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With Some Help From Friends, Mathias Uhlen Hopes to Map Human Proteome

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A new human antibody initiative led by Stockholm-based professor Mathias Uhlen will collaborate with two other European antibody initiatives and will likely join the umbrella HUPO antibody initiative, ProteoMonitor has learned. Uhlen’s initiative seeks to use antibodies to systematically map the entire human proteome and create a public database of the results.

Uhlen, a professor at Albanova University Center at the Royal Institute of Technology in Stockholm, launched the Human Proteome Resource on Jan. 1 with $28 million over four years in initial funding from the Swedish non-profit Wallenberg Foundation. Uhlen wants to stretch this funding to analyze the proteins expressed by 10,000 genes, at a rate of 100 genes per week, although he admits that this is a lofty goal that may not be met. He hopes that Wallenberg or other collaborators will eventually dish out a total of $80 million, which he estimates is the minimum amount required to look at the whole proteome. “If the endpoint is just to make antibodies and localize the proteins in their tissues, then with the $80 million we can lift the information content enormously,” Uhlen told ProteoMonitor in a personal interview in late August.

The Human Proteome Resource

Uhlen has spent the last several months touring the circuit of proteomics conferences, giving numerous talks promoting the initiative — including one that received mixed reviews at the recent mass spec conference in San Francisco (see PM 9-5-03). He is no neophyte in this business of promoting: in the 1990s he invented the gene sequencing method pyrosequencing and later founded the genotyping company of the same name. But now Uhlen views previous work as worthless without his present pet project. “The only justification for the human genome project is getting to the proteins,” he said. “In the end you have to do this project — otherwise sequencing the human genome is a waste of time.”

Uhlen terms his methods, which he details in a paper describing a pilot project that profiled the proteome of human chromosome 21 (Molecular & Cellular Proteomics, 2003 June 2(6):405-414), “affinity proteomics.” He defines this as “the systematic generation and use of protein-specific affinity reagents to functionally explore the proteome” — in other words, generating antibodies to profile all human proteins.

Uhlen further classifies affinity proteomics as a type of “genome-based proteomics,” which — in contrast to hypothesis-driven classical proteomics that rely on mass spec techniques to pick out proteins from a particular sample — consists of methodically expressing all the proteins encoded in the genome. “The majority of scientists go for hypothesis-driven approaches. But I think this is going to be the basis for all research. It is the proteomics community’s duty to provide a protein database,” Uhlen said.

Uhlen’s approach is somewhat different from those taken by other well-known antibody-focused proteomics scientists such as Marius Ueffing, leader of the German Society for Proteome Research’s monoclonal antibody initiative (see PM 1-27-03), and Dolores Cahill, who directs the Center for Human Proteomics at the Royal College of Surgeons in Dublin, Ireland and works on antibody arrays. First of all, rather than expressing the entire encoded protein, Uhlen uses homology search programs such as BLAST to find domains of the protein that contain the least homology to other proteins and the fewest transmembrane portions. He expresses in E. coli only the selected portion of the protein-producing protein fragments that he has dubbed Protein Epitope Signature Tags. Uhlen then immunizes rabbits or chickens with the PrESTs and collects polyclonal antibodies — which are easier and cheaper to generate and better at binding non-native proteins such as PrESTs than monoclonal antibodies. The antibodies will then be used for expression localization studies with tissue arrays.

The initial database will contain this localization information, but Uhlen hopes that — with the help and input of collaborators — the database will eventually also contain a wide range of information about every protein in the healthy human proteome and in several diseased ones, too — including such details as size, structure, modifications, and interactions. So far, Uhlen has done “one pass of the genome,” with a 50 percent overall success rate for generating workable antibodies to a given PrEST. A second pass is on the way, though Uhlen admitted that it is unlikely he would be able to generate antibodies to every encoded protein this way. “Some proteins are very hard to get — maybe 5 percent of the proteome we won’t be able to study this way,” he said. “But these are maybe not the most interesting proteins.”

Bringing it all Together

Uhlen has repeatedly expressed his desire to collaborate, which he views as key to accomplishing such a broad task. “I would love to join an international consortium that goes through all proteins and gets interactions … there is much more to be done,” he said in the August interview with ProteoMonitor.

Since then, both Ueffing and Cahill have answered his call. In an e-mail interview from the Proteomic Forum 2003 held this week in Munich — where all three scientists gave talks — Ueffing said, “We [Uhlen and I] have decided to establish a fully integrated structure under the roof of the European Proteome Initiative for both of our projects … We aim at jointly forming a [European Union 6th Framework] consortium to propose a common platform for R&D with respect to protein expression, protein structure, and antibody production. Both our initiatives will be presented in Montreal at the HUPO conference.” The EU 6th FW Program is a large European funding initiative for science and technology development.

In a phone interview with ProteoMonitor from the same conference, Cahill had similar news. “I just met [Uhlen] yesterday [Sept. 15] — now I’m going to be on [his] scientific advisory board and we’re going to collaborate,” she said. Cahill emphasized that nothing was official until she and Uhlen had found funding for such a collaboration, but she expressed a clear vision of how she could coordinate both with Uhlen and with Ueffing. “What we would like to do is supply both [initiatives] with recombinant proteins,” Cahill said. “We would express some of our proteins and give [Uhlen] the recombinant protein, which he would then use to immunize his rabbits or chickens. Then the other thing we can do is put his antibodies on our protein arrays and see how specific the antibodies are and look at the specificity and cross-reactivity. [With the German initiative] it would be the same thing — we would supply the recombinant proteins and then we would screen their monoclonal antibodies,” she said. She added that Protagen, a spinoff company that she started, would probably be the official collaborator for the German initiative while her academic group would collaborate directly with Uhlen.

Ueffing’s initiative aims at establishing a European protein and antibody bank with the goal of generating monoclonal antibodies against subsets of recombinant human proteins (see PM 1-27-03). Ueffing said that his group has recently established a production center for this work in Rijeka, Croatia, and that representatives from Roche and Amersham Biosciences — two companies that Ueffing said expressed interest in the project — visited the center a couple weeks ago. Ueffing said he hopes to entice big pharma into investing in the project in exchange for access to the antibody bank.

HUPO's Umbrella

Ueffing’s reliance on corporate money is a criticism that Matthias Mann, professor of biochemistry and molecular biology at the University of Southern Denmark and head of the umbrella HUPO antibody initiative, has for his initiative. “[Ueffing’s group’s] main problem is that they still don’t have the money, and if they want to get it partially from companies, there’s some tension between making [the results] available to everybody and giving the companies something in turn for their investment,” Mann said. Since Uhlen’s project is funded by a non-profit, his group has fewer obstacles to making information public, Mann said. Also, “the exciting thing is that they have already gotten the money, and at least [with Uhlen’s project] we have something on our hands fairly soon, in terms of reagents,” Mann said.

Still, Mann sees Ueffing and Uhlen’s approaches as very complementary, and he intends to use HUPO as a vehicle for coordinating them. “This is not really a HUPO antibody initiative as such — it’s really to encourage the different initiatives to work together and to avoid duplication so it’s not everyone doing the same thing. In return, HUPO can assure funding bodies that this makes sense internationally — that it’s not just something that this particular researcher wants to do,” Mann said. He noted, however, that it was difficult for HUPO itself to get funding from usual sources like the NIH, due to its broad, international nature. Nonetheless, HUPO president Samir Hanash announced at the Munich meeting this week that HUPO’s Human Plasma Proteome Project had raised a total of $1 million, and that the NIH had matched corporate funds in contributing to this sum, according to Mann. “In theory, that could happen with the antibodies also, but I’m not optimistic,” Mann said.

Although Uhlen is not yet officially working with HUPO on the Human Proteome Resource, Mann definitely sees such a role in his future. “Uhlen said that he wanted to become more active in HUPO Europe. If he [does that], it would only be natural that his antibody initiative becomes more a part of HUPO than it is now,” Mann said.

— KAM

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