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.
The article, "A New Strategy for the Synthesis of Glycoproteins" was authored by Zhiwen Zhang, Jeff Gildersleeve, Yu-ying Yang, Ran Xu, Joseph A. Loo, Sean Uryu, Chi-Huey Wong, and Peter G. Schultz and will appear in the January 15, 2003 issue of the journal Science. See: http://www.sciencemag.org.
This work was supported by the Department of Energy, the National Institutes of Health (NIH), and the Skaggs Institute for Research. Individual scientists involved in this study were sponsored through NIH and National Research Service Award fellowships.
Supporting Material: The Basis of the Technology
The technology that allows this advance is a methodology that Schultz and his colleagues have developed that exploits the redundancy of the genetic code of organisms like E. coli or yeast that allow these cellular factories to mass produce proteins with unnatural amino acids.
Scientists have for years created proteins with such unnatural amino acids in the laboratory, but until Schultz and his colleagues began their work in this field, nobody had ever found a way to get organisms to add unnatural amino acids into their genetic code.
When a protein is expressed, an enzyme reads the DNA bases of a gene (A, G, C, and T), and transcribes them into RNA (A, G, C, and U). This so-called "messenger RNA" is then translated by another protein-RNA complex, called the ribosome, into a protein. The ribosome requires the help of transfer RNA molecules (tRNA) that have been "loaded" with an amino acid, and that requires the help of a "loading" enzyme.
Each tRNA recognizes one specific three-base combination, or "codon," on the mRNA and is loaded with only the one amino acid that is specific for that codon.
During protein synthesis, the tRNA specific for the next codon on the mRNA comes in loaded with the right amino acid, and the ribosome grabs the amino acid and attaches it to the growing protein chain.
The redundancy of the genetic code comes from the fact that there are more codons than there are amino acids used. In fact, there are 4x4x4 = 64 different possible ways to make a codon-or any three-digit combination of four letters in the mRNA (UAG, ACG, UTC, etc.). With only 20 amino acids used by the organisms, not all of the codons are theoretically necessary.
But nature uses them anyway. Several of the 64 codons are redundant, coding for the same amino acid, and three of them are nonsense codons-they don't code for any amino acid at all. These nonsense codons are useful because normally when a ribosome that is synthesizing a protein reaches a nonsense codon, the ribosome dissociates from the mRNA and synthesis stops. Hence, nonsense codons are also referred to as "stop" codons. One of these, TAG, played an important role in Schultz's research.
Schultz and his colleagues knew that if they could provide their cells with a tRNA molecule that recognizes TAG and also provide them with a synthetase "loading" enzyme that loaded the tRNA with a glycosylated form of the amino acid serine, the scientists would have a way to site-specifically insert that glycosylated amino acid into any protein they wanted.
They needed to find a functionally "orthogonal" pair-a tRNA/synthetase pair that react with each other but not with the normal pairs. They then devised a methodology to evolve the specificity of the orthogonal synthetase to selectively accept glycosyl amino acids.
They created a library of cells, each encoding a mutant synthetase, and they devised a positive selection whereby only the cells that load the orthogonal tRNA with any amino acid would survive. Then they designed a negative selection whereby any cell that recognizes TAG using a tRNA loaded with a natural amino acid dies. In so doing, they found their orthogonal synthetase mutants that load the orthogonal tRNA with only the desired amino acid-which is called N-acetylglucosamine modified serine.
With this system, a ribosome that was reading an mRNA would insert the modified serine when it encountered TAG. Furthermore, any codon in an mRNA that is switched to TAG will encode for the new amino acid in that place, giving Schultz and his colleagues a way to site-specifically incorporate glycosylated amino acids into proteins expressed by the E. coli.
About The Scripps Research Institute
The Scripps Research Institute in La Jolla, California, is one of the world's largest, private, non-profit biomedical research organizations. It stands at the forefront of basic biomedical science that seeks to comprehend the most fundamental processes of life. Scripps Research is internationally recognized for its research into immunology, molecular and cellular biology, chemistry, neurosciences, autoimmune diseases, cardiovascular diseases and synthetic vaccine development.
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