Engineering intracellular sialylation pathways

a technology of intracellular sialylation and sialylation pathway, which is applied in the direction of transferases, enzymology, organic chemistry, etc., can solve the problems of limiting the wider application of this expression system, insect cell lines generating complex carbohydrates, and specific and limiting carbohydrate processing, so as to enhance the production of limiting components

Inactive Publication Date: 2005-12-29
BETENBAUGH MICHAEL +3
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Problems solved by technology

Thus, carbohydrate processing is specific and limiting in a wide variety of organisms including insect, yeast, mammalian, and plant cells.
The inability of insect cell lines to generate complex carbohydrates comprising sialic acid significantly limits the wider application of this expression system.
Unfortunately, Man and GlcNAc are the residues most commonly found on the termini of glycoproteins produced by insect cells.
However, the in vivo activity of the insect cell-derived product was substantially lower due to its rapid clearance from injected rats.
In addition, sialic acid and CMP-sialic acid are not permeable to cells so these substrates can not be provided directly to the medium of the cultures (Bennett et al.

Method used

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  • Engineering intracellular sialylation pathways
  • Engineering intracellular sialylation pathways
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Examples

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example 1

Evaluation of N-glycosylation Pathway Enzymes

[0223] The levels of N-linked oligosaccharide processing enzymes are measured using analytical assays to characterize carbohydrate processing in native and recombinant insect cells. These assays are used to compare the N-glycan processing capacity of different cell lines and to evaluate changes in processing and metabolite levels following metabolic engineering modifications.

[0224]High Performance Anion Exchange Chromatography (HPAEC) Assay For Galactose Transferase

[0225] HPAEC is used in combination with pulsed amperometric detection (HPAEC-PAD) or conductivity to detect metabolite levels in the CMP-SA pathway and to evaluate N-linked oligosaccharide processing enzymes essentially as described by (Lee et al. (1990) Anal. Biochem. 34:953-957, Lee et al. (1996) J. Chromatography A 720:137-149). Shown in FIG. 9 is an example of the use of HPAEC-PAD for measuring Gal T activity by following the lactose formation reaction:

UDP−Gal+Glc GalT...

example 2

Enhancing SA Levels By Substrate Addition

[0233] Because the conventional substrates in insect cell media are not efficiently converted to CMP-SA in insect cells as demonstrated by the low levels of CMP-SA, alternative substrates are added to the culture medium. Because sialic acid and CMP-SA are not permeable to cell membranes (Bennetts et al. (1981) J. Cell. Biol. 88:1-15), they are not considered as appropriate substrates. However, other precursors in the CMP-SA pathway are incorporated into cells and considered as substrates for the generation of CMP-SA in insect cells.

[0234] Incorporation And Conversion of N-acetylmannosamine (ManNAc)

[0235] ManNAc has been added to maimmalian tissue and cell cultures and enzymatically converted to SA and CMP-SA (Ferwerda et al. (1983) Biochem. J. 216:87-92, Gal et al. (1997) Improvement of the interferon-gamma sialylation in Chinese hamster ovary cell culture by feeding N-acetylmannosamine, Thomas et al. (1985) Biochim. Biophys. Acta 846:37-4...

example 3

Purification And Cloning of CMP-SA Synthetase

[0239] A bioinformatics search of the cDNA libraries of HGS revealed a novel human CMP-sialic acid synthetase (CMP-SA synthetase, or CMP-SAS) gene based on its homology with the E. coli DNA sequence. The bacterial enzyme includes a nucleotide binding site for CTP. This binding site contains a number of amino acids that are conserved among all known bacterial CMP-SAS enzymes (See Stoughton et al., Biochem J. 15:397-402 (1999). The identity of the human cDNA as a CMP-SA synthetase gene was confirmed by the presence of significant homology within this binding motif:

bacterial sequence:IIAIIPARSGSKGLidentity / homology+ A+I AR GSKG+human cDNA:LAALILARGGSKGI

[0240] This human homologue commercially, publicly, or otherwise available for the purposes of this invention is cloned and expressed in insect cells. The nucleotide and amino acid sequences of human CMP SA synthetase are shown in FIGS. 29 and 30 respectively. The characterization of CMP-SA...

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Abstract

Methods for manipulating carbohydrate processing pathways in cells of interest are provided. Methods are directed at manipulating multiple pathways involved with the sialylation reaction by using recombinant DNA technology and substrate feeding approaches to enable the production of sialylated glycoproteins in cells of interest. These carbohydrate engineering efforts encompass the implementation of new carbohydrate bioassays, the examination of a selection of insect cell lines and the use of bioinformatics to identify gene sequences for critical processing enzymes. The compositions comprise cells of interest producing sialylated glycoproteins. The methods and compositions are useful for heterologous expression of glycoproteins.

Description

[0001] This application claims benefit under 35 U.S.C. section 119(e) based on copendino U.S. Provisional Application Ser. No. 60 / 227,579, filed Aug. 25, 2000, which is herein incorporated by reference in its entirety, and also claims benefit under 35 U.S.C. section 120 to U.S. application Ser. No. 09 / 516,793, filed Mar. 1, 2000, which is herein incorporated by reference in its entirety and which claims benefit under 35 U.S.C. section 119(e) based on U.S. Provisional Applications Nos. 60 / 169,624, filed Dec. 8, 1999, and 60 / 122,582, filed Mar. 2, 1999, both of which are herein incorporated by reference in their entireties.[0002] Part of the work performed during the development of this invention utilized U.S. Government funds in the form of grants from the National Science Foundation, Grant Numbers BES9814157, BES9814100, and the National Institutes of Health, Grant Number RO1-GM-49734. The U.S. Government has certain rights in this invention.FIELD OF THE INVENTION [0003] The inventi...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/52C12P21/00
CPCC12P21/005C12N15/52
Inventor BETENBAUGH, MICHAELLAWRENCE, SHAWNLEE, YUANCOLEMAN, TIMOTHY
Owner BETENBAUGH MICHAEL
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