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Enzymes for glycan analysis

A technology for glycosidase and glycoprotein, which is applied in the field of tools for glycan analysis, and can solve the problems of difficulty, reduction, and function of glycoprotein research.

Active Publication Date: 2020-05-19
GENOVESE CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

They are also often negatively charged, complicating mass spectrometry analysis
This makes research on glycoproteins difficult and reduces the manufacturer's ability to confirm that glycoprotein lots will function in homologous material
To overcome these problems, attempts have been made to genetically engineer CHO cells to reduce glycan complexity, although this may affect function
Chemical methods have also been used, but these usually destroy the protein

Method used

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  • Enzymes for glycan analysis
  • Enzymes for glycan analysis
  • Enzymes for glycan analysis

Examples

Experimental program
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Effect test

preparation example Construction

[0047] Polypeptides can be produced by suitable methods, including recombinant or synthetic methods. For example, polypeptides can be synthesized directly using standard techniques known in the art, such as Fmoc solid phase chemistry, Boc solid phase chemistry, or by solution phase peptide synthesis. Alternatively, a polypeptide may be produced by transforming a cell, usually a bacterial cell, with a nucleic acid molecule or vector encoding the polypeptide. Methods for producing polypeptides by expression in bacterial host cells are described below and exemplified in the Examples. The invention provides nucleic acid molecules and vectors encoding polypeptides of the invention. The invention also provides host cells comprising such nucleic acids or vectors. An exemplary polynucleotide molecule encoding a polypeptide disclosed herein is provided as SEQ ID NO:13. The sequence includes codons for an N-terminal methionine (ATG) at the 3' end and codons for a GSGLE linker and a 6...

Embodiment 1

[0128] Example 1 - Sialidase

[0129] Materials and Methods

[0130] Expression and purification of sialidase

[0131] The genes identified in Ekkermansia muciniphila (Am0705, Am0707, Am1757, Am2058) were codon optimized for good expression in E. coli in the vector pET21a(+). This vector was transformed into BL21(DE3)Star cells. E. coli was routinely grown in LB at 37°C, 200 rpm. In the presence of the plasmid, add 100 µg / mL kanamycin. After overnight incubation, dilute the culture 1:20 in fresh LB(amp) and grow the culture until OD 620 About 0.7 to 0.8, after which recombinant protein expression was induced by adding 1 mM IPTG, and the expression lasted for 5 hours, and then the cells were collected and frozen. Frozen cells were thawed and dissolved in His-binding buffer (20 mM NaP pH 7.4, 500 mM NaCl, 20 mM imidazole) and sonicated to release intracellular proteins. Cell debris was removed by centrifugation. The filtered sterile supernatant was affinity purified on ...

Embodiment 2-O

[0159] Example 2-O-glycosidase

[0160] Materials and methods

[0161] Expression and purification of endo-O-glycosidase

[0162] The endo-N-acetylgalactosaminidase of Streptococcus oralis was codon optimized for good expression in E. coli in the vector pET21a(+). This vector was transformed into BL21(DE3)Star cells. E. coli was routinely grown in LB at 37°C, 200 rpm. In the presence of the plasmid, add 100 µg / mL kanamycin. After overnight incubation, dilute the culture 1:20 in fresh LB(amp) and grow the culture until OD 620 About 0.7 to 0.8, after which recombinant protein expression was induced by adding 1 mM IPTG, and the expression lasted for 5 hours, and then the cells were collected and frozen. Frozen cells were thawed and dissolved in His-binding buffer (20 mM NaP pH 7.4, 500 mM NaCl, 20 mM imidazole) and sonicated to release intracellular proteins. Cell debris was removed by centrifugation. Filtered sterile supernatants were affinity purified on a nickel colum...

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Abstract

The present invention relates to enzymes and combinations thereof useful for studying glycoproteins, and corresponding methods of use. In particular, the invention relates to a sialidase composition comprising an additional protease and / or glycosidase, preferably an O-glycoprotein-specific endoprotease and / or an O-glycosidase.

Description

technical field [0001] The present invention relates to enzymes and combinations thereof for studying glycoproteins, and corresponding methods of use. Background technique [0002] Glycosylation of proteins plays a key role in many physiological functions in humans, including signal transduction, transport, protection from proteolytic activity, adhesion, inflammatory response, microbial colonization, etc. Most glycan chains attached to proteins, whether O-linked or N-linked, are modified with terminal sialic acid. As the outermost glycans on glycoproteins, their presence / absence is often critical for downstream effects including, for example, the inflammatory potential of immunoglobulins. Sialic acids on proteins are heterologous in terms of their presence / absence and individual structural modifications on a given protein. They also often carry a negative charge, complicating mass spectrometry analysis. This makes research on glycoproteins difficult and reduces the manufa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24G01N33/68
CPCC12N9/2402C12N9/52G01N33/84G01N2333/924G01N33/68C12Y304/24057G01N2333/952
Inventor 弗雷德里克·里奥罗尔夫·卢德史蒂芬·比约克马林·梅贾尔弗雷德里克·奥尔森
Owner GENOVESE CORP
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