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Novel Array Based on cDNA Vectors Could Rival Invitrogen s ProtoArray

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San Diego — A novel array called the Nucleic Acid-Programmable Protein Array received positive reviews from researchers attending the PEPTALK Protein Arrays conference here this week.

Developed by Joshua LaBaer’s research group at Harvard University, the NAPPA array consists of cDNA vectors coupled with a capture antibody, and could be advantageous over traditional protein arrays in that proteins do not have to be purified. Instead, proteins are made at the time of the assay by bathing the cDNA array in a cell-free transcription/translation mix called a reticulocyte lysate, said LaBaer, who gave the keynote presentation during PEPTALK.

If the array enters the market — and it is unclear whether this will be the case — it would compete with Invitrogen’s ProtoArray, according to at least one industry expert.

“We would like to avoid purifying proteins because we’re small [as a group], and we’re lazy,” joked LaBaer. “With NAPPA, proteins are made at the time they are assayed.”

Each cDNA on a NAPPA array is fused with a GST protein tag at the C-terminus. After the cDNA-GST is expressed, the protein is captured by a neighboring GST antibody.

“LaBaer said he doesn’t think his group has done anything special, but I think it’s a really big deal,” said Todd Martinsky, the founder of TeleChem International’s ArrayIt Division when asked to comment on the NAPPA array. “The idea is if proteins are so hard to make, then just make it on the chip. It’s a much faster route to content.”

Aside from avoiding protein purification, another potential advantage of the NAPPA array is that it is cheaper to manufacture. While every proteinx typically costs $5,000 to $10,000 to make, one cDNA spot typically costs only 10 cents to make, LaBaer pointed out during his talk.

“It’s a hell of a research tool,” Neal Gordon, the president of Epitome Biosystems, a Massachusetts-based company that manufactures peptide arrays, said following the presentation. “It’s obviously a cost-effective way of doing it.”

In addition to being cost-effective, the system is also clever in that it has a built-in quality control for proteins, Gordon pointed out. Since the GST protein is attached to the C-terminal end of the target protein, the protein will only be captured if the entire protein is transcribed and translated.

“If it’s terminated early, there’ll be no GST, and there’ll be no binding to the antibody,” Gordon explained. “It’s an inbred quality control.”

Steven Bodovitz, the principal consultant for BioPerspectives who has written numerous reports on the protein array market, said that the NAPPA array is also advantageous in that cDNA is more stable than protein, and that more protein can be made at the time of an assay than can be spotted onto a regular protein array.

However, a drawback to the NAPPA array is that proteins may not fold properly in a cell-free medium, said Bodovitz.

The closest thing to the NAPPA array on the market is Invitrogen’s ProtoArray products, said Bodovitz. “Both of those arrays have full-length proteins, but with the ProtoArray, the proteins are made in yeast, and with the NAPPA they’re made in cell-free lysate,” he said. “If this [NAPPA array] were to make it out onto the market, it would compete with ProtoArray.”

Barry Schweitzer, the director of operations for Protometrix/Invitrogen called the NAPPA array “interesting technology.”

“With this array, it’s kind of like ‘Add water and use’, and the protein is available right away,” said Schweitzer.

Schweitzer agreed that the NAPPA array is cheaper to make in theory. However, he pointed out that it is more difficult to prototype, with particular users needing different cDNA content.

“It’s cheaper to make in one sense. On the other hand you have to print out a different array for each particular user,” said Schweitzer.

Another potential drawback of the NAPPA array is “cross-talk” between different spots, said Schweitzer.

Since each cDNA-derived protein is linked to a GST, they may overlap in solution and be captured by a GST antibody on another spot, he pointed out.

Marius Ueffing of the National Research Center in Munich said he was impressed by LaBaer’s data showing the results of protein-protein interactions.

In his talk, LaBaer described detection of interaction between transcription factors Jun and Fos. Fos-GST was expressed and immobilized by GST capture on the NAPPA array. He also managed to show that the Jun-Fos interaction was detected clearly above background signal.

Other protein-protein interactions that were investigated using NAPPA included those between p16 and cyclin-dependent kinases. LaBaer showed that interactions were detected among CDK4 and p16, as well as CDK6 and p16. However, no interaction was observed between CDK2 and P16. These observations are in accordance with previously published data, said LaBaer.

Ueffing said the NAPPA technology has excellent potential, especially because Harvard’s repository of cDNA clones is considered extensive.

“One could imagine that a company built on this platform would be successful,” said Ueffing. “This technology is very much controlled and has pre-defined components. However, I have not tried this array so I don’t know what problems would have to be overcome.”

— TSL

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