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High temp. resistant Nus G protein gene and coded polypeptide and preparation process thereof

A high temperature, protein technology, applied in the field of mutation or genetic engineering

Inactive Publication Date: 2003-05-21
HUADA GENE RES & DEV CENT HANGZHOU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with E.coli, the rabbit antiserum after the expression product was immunized with rabbits, no cross-antiserum was found in the Western blotting method; however, it was found that the NusG gene of the two had 45% identity, 22.5% similar amino acid sequence, and most of the similarity C-terminal region

Method used

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  • High temp. resistant Nus G protein gene and coded polypeptide and preparation process thereof
  • High temp. resistant Nus G protein gene and coded polypeptide and preparation process thereof
  • High temp. resistant Nus G protein gene and coded polypeptide and preparation process thereof

Examples

Experimental program
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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 hot spots of breakage to the greatest extent, a variety of methods and principles 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 were used to process samples during physical shearing, and samples were processed by setting enzyme gradients during enzyme digestio...

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

The present invention relates to coding separated DNA with activity or its functional identical varianjt and polypeptide with high-temp. resisting NusG protein activity which is produced by utilizingrecombinant DNA technology and the described separated DNA or its functional identical variant. By using tengchong thermophilic anaerobe whole genome sequencing and analysis as basis said invention clones and separates the high-temp. resisting NusG protein gene. Said gene is useful for preparing transgenic microbe or animal and plants to produce high-temp. resisting NusG protein and recovering and obtaining the enzyme coded by said gene. Besides, said invention also provides the amino acid sequence of polypeptide with high-temp. resisting NusG protein activity for preparing, separating and purifying the polypeptide with high-temp. resisting NusG protein activity.

Description

technical field [0001] The present invention relates to mutation or genetic engineering, in particular to a high-temperature-resistant NusG protein gene sequence, encoded polypeptide and a preparation method. Background technique [0002] NusG gene is a coding gene that exists in most microorganisms, it is a kind of transcription anti-terminator, and it is also a kind of elongation factor. It interacts with many other factors to regulate the elongation and termination of the transcription chain. In E.coli, the NusG gene encodes a polypeptide chain of 181 amino acids in length, and the encoded protein product is of great significance to the growth and development of bacteria. NusG starts to be transcribed from the PEG promoter, terminates at the rho-dependent terminator in the VP1K region, and is functionally linked to SEC. The NusG protein is 21Kd and is an indispensable E.coli protein that regulates the rho-dependent transcriptional termina...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N15/63
Inventor 于军李蔚李杰胡松年田宇清
Owner HUADA GENE RES & DEV CENT HANGZHOU