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Production of complex carbohydrates

Inactive Publication Date: 2006-05-11
UNIV OF IOWA RES FOUND +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0020] The invention also provides a process for the production of a complex carbohydrate, comprising culturing production cells comprising a chimeric DNA sequence encoding a glycotransferase, so as to yield production cells comprising an altered level of complex carbohydrate, wherein the production cells are bacteria comprising a core lipid structure containing a terminal heptose molecule and encoding an enzyme capable of adding an acceptor molecule to the heptose molecule. The invention also provides a process further comprising recovering the complex carbohydrate.

Problems solved by technology

However, most pathogenic bacteria are fastidious in their growth requirements and slow growing, making this mode of production impractical.
For example, Haemophilus influenzae is known to require a carbon dioxide atmosphere and brain / heart extract for growth Helicobacter pylori grows very poorly in broth cultures required for OS and PS production.
In addition, many of these bacterial pathogens (for example, Neisseria meningitidis) can be dangerous to grow in large volumes because of the risk of aerosol and possible infection spread.
Bacteria do not glycosylate peptides and proteins efficiently or in a manner equivalent to that of eucaryotes.
Classic organic syntheses of complex carbohydrates have long been known, but with limited practical application.
In addition to the difficulties inherent in the complexity of the glycopolymer molecule, many glycosidic bonds are labile and must be protected and deprotected during chemical synthesis, adding to the difficulty of synthesis and reducing the yield of product.

Method used

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Examples

Experimental program
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Effect test

example 1

Selection of a Hib Production Cell

[0030] Capsular strains of Haemophilus influenzae type b (Hib) are responsible for various invasive and bacteraemic infections in humans, including meningitis and pneumonia. The surface lipooligosaccharides (LOS) of Hib are known to be important factors in microbial virulence and pathogenesis. Structural studies of Hib LOS from wild-type and mutant strains have shown that the LOS contains a conserved heptose trisaccharide core which can be extended with additional sugars on each heptose. Recently, a revised structure of the E. coli K-12 core region was reported which also contains a heptose trisaccharide inner core and a fourth heptose present on the terminus of the main oligosaccharide branch:

[0031] Previous work showed that the core region of E. coli transformed with synthetic enzyme genes could be elongated by the addition of saccharide monomers under the direction of H. influenzae genes to produce a modified E. coli LPS that was elongated by ...

example 3

Transformation of the Hib Production Cell

[0038] Restriction fragments of EMBLOS-I were used to make a series of plasmids which modified E. coli strain JM 109 to give clones which produced a proposed chimeric series of higher mass LPS species. The transformants termed pGEMLOS-4, pGEMLOS-5, and pGEMLOS-7 generated modified or chimeric LPS of 5.5, 5.1, and 4.5 kDa, respectively. All three apparently modified the 4.1 kDa LPS species from E. coli, although only the LPS from pGEMLOS-4 expressed the 6E4 epitope. The LPS from strain pGEMLOS-5 was found to react positively with MAb 3F 11, suggesting the presence of terminal N-acetyllactosamine. The epitope recognized by MAb 6E4 is also present in the LOS of H. influenzae nontypable strain 2019, as well as the LPS from Salmonella minnesota Re mutant. Binding of this monoclonal antibody to H. influenzae LOS can be inhibited by Kdo and the Kdo trisaccharide from the Re mutant of S. minnesota. Because the 6E4 epitope has been associated with th...

example 4

Isolation and Purification of Oligosaccharides

[0040] The LPS from PGEM (31 mg), pGEMLOS-4 (25 mg), pGEMLOS-5 (15 mg), and pGEMLOS-7 (4.4 mg) was hydrolyzed in 1% acetic acid (2 mg LPS / ml) for 2 hours at 100° C. The hydrolysates were centrifuged at 5000 g for 20 min at 4° C. and the supernatants removed. The pellets were washed with 2 ml of H2O and centrifuged again (5000 g, 20 min, 4° C.). The supernatants and washings were pooled and lyophilized to give the oligosaccharide fractions. As a standard, 10 mg of LPS from Salmonella typhimurium TV 119 Ra mutant (Sigma, St. Louis) was treated in the same fashion.

[0041] To prepare desalted oligosaccharide pools for ESI-MS analysis, small aliquots of the crude oligosaccharide fractions (<2 mg) were chromatographed on two Bio-Select SEC 125-5 HPLC columns (Bio-Rad, Richmond, Calif.) connected in series, using 0.05 M pyridinium acetate (pH 5.2) at a flow rate of 1 ml / min. A refractive index detector was used to monitor column effluent and c...

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Abstract

Compositions and methods for making complex carbohydrates in a bacterial production cell are disclosed. The complex carbohydrates that can be made include oligosaccharides and polysaccharides of bacterial or mammalian origin.

Description

CLAIM OF PRIORITY [0001] This application claims priority under 35 U.S.C. 119(e) from U.S. Provisional Application Ser. No. 60 / 134,756, filed May 18, 1999, which application is incorporated herein by reference.GOVERNMENTAL RIGHTS [0002] The United States Government retains certain rights in this invention. Financial support was provided by the National Institute of Allergy and Infectious Diseases under Grant Number A124016 and from the NIH National Center for Research Resources under Grants Number RR01614 and RR04112.FIELD OF THE INVENTION [0003] This invention relates to a method for the production of complex carbohydrates on an LPS backbone structure in Gram-negative bacteria BACKGROUND OF THE INVENTION [0004] Complex carbohydrates occur in nature and are involved in a wide array of biological functions, including viral, bacterial and fungal pathogenesis, cell-to-cell and intracellular recognition, binding of hormones and pathogens to cell-surface receptors and in antigen-antibody...

Claims

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Application Information

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IPC IPC(8): C12P19/28C12N1/21C12N15/09A61K31/702A61P19/02A61P29/00A61P31/04A61P31/18C07H3/06C12N9/10C12N15/54C12P19/00C12P19/04C12P19/18C12R1/19
CPCC12N9/1051C12P19/04C12P19/18A61P19/02A61P29/00A61P31/04A61P31/18
Inventor APICELLA, MICHAEL A.GIBSON, BRADFORD W.PHILLIPS, NANCY J.
Owner UNIV OF IOWA RES FOUND
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