Scientists at Scripps Research describe new strategy for the synthesis of glycoproteins

01/13/04

A new way to make therapeutic proteins

A team of investigators at The Scripps Research Institute and its Skaggs Institute for Chemical Biology in La Jolla, California has developed a new way of making glycoproteins­-proteins with carbohydrates (sugars) attached.

Methods for making glycosylated proteins are important to scientists who want to understand the role of carbohydrates in protein structure and function, since the human body contains many heavily glycosylated proteins, including antibodies, hormones, and immune system proteins like cytokines and interleukins. These methods are also of interest to doctors, since pharmaceuticals are often heavily glycosylated proteins (e.g., erythropoietin, which is useful for treating anemia, cancer, and AIDS).

"In the future, you will see more and more proteins [coming forth] as drug candidates, mainly because of advances in genomic research," says Chi-Huey Wong, Ph.D, who is Ernest W. Hahn Professor and Chair in Chemistry at Scripps Research, "and most of these proteins have sugars on them."

Led by Professor Wong, Professor Peter G. Schultz, Ph.D., who holds the Scripps Family Chair in Chemistry at Scripps Research, and Scripps Research Associate Zhiwen Zhang, Ph.D., the team of scientists discovered a new way of synthesizing glycoproteins, and they report their strategy in the latest issue of the journal Science.

The strategy, which avoids some of the bottlenecks of previous methods, involves using a modified form of the bacterium Escherichia coli to express a glycosylated form of the protein myoglobin. The E. coli was evolved so that it would insert a glycosylated amino acid into the sequence of the myoglobins as they were being produced.

The Tough Task of Making Glycoproteins
Glycoproteins are basically proteins that have been modified so that one or more carbohydrates (sugars) are attached to nitrogen or oxygen atoms within the protein's amino acids.

The modifications on glycoproteins are very much a part of the language of life, and some even call carbohydrates the third alphabet, behind DNA and proteins. Sugars on proteins are like the accents on spoken words­-they change the meaning without changing the spelling. If the correct sugars are not there, the biology is altered.

Some of the most intriguing problems in modern biology and medicine require scientists and doctors to synthesize proteins that have been modified with particular sugars attached in particular places. This presents a sticky problem because in the human body, the proteins are usually made first and then modified, and this modification is handled by a number of intricate mechanisms, not all of which may be reproduced in the test tube. Producing glycoproteins in the laboratory has been especially problematic.

Even when it is possible to directly synthesize particular glycosylated proteins in the test tube, producing them may be expensive, difficult, time-consuming, and not at all practical. Some glycosylated proteins are produced in microorganisms or cultures of eukaryotic cells, like yeast or Chinese hamster cells­-an expensive and sometimes inexact process, which often involves difficult and expensive purification schemes.

Zhang, Schultz, Wong, and their colleagues have found another way­-making homogeneous pools of glycosylated proteins in E. coli.

Bacterial cultures like E. coli have been used to produce proteins cheaply and easily for years, but it has never been possible to produce glycosylated proteins in them because bacteria don't normally have the same ability to attach sugars to proteins as eukaryotic cells do. The Scripps researchers solved this problem by modifying a form of E. coli to make homogeneous pools of glycosylated myoglobin protein with sugars attached at one desired position (see below: The Basis of the Technology).

Once the protein with the glycosyated amino acid was made and isolated, the Scripps Research team was able to add additional sugars to the same site by using a "transfer enzyme," called glycosyltransferase, which attached the extra sugars.

The use of E. coli to make the myoglobin is a significant advance because it is a general and versatile method and it opens up the gates for using bacterial cultures to put other sugars on other proteins. The method is also scalable, and should be cheaper than other current technologies.

Source: Eurekalert & others

Last reviewed: By John M. Grohol, Psy.D. on 21 Feb 2009
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