Refractory glutamic imine methyl transferase gene, polypeptide coded by it and preparing process

A glutamic acid imine methyl, high temperature-resistant technology, applied in the field of mutation or genetic engineering, can solve problems to be studied, etc.

Inactive Publication Date: 2002-08-28
HUADA GENE RES & DEV CENT HANGZHOU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although the structure of glutamate imine methyltransferase is known, its role in biological function remains to be investigated

Method used

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  • Refractory glutamic imine methyl transferase gene, polypeptide coded by it and preparing process
  • Refractory glutamic imine methyl transferase gene, polypeptide coded by it and preparing process
  • Refractory glutamic imine methyl transferase gene, polypeptide coded by it and preparing process

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Construction of a sequencing library

[0036] 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 hot spots of breakage to the greatest extent, a variety of methods and principles of library construction under different conditions were adopted. . Firstly, physical shearing method (including ultrasonic method and shearing with Hydroshear Machine) was used, and then AluI was selected for random partial enzyme digestion according to the genome characteristics of the bacteria. Different intensities were used to process samples during physical shearing, and samples were processed by setting enzyme gradients during enzyme di...

Embodiment 2

[0037] Example 2: Genome Sequencing

[0038] 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

[0039] Example 3: Basecalling and sequencing quality monitoring

[0040] 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.

[0041] 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 refractory glutamic imine methyltransferase gene, the polypeptide coded by it and its preparing process are disclosed. On the basis of full-genom sequencing and analyzing of a thermophilic anaerobe, the said refractory glutamic imine methyltransferase gene is cloned and separated. It can be used to prepare the transgenic microbes or animal or plant for producing the said gene and recovering theenzyme coded by the said gene. The amino acid sequence for the polypeptide with activity of the said gene, the function-equal object and a process for preparing, separating and purifying the said polypeptide are also disclosed.

Description

technical field [0001] The invention relates to mutation or genetic engineering, in particular to a gene sequence of a high-temperature resistant glutamic acid imine methyltransferase, a coded polypeptide and a preparation method. Background technique [0002] This gene encodes a thermostable glutamate imine methyltransferase. Glutamate imine methyltransferase, also known as glutamate formyltransferase, catalyzes formate metabolism (from 5-formyltetrahydrofolate to L-glutamate). The function of glutamate imine methyltransferase is to maintain the balance of conversion from 5-formyltetrahydrofolate to leucovorin. It is a member of the glutamate-determined iminomethyltransferase family. Glutamate imine methyltransferase has a theoretical pH value: 5.58, and the protein is estimated to be an unstable compound (instability index 42.55). Estimated half-lives of glutamate imine methyltransferase in vitro are 30 hours for mammals, less than 20 hours for y...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/63
Inventor 汪建李蔚王俊田微包其郁
Owner HUADA GENE RES & DEV CENT HANGZHOU
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