US20110165626A1 – High Yield Production of Sialic Acid (Neu5ac) by Fermentation – Google Patents


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We stained cell lines with lectins that have been used to recognize the following three different epitopes: (i) WGA recognizes all sialic acids, (ii) MAA recognizes α2,3 sialic acid, and (iii) SNA recognizes α2,6 sialic acid. In this report, we demonstrate that AAV1 and AAV6 use both α2,3 and α2,6 N-linked sialic acids for binding and infection. To analyze if the sialylated receptors of AAV1 and AAV6 are glycoproteins or glycolipids, we treated Cos-7 and Pro-5 cells with 200 μg/ml proteinase K. Cell viability was assessed immediately after proteinase K treatment, and no cytotoxic effect was observed. Sialic acids are abundant on the surfaces of muscle cells, which may partly explain the high transduction efficiency of AAV1 and AAV6 vectors for this tissue. To determine if N-linked sialic acid is necessary for efficient AAV1 and AAV6 transduction, we tested AAV1 and AAV6 transduction on another Pro-5-derived cell line, Lec-1 (40), that is deficient in N-linked glycans but is not deficient in O-linked glycans (Fig. (Fig.7C).7C). Resialylation by α2,3(N)-sialyltransferase and α2,6(N)-sialyltransferase increased AAV6 transduction by 11-fold and 6-fold, respectively, but only 2.5-fold for AAV1. Therefore, Pro-5 cells and HepG2 cells display α2,3 sialic acid or α2,6 sialic acid on their surfaces, respectively, while Cos-7 cells display α2,3 sialic acid and a relatively small amount of α2,6 sialic acid.

Royal Republic. Weekend Man. 24 The following sialyltransferases were used to add specific sialic acids to the surfaces of Lec-2 cells: α2,3(O)-sialyltransferase, α2,3(N)-sialyltransferase, and α2,6(N)-sialyltransferase. The page “Sialic acid manufacturer” does not exist. In case you loved this post and you would want to be given more details regarding Supplier of sialic acid powder as Raw Material for Supplements,Supplier of sialic acid powder as Raw Material for food,Supplier of sialic acid powder as Raw Material for drinks,Supplier of sialic acid powder as Raw Material for beverages,Supplier of sialic acid powder as Raw Material for cosmetics,Supplier of sialic acid powder as Raw Material for pharmaceuticals,manufacturer of sialic acid powder as Raw Material for Supplements,manufacturer of sialic acid powder as Raw Material for food,manufacturer of sialic acid powder as Raw Material for drinks,manufacturer of sialic acid powder as Raw Material for beverages,manufacturer of sialic acid powder as Raw Material for cosmetics,manufacturer of sialic acid powder as Raw Material for pharmaceuticals,Supplier of sialic acid powder for Supplement Ingredients,Supplier of sialic acid powder for food Ingredients,Supplier of sialic acid powder for drink Ingredients,Supplier of sialic acid powder for beverage Ingredients,Supplier of sialic acid powder for cosmetic Ingredients,Supplier of sialic acid powder for pharmaceutical Ingredients,manufacturer of sialic acid powder for Supplement Ingredients,manufacturer of sialic acid powder for food Ingredients,manufacturer of sialic acid powder for drink Ingredients,manufacturer of sialic acid powder for beverage Ingredients,manufacturer of sialic acid powder for cosmetic Ingredients,manufacturer of sialic acid powder for pharmaceutical Ingredients kindly visit our own web page. ’ says the swine-bird-human flu strain, reported to be found first in Mexico in late-March 2009, could have only come from Dr James S. Robertson and his colleagues in association with the US Centre for Disease Control and vaccine manufacturer Novavax, Inc, which was ready to profit from the release he says. Sialic Acid or N-Acetylneuraminic Acid Sialic Acid or N-Acetylneuraminic Acid factory, Supplier, Manufacturer. All glycans recognized contain a common motif: sialic acid linked to N-acetyl-lactosamine. A total of 264 glycans were screened for binding to the capsids as described in Materials and Methods. For example, methods of transforming Bacillus species and promoters that can be used to express proteins are taught in U.S. Such promoters can be obtained from genes that have been cloned from the species, or heterologous promoters can be used. Promoters for use in E. coli include the T7, trp, or lambda promoters.

Preferably, NeuB and NeuC are isolated from bacterial strains that contain sialylated structure in their cells envelope, such as C. jejuni strain ATCC Accession No. 43438. It is also within the scope of the invention to use substantially identical sequences, and/or conservatively modified variations of said sequences as defined hereafter. Using competition, genetic, inhibitor, and enzymatic reconstitution experiments we demonstrate that a glycoprotein(s) with N-linked α2,3 and/or α2,6 sialic acids serves as a receptor(s) for AAV1 and AAV6 transduction. The glycan array binding data provide independent support of AAV1 interaction with α2,3 and α2,6 trisaccharides. Resialylation experiments confirmed that AAV1 and AAV6 use α2,3 or α2,6 N-linked sialic acid for efficient transduction. The above results (Fig. (Fig.2)2) show that AAV1 and AAV6 transduce Pro-5, Cos-7, and HepG2 cells efficiently; therefore, we tested the sialic acid linkages on the surfaces of these cell lines. N-linked, not O-linked, sialic acid facilitates AAV1 and AAV6 transduction. Consistent with the results shown in Fig. Fig.22 and and3,3, transduction by AAV6 appears to be more dependent on sialic acid than AAV1. To confirm if both α2,3 and α2,6 sialic acids facilitate AAV1 and AAV6 transduction, we carried out a lectin competition assay on these three cell lines (Fig. 5A to C).

Proteinase K treatment reduced AAV1 and AAV6 transduction. Similarly, SNA blocked AAV1 and AAV6 transduction on HepG2 cells and, to a lesser extent, on Cos-7 cells but not on Pro-5 cells. AAV1 and AAV6 transduction is not inhibited by mucin. Since AAV1 and AAV6 transduced all these cell lines efficiently, it appears that there is no obvious preference for a particular sialic acid linkage(s). AAV1 capsid glycan specificity on glycan array. In contrast, AAV4, which uses O-linked sialic acid for transduction, transduced Lec-1 cells fourfold more efficiently than Pro-5 cells, suggesting that removal of the N-linked glycan facilitates AAV4 interaction with O-linked glycan. At 0.5-mg/ml mucin concentration, 50% inhibition of AAV4 transduction was observed. As shown in Fig. Fig.9,9, mucin inhibited AAV4 transduction in a dose-dependent manner. Although both AAV4 and AAV5 bind to α2,3-linked sialic acid for transduction, AAV4 binds sialic acid present on O-linked oligosaccharides, whereas AAV5 binds sialic acid present on N-linked oligosaccharides (19). To determine whether O-linked or N-linked sialic acid is used by AAV1 and AAV6 for transduction, Cos-7 cells were cultured with inhibitors of O-linked (N-benzyl GalNac) or N-linked (tunicamycin) glycosylation. Sialic acid saccharides are expressed on both glycoproteins and glycosphingolipids. PPMP is a glucosylceramide synthase inhibitor, which acts to deplete glycosphingolipids from the cell membrane (22). To test if glycolipids on cell membrane are also required for AAV1 and AAV6 transduction, Cos-7 cells were incubated with PPMP for 40 h prior to transduction with AAV1, AAV6, AAV2, or AAV5.

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