Nature Methods a la Frederick
Posted by Jim H on November 25, 2008
At the last BioBeers, I didn’t have a lot of time to talk to Jim Hartley. He’s now with SAIC/NCI-Frederick working in the Protein Expression group with Deb Chatterjee. It did give me great pleasure, though, as I was scrambling to get my laptop to communicate with the PC projector, to eavesdrop on a conversation Jim was having with Mike Smith about this new cell-free system they’ve come up with for screening genome wide protein expression. An “all DNA” protein microarray, of sorts. This was described in a September PLoS publication:
Protein Microarray On-Demand: A Novel Protein Microarray System
1 Protein Expression Laboratory, SAIC-Frederick, Inc., NCI-Frederick, Frederick, Maryland, United States of America, 2 Laboratory of Molecular Technology, SAIC-Frederick, Inc., NCI-Frederick, Frederick, Maryland, United States of America, 3 Advanced Technology Program, SAIC-Frederick, Inc., NCI-Frederick, Frederick, Maryland, United States of America, 4 Department of Microbiology and Immunology, School of Medicine and Health Sciences, University of North Dakota, Grand Forks, North Dakota, United States of America
We describe a novel, simple and low-cost protein microarray strategy wherein the microarrays are generated by printing expression ready plasmid DNAs onto slides that can be converted into protein arrays on-demand. The printed expression plasmids serve dual purposes as they not only direct the synthesis of the protein of interest; they also serve to capture the newly synthesized proteins through a high affinity DNA-protein interaction. To accomplish this we have exploited the high-affinity binding (~3-7×10 -13 M) of E. coli Tus protein to Ter, a 20 bp DNA sequence involved in the regulation of E. coli DNA replication. In our system, each protein of interest is synthesized as a Tus fusion protein and each expression construct directing the protein synthesis contains embedded Ter DNA sequence. The embedded Ter sequence functions as a capture reagent for the newly synthesized Tus fusion protein. This “all DNA” microarray can be converted to a protein microarray on-demand without need for any additional capture reagent..
To take it back a step further, I saw Jim in a professional capacity at LTI frequently primarily because our names are nearly homophones (Hardy vs Hartley). Seems like we were always swapping mail or phone messages (back in the days before e-mail). And just to let you know, back in thise days Jim was working in R&D “cloning” the native restriction enzymes everyone uses today (which some bone-headed marketing guy dcided he’d share with NEB, but that’s a whole other story), working on recombinant TdT (a real popular enzyme back in the day we were making from frozen calf thyroids we bought from a meat packing plant. Talk about variable yield..). He may be best know for working on the team that discovered/commercialized the Gateway Cloning system. he is also the inspiration for me starting BioBeers, and this is the first one he’s made it to.
Mike Smith worked on creating all the Competent Cells at LTI. Things like DH5 alpha, Electromax, LE cells. When IVGN laid us all off (yet another example of wasting talent, but don’t get me going again), he started GeneChoice (now a shell of its former self after being peddled around like rubbish by another bunch of marketing geniuses), making better comp cells than the one’s he created 10 years earlier.
Anyway, back to my original story. Just reading the abstract in Nature Methods this week (you have to subscribe to Nature our buy the article on-line, despite this research being funded by our tax dollars on a Federally owned land). The abstract is pretty sparse, but here it is (proper citation: Nature Methods 5, 1001 – 1002 (2008) doi:10.1038/nmeth1208-1001):
Comprehensive sets of clones and improved high-throughput methods for production of functional proteins now allow proteome-scale in vitro experiments on nearly 15,000 human genes
Identification of highly expressed, soluble proteins using an improved, high-throughput pooled ORF expression technology.
Waybright T, Gillette W, Esposito D, Stephens R, Lucas D, Hartley J, Veenstra T.Laboratory of Proteomics and Analytical Technologies, Advanced Technology Program, SAIC-Frederick, National Cancer Institute at Frederick, Frederick, MD 21702, USA.
This article describes an improved pooled open reading frame (ORF) expression technology (POET) that uses recombinational cloning and solution-based tandem mass spectrometry (MS/MS) to identify ORFs that yield high levels of soluble, purified protein when expressed in Escherichia coli. Using this method, three identical pools of 512 human ORFs were subcloned, purified, and transfected into three separate E. coli cultures. After bulk expression and purification, the proteins from the three separate pools were digested into tryptic peptides. Each of these samples was subsequently analyzed in triplicate using reversed-phase high-performance liquid chromatography (LC) coupled directly online with MS/MS. The abundance of each protein was determined by calculating the average exponentially modified protein abundance index (emPAI) of each protein across the three protein pools. Human proteins that consistently gave high emPAI values were subjected to small-scale expression and purification. These clones showed high levels of expression of soluble protein. Conversely, proteins that were not observed by LC-MS/MS did not show any detectable soluble expression in small-scale validation studies. Using this improved POET method allows the expression characteristics of hundreds of proteins to be quickly determined in a single experiment.