System and method for the production of recombinant glycosylated proteins in a prokaryotic host

Abstract

A system and a method for the production of recombinant N-glycosylated target proteins. The system comprises a prokaryotic organism (e.g. Escherichia coli) into which is introduced a genetic information encoding for a metabolic apparatus capable of carrying out the requested N-glycosylation of the target protein. Said prokaryotic organism also contains the genetic information required for the expression of one or more recombinant target proteins. The metabolic apparatus preferably comprises specific glycosyltransferases for the assembly of the oligosaccharide on a lipid carrier and an OTase that covalently links this oligosaccharide to specific residues of the desired protein.

Description

[0001]This application is a continuation of U.S. application Ser. No. 10 / 506,917, filed Sep. 3, 2004, which is a national stage application of PCT / CH03 / 00153, filed Mar. 5, 2003, which claims the benefit under 35 U.S.C. §119(e) of 60 / 364,655, filed Mar. 14, 2002, and which claims priority under 35 U.S.C. §119(a) to CH 2002 0394 / 02, filed Mar. 7, 2002. Each of the foregoing applications is incorporated by reference herein in its entirety.FIELD OF INVENTION[0002]The present invention relates to an expression system and a method for the production of recombinant human and / or animal and / or plant and / or prokaryotic and / or fungal glycoproteins. Such glycoproteins may serve as nutrition or medical drugs for human or animals or plants because of their identical structure to the glycoproteins normally produced in these organisms.TECHNICAL BACKGROUND[0003]Glycosylation constitutes one of the most important of all post-translational protein modifications in eukaryotic cells and may have numero...

AI Technical Summary

Problems solved by technology

The importance of a highly defined oligosaccharide structure on recombinant glycoproteins contrasts sharply to the inability of presently used biotechnological processes to generate glycoproteins.

However, the plethora of glycosyltransferases active in the Golgi compartment of eukaryotes makes such an approach very difficult.

Additional problems with the use of eukaryotic expression systems are the following: In general, the mammalian expression system has its drawbacks in the use of growth medium, which contains calf serum.

This raises concern about biosafety because of possible contamination with bovine spongiform encephalopathy (BSE).

Furthermore human cell line cultures are much more difficult to keep sterile, these cells grow slowly and require expensive process control.

Otherwise host cells must be adapted by genetic engineering of the glycosylation pathway in the Golgi (FIG. 1B), and this represents the major drawback of human cell lines in the expression of recombinant glycoproteins.

Furthermore, large scale Insect cell culture offers particular challenges to the biotechnologist due to the higher oxygen consumption and higher shear sensitivity of the cells as compared to mammalian cells.

Like in mammalian cells, the major drawback in the heterologous expression of glycoproteins resides in the different structure of the N-glycan as described before.

Especially the lack of terminal sialic acid residues is detrimental, because these sugars play important roles in glycoprotein biology.

However, the inability of E. coli cells to exert post-translation& modifications of proteins remains the strongest drawback for its use as the preferred host for the production of human proteins.

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