Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

cmp-dependent sialidase activity

A technology of sialyltransferase and enzymatic activity, applied in the field of controlled hydrolysis of α2,6 glycosidic bonds, can solve the problems of low expression yield of hST6Gal-I, limited availability of hST6Gal-I, poor activity, etc.

Active Publication Date: 2021-09-28
F HOFFMANN LA ROCHE & CO AG
View PDF4 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to recombinant expression in different hosts (methylotrophic yeast Pichia pastoris, cultured Spodoptera frugiperda cells, E. coli-based expression system) Low expression yield and / or poor activity of expressed hST6Gal-I, the availability of recombinant hST6Gal-I for such applications is still limited

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
  • cmp-dependent sialidase activity
  • cmp-dependent sialidase activity
  • cmp-dependent sialidase activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0174] Tests for sialyltransferase enzyme activity

[0175] Asialo-fetuin (desialylated fetuin, Roche Applied Science) was used as acceptor and CMP-9-fluoro-NANA (CMP-9-fluoresceinyl-NeuAc) was used as donor. Enzymol substrates (Brossmer, R. and Gross H. J. (1994) Meth. Enzymol. 247, 177-193). The enzymatic activity of sialyltransferase was determined by measuring the transfer of sialic acid from the donor compound to asialo-fetuin. Reaction mixture (35 mM MES, pH 6.0, 0.035% Triton X-100, 0.07% BSA) contained 2.5 µg enzyme sample, 5 µL asialo-fetuin (20 mg / mL) in a total volume of 51 µL and 2 µL of CMP-9-fluoro-NANA (1.0 mg / mL). The reaction mixture was incubated at 37°C for 30 min. Stop the reaction by adding 10 µL of the inhibitor CTP (10 mM). Load the reaction mixture onto a PD10 desalting column equilibrated with 0.1 MTris / HCl (pH 8.5). Fetuin was eluted from the column using equilibration buffer. Fraction size is 1 mL. Determine the concentration of fetuin formed ...

Embodiment 2

[0177] SDS gel electrophoresis

[0178] Analytical SDS gel electrophoresis was performed using NuPAGE gels (4-12%, Invitrogen). Samples (36 µL) were diluted with 12 µL of NuPAGE LDS sample buffer (Invitrogen) and incubated at 85°C for 2 min. Load an aliquot (typically containing 5 µg protein) onto the gel. Gels were stained using SimplyBlue SafeStain (Invitrogen).

Embodiment 3

[0180] N-terminal sequencing by Edman degradation

[0181] The N-terminal sequences of the expressed variants of human ST6Gal-I were analyzed by Edman degradation using reagents and devices from Life Technologies. Sample preparation was performed as described in the instruction manual for the Life Technologies ProSorb Sample Preparation cartridge (Cat. No. 401950) and the Life Technologies ProBlott Mini PK / 10 Membrane (Cat. No. 01194). For sequencing, the Procise Protein Sequencing Platform (Procise Protein Sequencing Platform) was 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
Login to View More

PUM

No PUM Login to View More

Abstract

The present invention relates to the properties of certain glycosyltransferase variants with N-terminal truncating deletions or internal deletions. Any of the mutants disclosed herein exhibit α-2,6-sialyltransferase enzymatic activity in the presence of CMP-activated sialic acid as a co-substrate and in the presence of a suitable acceptor site. A fundamental finding documented in the present invention is that such enzymes are not only able to catalyze the transfer of sialidyl moieties, but they are also able to catalyze the hydrolytic cleavage of terminally bound sialic acids from glycans. Specifically, it was found that in the presence of cytidine-5'-monophosphate (CMP), glycosyltransferase activity is inhibited and sialidase activity is stimulated. Sialidase activity was found to be dependent on the presence of a specific stretch of amino acids (positions 90-108) in the polypeptide sequence of the reference (wild-type) hST6 Gal-I polypeptide. Deletion of part of this sequence in N-terminal truncated variants was found to abolish sialidase activity, especially in the presence and absence of CMP. Thus, compositions, uses and methods employing the CMP-mediated feedback regulation documented herein are disclosed.

Description

[0001] The present invention relates to the properties of certain glycosyltransferase variants with N-terminal truncating deletions or internal deletions. Any of the mutants disclosed herein exhibit alpha-2,6-sialyltransferase enzymatic activity in the presence of CMP-activated sialic acid as a co-substrate and in the presence of an appropriate acceptor site. A fundamental finding documented in the present invention is that such enzymes are not only able to catalyze the transfer of sialidyl moieties, but they are also able to catalyze the hydrolytic cleavage of terminally bound sialic acids from glycans. Specifically, it was found that in the presence of cytidine-5'-monophosphate (CMP), glycosyltransferase activity is inhibited and sialidase activity is stimulated. Sialidase activity was found to be dependent on the presence of a specific stretch of amino acids (positions 90-108) in the polypeptide sequence of the reference (wild-type) hST6Gal-I polypeptide. It was found that d...

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 Patents(China)
IPC IPC(8): C12N9/10C12P19/18
CPCC12N9/1081C12P19/18C12P19/44C12Y301/03C12P21/005C12Y204/99001C07K16/00C07K2317/41
Inventor H.佐贝克M.格赖夫M.托曼S.马利克
Owner F HOFFMANN LA ROCHE & CO AG
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com