Editorial Article
Monday January 11, 2010
by Catherine Shaffer
Protein expression, once the province of specialized proteomics laboratories, is now a versatile tool used in many disciplines within the life sciences. Advances in genomics have generated increased interest in functional studies of proteins. New products and technologies in proteomics make protein expression accessible to scientists who do not have specific molecular biology training. Traditional methods of protein expression include bacterial and mammalian cell expression; new innovations on these older methods are now available.
One of the most exciting advances is cell-free expression, which puts all of the apparatus of gene transcription and translation in a single bench-top reaction. Since it is an open system, it allows the addition of auxiliary components like modified tRNAs. Applications for cell-free expression include any situation where it is useful to have small amounts of protein made quickly and on demand, such as protein-protein interaction and protein modification studies.
The TNT® T7 and TNT® SP6 Quick Coupled Transcription/Translation Systems from Promega are used frequently in cell biology studies where the focus is on genes and pathways. This technology is based on rabbit reticulocyte lysate, and requires only the addition of template DNA to get the reaction going. This makes it ideal for casual or one-off use where proteomics are not the main subject of investigation. Gary Kobs, strategic marketing manager for Promega, recounts a visit he made to a customer's laboratory. “I visited a drosophila lab at University of Michigan in Ann Arbor last summer. They were interested in seeing how their protein interacts with other proteins in the drosophila genome, but they were not protein experts. They were drosophila people. They used our TnT system, which is very simple. They added the gene of interest; it transcribed, translated and made protein for them in one hour. They can use it to look at how that protein interacts with other proteins very easily without having to delve into optimization of expression.”
Cell-free expression is not only for the novice, though. It has much to offer proteomics experts as well. Taking expression out of living cells allows the expression of toxic proteins, or the incorporation of non-natural amino acids. A number of these systems are available on the market, based on everything from E. coli to rabbit reticulocytes. However, for some applications, there is simply no substitute for human host expression. The Human In Vitro Protein Translation System from Thermo Scientific Pierce Protein Research Products fulfills this need. Based on extracts of immortalized human cell lines, this system excels in applications that require authentic post-translational modification. Researchers studying human diseases can express proteins in human cell-free extracts instead of traditional E. coli, wheat germ, insect, or rabbit reticulocyte systems. Depending on the expression system, in vitro translation reactions generate approximately 2 - 40 ug of protein per 25 ul reaction. The small scale of in vitro expression systems is also well suited to the microplate format, where dozens of parallel reactions can be combined or compared side-by-side.
On a per-milligram basis, in vitro translation does not seem very economical. Conventional protein expression (i.e. in vivo expression) in prokaryotes costs, on average, 20 dollars per milligram, according to Atul Deshpande, technical product manager at Thermo Fisher Scientific. The price for cell-free expression (i.e. in vitro expression) can be as much as $40,000 per milligram. “When you look at that, one quickly realizes that in vivo and in vitro protein expression systems are used for different purposes. Why people use in vitro translation, even though expensive for a reaction, is because it is simple, reliable, and enables rapid and convenient production of recombinant proteins for downstream applications such as protein engineering, protein-protein, and protein-nucleic acid interactions.”
For those who need larger quantities of protein, the new cell-based expression systems have much to offer. Choices of protein expression system depend on a number of factors, including the size of the protein, the glycosylation pattern, enzymatic activity, antigenicity, folding, as well as downstream applications.
Typically, eukaryotic expression systems are chosen when the end product is a glycosylated protein with functional activity. But mammalian cell-based expression systems are notoriously sensitive, need constant attention, and are difficult to scale up. An E. coli expression system from Genway solves the simultaneous problems of scale-up as well as generating functional, properly folded human proteins. Genway develops protein expression systems for the research and diagnostic market, so their technologies must perform to very high standards in order to be used in diagnostic tests. The company has recently demonstrated this with the expression of TGF-beta2 (transforming growth factor beta 2) in E. Coli. TGF-beta2 is a cytokine secreted during embryonic development. It has significance in the development of cardiac, lung, and other tissues. Says Sergey Sikora, PhD, vice president of business development for Genway, “We achieved a lot of success in E. coli for functional human proteins. E. coli expression is usually, for manufacturing at least, an expensive route.” Functional proteins like cytokines are difficult to express, and tend to come out misfolded. “We have our proprietary technology for the refolding of proteins to make them function.”
Another important tool in protein expression is epitope labeling. This can be done for purposes of purification or analysis. The epitope tag is created by inserting short, antigenic peptide sequences into the protein through recombinant DNA technology. The foreign peptide sequence can then be recognized by highly specific antibodies that can be used to immunoprecipitate or immunoaffinity purify the protein. Common epitope tags that are supported commercially include the his-tag, c-MYC, and FLAG.
Sigma-Aldrich is the originator of one of the most widely used of these systems, the FLAG epitope system. FLAG is a very small, hydrophobic epitope tag that is also cleavable by enerokinase protease, making it “removable.” Because it is very small, its interference in protein folding is minimal. FLAG epitope has been used heavily in studying proteins, but Sigma has also found emerging applications in the field of epigenetics. Says Angela Crawford, product manager for Sigma, “We're finding that they have a lot of genes that there aren't any good antibodies for, and what they like to do is use antibodies to pull down the chromatin that they're studying and look at interaction partners, whether they be proteins or nucleic acid type molecules.... What we're finding is that since the FLAG tag is smaller and does not interfere with the domains or functions or that sort of thing, a lot of our customers are using the FLAG epitope system to purify those chromatin-interesting genes. We're very excited to see a lot of that, and actually we will be launching products in February that are FLAG tagged genes specifically for epigenetic studies.”
Another entry in the epitope tagging field is the Profinity eXact Fusion-tag system from Bio-Rad Laboratories. This is an E. coli-based tool for single-step purification of affinity-tagged proteins with the use of a protease. The system uses the protease subtilisin immobilized on a chromatographic matrix, which targets the prodomain sequence of subtilisin. The mature subtilisin and the prodomain interact strongly, creating an effective system for tagging expressed proteins. The advantage of this system is that the tagging is highly specific and only happens once the protein has been captured on the affinity column. This helps to overcome one of the most persistent challenges in proteomics: effectively handling the broad dynamic range of proteins in nature, as well as improving the overall quality of results, through increased sensitivity and reproducibility.
Standard techniques for protein expression have been updated through advances in understanding of the mechanisms of transcription and translation from the fields of genomics and proteomics. Updated technologies include cell-based and cell-free protein expression as well as methods for labeling, capturing, and imaging expressed proteins. This has made benchtop protein expression more accessible to scientists outside of the field of molecular biology, while at the same time adding powerful functionality to users within the field.