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Polysialic acid, blood group antigens and glycoprotein expression

A technology of antigens and oligosaccharides, applied in the direction of glycosyltransferases, enzymes, biochemical equipment and methods, etc., can solve the problems of expensive PEGylation process, molecular structure change, and biological activity reduction

Inactive Publication Date: 2016-03-09
GLYCOBIA +4
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Third, the often observed increase in the half-life of PEGylated proteins can be accompanied by a decrease in biological activity related to changes in molecular structure caused by conjugation[2]
Fourth, the PEGylation process is expensive and requires many in vitro chemical reactions and multiple purifications [7]
However, this requires the addition of multiple C-terminal thiols, which are difficult to express in E. coli fermentation and require a mammalian expression system [14]

Method used

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  • Polysialic acid, blood group antigens and glycoprotein expression
  • Polysialic acid, blood group antigens and glycoprotein expression
  • Polysialic acid, blood group antigens and glycoprotein expression

Examples

Experimental program
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Embodiment 1

[0226] Plasmid construction

[0227] In this study, plasmids were constructed in yeast using standard homologous recombination (ShanksRM, CaiazzaNC, HinsaSM, ToutainCM, O'TooleGA: A Saccharomyces cerevisiae-based molecular toolkit for manipulating genes in Gram-negative bacteria, Applied Environmental Microbiology (ApplEnvironMicrobiol) 2006, 72(7):5027-5036). Plasmids were recovered from yeast and confirmed by transfer into E. coli strain DH5α via PCR and / or sequencing. The list below describes the plasmids constructed during the course of this study. The plasmid name is followed by the inserted gene / sequence in order from 5' to 3', followed by the vector in brackets. Glycan expression plasmids were constructed in vector pMW07 (Vaderrama-Rincon et al.). Protein expression plasmids are typically constructed in the vector pTRCY. The sugar nucleotide synthesis plasmid was cloned in pTrcY, pMQ70.

[0228] In the order of the pictures:

[0229] pMW07: (Vector) pBAD, ChlorR, ...

Embodiment 2

[0271] Engineered Escherichia coli for expression of human Thomsen-Friedenreich antigen (T antigen)

[0272] T-antigen glycans (T-antigen, Galβ1,3GalNAcα-) are structures found at the core of many human-related human glycans. Construction of a plasmid for the production of glycosyltransferase and sugar nucleotide epimers necessary for constructs using native UndPP-GlcNAc as substrate for the assembly of human T-antigen-containing glycans in E. coli Expression of enzyme activity. Plasmid pMW07 (Valderrama-Rincon et al.) was used as a vector because it contains a low copy number origin of replication (ORI), an inducible pBAD promoter, and allows cloning in Saccharomyces cerevisiae via homologous recombination Yeast ORI. The sequence of pMW07 is provided as SEQ ID NO:1.

[0273] To generate disaccharide glycans with the GalNAcα1,3GlcNAc structure, a plasmid was constructed to express the Campylobacter jejuni GalNAc transferase PglA and the epimerase GalE to facilitate the synt...

Embodiment 3

[0280] In vivo synthesis of proteins bearing V-glycans terminated in human T antigens

[0281] Transfer of UndPP-linked oligosaccharides to specific asparagine residues using OSTPglB. This requires a D / EX1NX 2 The target protein consisting of the S / T sequon to be localized into the periplasm, bearing the PglB recognition site, and the presence of an appropriate glycan substrate. In this study, we also constructed the vector pTRCY for glycoprotein expression.

[0282] pTRCY was cloned in S. cerevisiae via homologous recombination by adding the URA3 gene and yeast 2 micron ORI to pTRC99a, resulting in a novel vector capable of replicating in yeast. The URA3 gene and the 2 micron ORI were amplified using primers containing homology to pTRC99a for insertion between the pBR322 ORI and the lacI gene. The sequence of vector pTRCY is SEQ ID NO:6.

[0283] hGH was cloned as a C-terminal translational fusion following the signal peptide, MBP, hexahistidine tag, and tev cleavage site...

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Abstract

The invention described herein generally relates to glycoengineering host cells for the production of glycoproteins for therapeutic use. Host cells are modified to express biosynthetic glycosylation pathways. Novel prokaryotic host cells are engineered to produce N-linked glycoproteins wherein the glycoproteins comprise polysialic acid or blood group antigens.

Description

[0001] Statement of Federally Sponsored Research and Development [0002] This invention was made with Government support under Grant Nos. 1R43GM093483-01, 5R43AI091336-01, and 5R43AI091336-02, National Institutes of Health. The government has certain rights in this invention. [0003] Cross References to Related Applications [0004] This application is related to US Provisional Application No. 61 / 801,948, filed March 15, 2013, which is hereby incorporated by reference in its entirety for all purposes. [0005] sequence listing [0006] This application contains a Sequence Listing that has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy created on [DATE] is named [.txt] and is [#######] bytes in size. technical field [0007] The disclosure herein relates generally to the fields of glycobiology and protein engineering. More specifically, embodiments described herein relate to the production of oligosaccharide compo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/00C12P19/08C12P19/28
CPCC12N9/1051C12N9/1081C12Y204/99004C12Y204/99008C12P21/005C12P19/02
Inventor 朱迪思·H·梅利特亚当·C·费舍尔布莱恩·S·汉密尔顿马修·P·德里萨
Owner GLYCOBIA
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