Biotech's new invaluable tool

In just two decades, the protein equivalent of an intron has carved out a significant niche in biotechnology -- and captured the interest of evolutionary biologists, who suspect these potentially ancient elements could provide clues to early enzymes.

Image: Wikimedia commons

With the ability to splice themselves out of proteins and paste the two loose ends of the protein back together, inteins are proving to be an invaluable tool in biotechnology. Just 20 years since their discovery, inteins are already being used to purify, manipulate, and even create proteins that were difficult (or impossible) to build using traditional techniques. "It's a very fast moving field," said chemical biologist linkurl:Fran Perler;http://www.neb.com/nebecomm/researchScientist.asp?id=FPerler of New England Biolabs, which currently has an intein-based purification method on the market. "The technology is all based on being able to introduce interesting chemical modifications into proteins and study how these modifications change the protein." The first intein was discovered in 1990 in the VMA1 gene of yeast, when researchers noticed that the gene, which codes for a membrane enzyme, appeared to be much larger than the polypeptide it produced. Upon closer examination of the nucleotide sequence, researchers noticed a segment in the middle of the gene that was missing from homologous genes. Further research revealed that the unusual sequence was excised before the final protein product, but unlike the well-known introns, the excision happened after the mRNA sequence had been translated into a protein. At the time, it was considered nothing more than "a molecular oddity," said molecular biologist linkurl:Xiang-Qin (Paul) Liu;http://www.biochem.dal.ca/faculty/facultypages/liu/ of Dalhousie University. But just two decades later, nearly 600 naturally occurring inteins have been found in all three domains of life, as well as in viruses, according to the InBase intein database, started by Perler. And while researchers are still trying to figure out what purpose they serve, if any, in the organisms that harbor them, more and more is being learned about how they work, and how they can best be put to use. "[Inteins have] many applications already realized in biotechnology and more and more emerging because it's a uniquely useful technology," Liu said. Specifically, it is the protein-splicing ability of the inteins that protein researchers find so useful. One way to take advantage of this quality, for example, is to insert a tag into or at the end of an intein, so that researchers can identify and separate the protein that contains the intein from solution. Once that's occurred, the intein can be activated to splice itself out, leaving only the mature protein behind. Indeed, systems based on this sort of technology are already commercially available. But inteins don't just cut; they also paste. In the late 1990s, Liu and his colleagues published the first example of split inteins, in which pieces of a gene are separated by some distance in the genome and come together by way of inteins that glue the pieces together as they splice themselves out. This so-called trans-splicing action generated excitement among protein biologists, who have used the technology to make synthetic proteins, or proteins with posttranslational modifications -- a feat that has linkurl:proven difficult;http://www.the-scientist.com/blog/display/56190/ using traditional protein synthesis methods. "[In] the current decade, we're realizing that DNA and that genomic information is only part of our hereditary information," Perler said. "There's all this epigenetic information [as well]. What [inteins] allow you to do is to synthesize proteins with these modifications and study the effect of these modifications both in vitro and in vivo. I think that's the most exciting application of inteins." In addition to their biotech applications, inteins have some unique characteristics that make them inherently interesting from an evolutionary perspective. Many inteins contain an endonuclease domain that allows them to jump between genomes. Indeed, phylogenetic analyses have suggested that they tend to be horizontally transmitted, with inteins at the same position in the same genes in different genomes showing high sequence similarity, "even if they're in different phyla," Perler said. Furthermore, many suspect that inteins are actually ancient elements that could perhaps provide some insight into some of the earliest enzymes. "Most of us that study inteins believe that the inteins we see now are remnants of the past," Perler said. It's possible, she explained, that the splicing domains initially existed without the endonucleases, which later "hijacked" the inteins by allowing them to jump to new genomic locations -- in other words, turning them into selfish DNA. Indeed, some inteins lack an endonuclease domain, and with a structure that suggests a duplication of two 65-amino acid domains -- the size of the first enzymes found in the primordial soup -- "inteins may have been involved in putting together protein domains," Perler said. "If you're trying to early in evolution figure out how to make better enzymes and better proteins, if these inteins allowed you to shuffle sequences, then it would be a really efficient way of building bigger proteins." Some also still wonder what function inteins might serve. "It's a really fascinating biological phenomenon, how selfish elements like this evolve," said microbiologist linkurl:Rahul Raghavan;http://www.biochem.arizona.edu/ochman/Personnel/raghavan.htm of Yale University. In the meantime, the evolving applications continue to drive the field, Perler said. With several technologies already on the market, and plenty more in the works, inteins are earning respect as a go-to technique in the protein toolbox. "The intein is not really the big deal," Perler said; "it's what you can do with it."
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