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EPO is a well-investigated glycoprotein with a known, clearly delineated biological function. It is thus favored as a model glycoprotein. Its protein component is made of 166 amino acids, and four complex sugar chains (oligosaccharides) are bound to it like branched antennas. They increase the lifetime of the protein in the blood. Glycoproteins are important pharmacological agents; however, they are difficult to produce. In cell cultures, no uniform saccharide chains are formed. In addition, bacteria are not able to recreate the complex oligosaccharides found in mammals. The alternative approach, chemical synthesis, is capable of producing tailored, uniform glycoproteins that can fulfill the function of glycoproteins as drugs and for research. However, this approach is very expensive.
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Highly expressive: Cell
proliferation was observed with concentrations of a
erythropoietin (EPO) analogue above 50 pg mL-1. The EPO analogue
has two human complex-type sialyloligosaccharides (see picture)
and was formed by the combined use of chemical synthesis and
protein expression in E. coli. Both the 24 and 30 positions are
glycosylated, but the two sialyloligosaccharides do not
interfere with binding of the EPO analogue to a receptor.
[Credit: Angewandte Chemie, Wiley-VCH] |
The team has found a happy medium: they used a biotechnology approach to produce only one part of the EPO protein chain in a bacterial culture. They used chemical synthesis to produce the second, shorter part of the protein chain with the sugar antennas. They introduced a synthetic anchoring site, to which sugars can be chemically attached. The saccharide used was a branched complex sialyloligosaccharide from egg yolks. In the final step, the researchers hooked together the two protein chains by a method known as native chemical ligation. Folding experiments demonstrated that, like natural EPO, the synthetic EPO analogue assumes a helical structure. Cell proliferation assays with bone marrow cells yielded a biological activity equal to that of the original. In vivo, the analogue could not increase the red-blood-cell count in the same way as true EPO. The researchers hope that by using different, more highly branched oligosaccharides they will be able to create analogues that also demonstrate high bioactivity in vivo.
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