Novel protein and gene that codes therefor

A protein and coding technology, applied in the direction of anti-enzyme immunoglobulin, glycosylase, glycosyltransferase, etc., can solve the problems of no enzyme activity, no enzyme activity, loss of enzyme activity, etc.

Inactive Publication Date: 2012-11-28
JAPAN TOBACCO INC
View PDF4 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, the most known sialyltransferases derived from marine microorganisms show that their optimum reaction temperature is below 30°C, and the enzyme activity will be rapidly lost in the temperature range above this
Generally, the optimum temperature for the growth of mammalian cells is around 37°C. Therefore, it is necessary to transform animal cells with an expression vector incorporating sialyltransferase from marine bacteria, and allow the animal cells to produce and breed in a suitable environment. However, there is a problem that even if the sialyltransferase protein derived from marine microorganisms is expressed in these cells, the enzymatic activity cannot be obtained
In addition, the synthesis of sugar chains is important in elucidating the functions of new sialic acid-containing sugar chains, however, there are still many problems in the synthesis of sugar chains
As one of the means to solve this problem, it has been considered to combine various animal-derived glycotransferases and sugar acceptors with a wide range of substrate specificity and optimum temperature for which a large number of genes have been obtained and expressed as recombinant enzymes. It is a sialyltransferase derived from a microorganism around 37°C, but there is no report on a sialyltransferase derived from a marine microorganism with an optimum temperature around 37°C

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Novel protein and gene that codes therefor
  • Novel protein and gene that codes therefor
  • Novel protein and gene that codes therefor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0154] Example 1: Screening and strains of microorganisms producing β-galactoside-α2,6-sialyltransferase Identification

[0155] (1) screening

[0156] Use seawater, sea sand, sea mud or marine fish and shellfish as the inoculation source. This inoculum source was spread on a plate medium composed of Marine Broth Agar 2216 medium (manufactured by Becton Dickinson), and microorganisms grown at 15°C, 25°C or 30°C were obtained. After the obtained microorganisms were purely cultured according to a conventional method, each microorganism was cultured using a liquid medium composed of Marine Broth 2216 medium (manufactured by Becton Dickinson). After the microorganisms grow sufficiently, the cells are collected from the culture medium by centrifugation. To the collected bacterial cells, 20 mM cacodylate buffer (pH 6.0) containing 0.2% Triton X-100 (manufactured by Kanto Chemical Industry Co., Ltd.) was added to suspend the bacterial cells. The cell suspension was ultrasoni...

Embodiment 2

[0162] Embodiment 2: Gram of β-galactoside-α2,6-sialyltransferase gene derived from JT-SHIZ-119 strain Long, base sequence determination, and the expression of the gene in Escherichia coli

[0163] (1) Confirmation of the homologue of the β-galactoside-α2,6-sialyltransferase gene in the JT-SHIZ-119 strain exist

[0164] In order to confirm the presence of β-galactoside-α2,6-sialyltransferase derived from Photobacterium mermaidi JT0160 strain in the JT-SHIZ-119 strain which has been confirmed to have β-galactoside-α2,6-sialyltransferase activity Enzyme gene (Yamamoto et al. (1996) J Biochem 120:104-110), or JT-ISH-224 strain derived β-galactoside-α2,6-sialyltransferase gene (PCT / JP2006 / 315850) For homologues, genomic Southern hybridization was performed.

[0165] First, in order to improve the efficiency of hybridization, an attempt was made to use the β-galactoside-α2,6-sialyltransferase gene fragment of the JT-SHIZ-119 strain itself as a probe. Specifically, it is u...

Embodiment 3

[0204] Example 3: From the large expression vector pTrc99A carrying the SHIZ119-N1C0 clone Extraction and purification of β-galactoside-α2,6-sialyltransferase from Enterobacter TB1

[0205] (1) Extraction and Purification

[0206] From the colony of Escherichia coli TB1 carrying the expression vector pTrc99A integrated with SHIZ119-N1C0 clone subcultured on the LBAmp plate medium, the bacterial cells were collected with an inoculation loop, and inoculated in a medium containing 30 μL × 200 ampicillin (400 mg / 20 mL) In 6 mL of LB liquid medium and 10 mL, shake culture at 30°C and 180 rpm for 8 hours.

[0207] The main culture was carried out according to the following procedure. Inject 300ml of LB medium mixed with 1.5mL x 200 ampicillin (400mg / 20mL) and 300μL of 1M IPTG (1.192g / 5mL) into a 1000ml baffled flask, and prepare 9 bottles in total (2.7L in total) . Each flask was inoculated with 12 ml of the pre-culture solution, and cultured with shaking at 30° C. and 180 ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

Provided is a novel protein that exhibits neuraminidase activity and / or [beta]-galactoside-[alpha]2,6-sialyltransferase activity. Also provided are a nucleic acid that codes for said protein, a vector containing a nucleic acid that codes for said protein, a host cell transformed by said vector, a method for producing recombinant [beta]-galactoside-[alpha]2,6-sialyltransferase, and an antibody that specifically recognizes the aforementioned protein.

Description

technical field [0001] The present invention relates to a protein having neuraminidase activity and / or β-galactoside-α2,6-sialyltransferase activity, a nucleic acid encoding the protein, and a method for producing the enzyme using a microorganism transformed with a gene encoding the protein Method, and antibody specifically recognizing the protein. Background technique [0002] Sugar chains are compounds composed of various sugars, such as monosaccharides such as galactose and N-acetylglucosamine. Sugar chains such as glycoproteins and glycolipids (hereinafter also referred to as complex sugar sugar chains) have very important functions in living bodies. Based on previous studies, it is considered that, among sugar chains, particularly, sugar chains containing monosaccharides called sialic acid exhibit important functions. For example, it has been shown mainly in mammalian cells that sugar chains containing sialic acid are important molecules that function as differentiati...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C07K16/40C12N1/15C12N1/19C12N1/21C12N5/10C12N9/10C12N9/24
CPCC12N9/1081C12N9/24C12Y302/01018C12N9/2402C12Y204/99001C07K16/40C12N15/11C12N9/10
Inventor 峰利喜山本岳梶原一三塚本浩史
Owner JAPAN TOBACCO INC
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap