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NCI, UND Team Develops Way to Make Protein Microarrays Cheaper, More Stable

Researchers from the National Cancer Institute, Science Applications International Corporation–Frederick and the University of North Dakota have developed a new way to create protein microarrays that they say is cheaper, less labor-intensive, and improves the stability and integrity for extended periods.
Described in an online article published Sept. 24 in PLoS One, the method is based on printing expression-ready plasmid DNA onto slides that can be converted into protein arrays on demand, thereby eliminating the need for antibody or other capture reagents to immobilize newly synthesized proteins onto a microarray surface.
The technology is in its early stages of development and further testing is being done to determine its full functionality. But the lead author told ProteoMonitor this week that the method he and his colleagues created is the simplest technique yet for making protein microarrays and could help to push the technology out of the shadow of mass spectrometry as a research platform for proteomics work.
While DNA microarrays are commonly used in genomic work, in proteomics and protein research, protein microarrays have trailed mass specs as the technology of choice, though there are signs that the field may be gaining wider acceptance. In the paper, the authors write that in order to further knowledge of protein function, quantitative proteomics, molecular interactions, and protein profiling, the continued development of high-throughput platforms, such as protein microarrays, are necessary.
“Unfortunately, inherent cost and technical limitations, including the required production of large libraries of purified proteins and long-term maintenance of array stability and integrity, have caused protein microarray development to lag behind that of DNA microarrays,” the authors said.
According to Deb Chatterjee, lead author of the PLoS One article and associate director of the protein expression laboratory at NCI, SAIC-Frederick, the instability of the arrays, in particular, has been a major roadblock to wider adoption of the technology.
While DNA microarrays retain stability for months, “Protein microarrays are stable for a month at the most, I would say, sometimes even less,” Chatterjee said, adding that for optimal use, they should be stable for at least six months.
In addition, he said, in microarrays using human proteins, only about 10 percent of proteins can be expressed in a system such as E. coli to get a “good protein. Most of the human proteins are very hard to express in soluble form.”
The method developed by the researchers, however, bypasses these bottlenecks. With this technology ”we are making the proteins, expressing the proteins, and purifying the proteins at the same place without separately expressing them and purifying them,” Chatterjee said.

“In our system, the expression vector DNA not only directs the synthesis of each protein, but also serves to capture the protein at its designated location on the microarray surface.”

Their microarrays exploit the high-affinity binding of E. coli Tus protein to Ter, a 20 base-pair DNA sequence involved in the regulation of E. coli DNA replication by synthesizing each protein of interest as a Tus fusion protein. Each expression construct directing the protein synthesis contains embedded Ter DNA sequence, which functions as a capture reagent for the newly synthesized Tus fusion protein.
To create their microarrays, Chatterjee and his team spotted DNA onto a slide. An expression system such as rabbit reticulocyte, wheat germ, or E. coli was then put onto the slide and incubated. “Then you wash it off and off you go,” Chatterjee said.
“In our system, the expression vector DNA not only directs the synthesis of each protein, but also serves to capture the protein at its designated location on the microarray surface,” the authors write.
Because there is no need for purification, a major and labor-intensive step is also skipped. 
The method is a general one that can be modified to generate microarrays directed at specific research areas such as kinase research, disease detection, or drug discovery, Chatterjee said.
The research team is currently further testing the utility of the technology, looking at protein-protein interactions and making a human kinase library. Chatterjee declined to share any data.
While their technology is still in early development, it is another development in a field that in recent months has begun to simmer with activity. In August, Promega officials told ProteoMonitor that the company is preparing to debut functional arrays by the end of the year [See PM 09/04/08].
Another company, Auguron Biosciences, is also continuing to develop a technology called nucleic acid-programmable protein array, or NAPPA, for launch. Earlier this year, the researchers said that the next generation of the technology now enables the large-scale expression of proteins [See PM 05/15/08].
More recently, Akonni Biosystems this week said it will use a $296,316 grant from the National Institutes of Health to develop a protein array-based diagnostic test for respiratory infections.
Chatterjee and his colleagues have also begun extending their technology to surface plasmon resonance applications. In SPR work, a purified protein needs to be spotted on a chip, “so we’re … spotting the DNA and [making] the protein on the chip and do an SPR assay,” he said.
The researchers have applied for a patent with US Patent and Trademark Office to protect the underlying technology.

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