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ALPHA-1, 4-GALACTOSYLTRANSFERASE (CgtD) FROM CAMPYLOBACTER JEJUNI

Inactive Publication Date: 2009-11-19
NAT RES COUNCIL OF CANADA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0006]In one aspect the present invention provides a method of producing a galactosylated product saccharide by contacting an acceptor substrate with a donor substrate comprising a galactose moiety and an isolated or recombinant α-1,4-galactosyltransferase polypeptide with at least 80% identity to SEQ ID NO:2; and allowing transfer of a galactose moiety to the acceptor saccharide to occur, thereby producing the galactosylated product saccharide. In one embodiment the α-1,4-galactosyltransferase polypeptide comprises an amino acid sequence with at least 90% or 95% identity to SEQ ID NO:2. In another embodiment the α-1,4-galactosyltransferase polypeptide comprises an amino acid sequence of SEQ ID NO:2. In a further embodiment the method is performed at a commercial scale of production. In another embodiment the method includes a step of isolating the galactosylated product saccharide.

Problems solved by technology

The oligosaccharide structures involved in these and other processes are potential therapeutic agents, but they are time consuming and expensive to make by traditional chemical means.
However, production of glycosyltransferases in sufficient quantities for use in preparing oligosaccharide structures has been problematic.
Expression in E. coli has been achieved for mammalian glycosyltransferases, but these attempts have produced mainly insoluble forms of the enzyme from which it has been difficult to recover active enzyme in large amounts (Aoki et al.

Method used

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  • ALPHA-1, 4-GALACTOSYLTRANSFERASE (CgtD) FROM CAMPYLOBACTER JEJUNI
  • ALPHA-1, 4-GALACTOSYLTRANSFERASE (CgtD) FROM CAMPYLOBACTER JEJUNI
  • ALPHA-1, 4-GALACTOSYLTRANSFERASE (CgtD) FROM CAMPYLOBACTER JEJUNI

Examples

Experimental program
Comparison scheme
Effect test

example 1

Cloning Expression and Characterization of CgtD from C. jejuni LIO87

[0143]ORF #8 from C. jejuni LIO87 (SEQ ID NO:1) was cloned and expressed in Escherichia coli. The extracts from Escherichia coli that overexpress the protein of SEQ ID NO:2 were assayed with UDP-Glc, UDP-GlcNAc, UDP-Gal and UDP-GalNAc as donors and the fluorescent derivatives of Glc, Gal, Lac, LacNAc and Gal-α-1,4-Lac as acceptors. The protein product of ORF #8 (SEQ ID NO:2) was active with UDP-Gal as donor and either the LacNAc (Gal-β-1,4-GlcNAc) or the Lac (Gal-β-1,4-Glc) derivatives as acceptor. SEQ ID NO:2 was designated CgtD (Campylobacter glycosyltransferase; D) and further characterized.

example 2

Further Characterization of CgtD Activity

[0144]To further characterize the enzymatic activity of CgtD, the gene encoding CgtD from C. jejuni LIO87 was cloned in pCWori+ as a C-terminal fusion with the E. coli maltose-binding (MalE) protein (construct CJL-99). CJL-99 was electroporated in E. coli AD202. CgtD expressed well as a MalE fusion (about 150 units per liter). The activity was assessed on the donors and acceptors determined optimal in Example 1. Activity was optimal at pH 7 to 8. Inclusion of divalent cations increased activity. Some diva lent cations were more beneficial than others e.g., MnCl2 yielded about 15% better activity than MgCl2.

example 3

Determining the Regio- and Stereo-Specificity of CgtD

[0145]The regio- and stereo-specificity of CgtD were determined using purified MalE-CgtD (CJL-99). 19 mg of αGal-1,4-βGal-1,4-βGlcNAc-p-nitrophenyl were synthesized. The 1H NMR resonances were assigned to the trisaccharide compound through the use of 2D homonuclear COSY and TOCSY spectra as shown in FIG. 1. These assignments were used to identify cross-peaks in an 1H-13C HSQC spectrum, which correlates the chemical shift of a proton atom with its directly bonded carbon neighbor (FIG. 2). Because inter-residue connectivities can be established across glycosidic bonds using a 1H-13C HMBC pulse sequence, HMBC spectra of the trisaccharide compound were acquired to establish the covalent linkages between sugar residues. These data confirmed that CgtD transferred a Gal residue to a Gal on the disaccharide precursor through an α1→4 linkage. Consistent with the HMBC spectra, the carbon resonance at position C4 of βGal displayed a downfile...

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Abstract

α-1,4-galactosyltransferase (CgtD) polypeptides, nucleic acids that encode the polypeptides, including a polypeptide from Campylobacter jejuni strain LIO87 have been isolated and characterized A method of producing a galactosylated saccharide comprising contacting an acceptor saccharide containing a terminal galactose, a donor substrate comprising a galactose moiety and one of the CgtD polypeptides is described.

Description

CROSS-REFERENCES TO RELATED APPLICATIONS[0001]This application claims the benefit of U.S. Provisional Application No. 60 / 797,132, filed May 2, 2006, which is herein incorporated by reference for all purposes.FIELD OF INVENTION[0002]The invention relates to α-1,4-galactosyltransferase (CgtD) polypeptides, nucleic acids that encode the polypeptides, and methods of using the polypeptides.BACKGROUND OF THE INVENTION[0003]Carbohydrates are now recognized as being of major importance in many cell-cell recognition events, notably the adhesion of bacteria and viruses to mammalian cells in pathogenesis and leukocyte-endothelial cell interaction through selectins in inflammation (Varki (1993) Glycobiology 3: 97-130). Moreover, sialylated glycoconjugates that are found in bacteria (Preston et al. (1996) Crit. Rev. Microbiol. 22:139-180; Reuter et al. (1996) Biol. Chem. Hoppe-Seyler 377:325-342) are thought to mimic oligosaccharides found in mammalian glycolipids to evade the host immune respon...

Claims

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Application Information

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IPC IPC(8): C12P19/18C12N9/10C12N1/19C12N15/63C07H21/04
CPCC12N9/1051C12N9/107C12Y204/01038C12P19/18
Inventor GILBERT, MICHELWAKARCHUK, WARRENHOULISTON, SCOTT
Owner NAT RES COUNCIL OF CANADA