP+ADP. This step plays an important role in restoring cyclic GMP and regulating the balance of ATP and GMP in the cell. Therefore, guanylate kinase is considered to be a fundamental protease in second messenger information transduction, a core protease in nucleotide metabolism, and a reversible reaction that catalyzes the reversible reaction of terminal phosphoryl polymers from ATP to ADP. [0004] cGMP and nitrogen oxide signaling are associated with natriuretic polypeptide and guanylin-binding signaling, and intracellular targets of cGMP in...

Claims

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Application Information

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IPC IPC(8): C12N9/00C12N9/12C12N15/52C12N15/54C12N15/63C12N15/64
Inventor 于军李蔚张丽敏胡松年田宇清
Owner HUADA GENE RES & DEV CENT HANGZHOU
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