The completion of the human genome project and the development of high-throughput approaches herald a dramatic acceleration in the pace of biological research. One of the most compelling next steps will be learning the functional roles of all proteins. Achievement of this goal depends in part on the rapid expression and isolation of proteins at large scale. We exploited recombinational cloning to facilitate the development of methods for the high-throughput purification of human proteins. cDNAs were introduced into a master vector from which they could be rapidly transferred into a variety of protein expression vectors for further analysis. A test set of 32 sequenceverified human cDNAs of various sizes and activities was moved into four different expression vectors encoding different affinity-purification tags. By means of an automatable 2-hr protein purification procedure, all 128 proteins were purified and subsequently characterized for yield, purity, and steps at which losses occurred. Under denaturing conditions when the His 6 tag was used, 84% of samples were purified. Under nondenaturing conditions, both the glutathione S-transferase and maltose-binding protein tags were successful in 81% of samples. The developed methods were applied to a larger set of 336 randomly selected cDNAs. Sixty percent of these proteins were successfully purified under denaturing conditions and 82% of these under nondenaturing conditions. A relational database, FLEXProt, was built to compare properties of proteins that were successfully purified and proteins that were not. We observed that some domains in the Pfam database were found almost exclusively in proteins that were successfully purified and thus may have predictive character.W ith the application of large-scale and high-throughput (HT) approaches to biological and medical questions, biology has embraced a new era of technology development and information collection. The great task lying ahead is to elucidate the functions of all proteins encoded in the genomes of sequenced model organisms. This process involves collection of information about the temporal, spatial, and physiological regulation of proteins, their interaction partners, biochemical activities, posttranslational modifications, and the mutual influence of all these parameters on the physiology of the organism. Over the past several decades, biologists and biochemists have amassed a large collection of powerful tools for the study of individual proteins. However, compared with the study of nucleic acids, the HT study of proteins is still in its infancy. The next great challenge in biology will be to adapt these tools and develop new ones that enable the simultaneous and parallel study of thousands of proteins.The elucidation of biochemical activity and protein-protein interactions are central aspects of understanding protein function. Protein microarrays provide one platform for biochemical experiments to be carried out at extraordinary pace (1-3). However, this exciting technology calls attention to the quest...

The Escherichia coli genome carries seven rRNA (rrn) operons, each containing three rRNA genes. The presence of multiple operons has been an obstacle to many studies of rRNA because the effect of mutations in one operon is diluted by the six remaining wild-type copies. To create a tool useful for manipulating rRNA, we sequentially inactivated from one to all seven of these operons with deletions spanning the 16S and 23S rRNA genes. In the final strain, carrying no intact rRNA operon on the chromosome, rRNA molecules were expressed from a multicopy plasmid containing a single rRNA operon (prrn). Characterization of these rrndeletion strains revealed that deletion of two operons was required to observe a reduction in the growth rate and rRNA/protein ratio. When the number of deletions was extended from three to six, the decrease in the growth rate was slightly more than the decrease in the rRNA/protein ratio, suggesting that ribosome efficiency was reduced. This reduction was most pronounced in the Δ7 prrn strain, in which the growth rate, unlike the rRNA/protein ratio, was not completely restored to wild-type levels by a cloned rRNA operon. The decreases in growth rate and rRNA/protein ratio were surprisingly moderate in the rrndeletion strains; the presence of even a single operon on the chromosome was able to produce as much as 56% of wild-type levels of rRNA. We discuss possible applications of these strains in rRNA studies.

Cholera, an infectious disease with global impact, is caused by pathogenic strains of the bacterium Vibrio cholerae. High-throughput functional proteomics technologies now offer the opportunity to investigate all aspects of the proteome, which has led to an increased demand for comprehensive protein expression clone resources. Genome-scale reagents for cholera would encourage comprehensive analyses of immune responses and systems-wide functional studies that could lead to improved vaccine and therapeutic strategies. Here, we report the production of the FLEXGene clone set for V. cholerae O1 biovar eltor str. N16961: a completegenome collection of ORF clones. This collection includes 3,761 sequence-verified clones from 3,887 targeted ORFs (97%). The ORFs were captured in a recombinational cloning vector to facilitate high-throughput transfer of ORF inserts into suitable expression vectors. To demonstrate its application, Ϸ15% of the collection was transferred into the relevant expression vector and used to produce a protein microarray by transcribing, translating, and capturing the proteins in situ on the array surface with 92% success. In a second application, a method to screen for protein triggers of Toll-like receptors (TLRs) was developed. We tested in vitro-synthesized proteins for their ability to stimulate TLR5 in A549 cells. This approach appropriately identified FlaC, and previously uncharacterized TLR5 agonist activities. These data suggest that the genome-scale, fully sequenced ORF collection reported here will be useful for high-throughput functional proteomic assays, immune response studies, structure biology, and other applications.cholera ͉ functional proteomics ͉ immunity ͉ ORF clones ͉ protein microarray

Kinases catalyze the phosphorylation of proteins, lipids, sugars, nucleosides, and other important cellular metabolites and play key regulatory roles in all aspects of eukaryotic cell physiology. Here, we describe the mining of public databases to collect the sequence information of all identified human kinase genes and the cloning of the corresponding ORFs. We identified 663 genes, 511 encoding protein kinases, and 152 encoding nonprotein kinases. We describe the successful cloning and sequence verification of 270 of these genes. Subcloning of this gene set in mammalian expression vectors and their use in high-throughput cell-based screens allowed the validation of the clones at the level of expression and the identification of previously uncharacterized modulators of the survivin promoter. Moreover, expressions of the kinase genes in bacteria, followed by autophosphorylation assays, identified 21 protein kinases that showed autocatalytic activity. The work described here will facilitate the functional assaying of this important gene family in phenotypic screens and their use in biochemical and structural studies.kinome ͉ autophosphorylation ͉ cell-based screens ͉ high-throughput cloning ͉ survivin T he term kinase refers to a large number of mechanistically, structurally, and evolutionary distinct classes of enzymes. They catalyze the transfer of the ␥-phosphate from nucleoside triphosphates to a large number of molecules, including proteins, sugars, nucleosides, and lipids, and affect the activity and fate of those molecules and the cell.Phosphorylation is a common posttranslational modification of proteins and plays a key role on protein structure and function and in all aspects of cell physiology. Protein kinases contain well conserved motifs and constitute the largest family of proteins in the human genome (1-3). Mutations of protein kinases are involved in carcinogenesis and several other pathological conditions (4-6). Phosphorylations of other biomolecules also play a critical role in the physiology and pathology of cells. Lipid kinases such as the phosphoinositide-3 kinase family members are key modulators of the cellular response to growth factors, hormones, and neurotransmitters and are involved in cancer (7). Nucleotide and nucleoside kinases regulate the intracellular levels of phosphate donors and nucleic acid precursors and are involved in the cellular response to damage and ischemia (8, 9). Sugar kinases regulate the rates of sugar metabolism, energy generation, and transcription activation and are involved in the process of cellular transformation and apoptosis (10-12).The near completion of the Human Genome Project, the ongoing annotation projects, and the availability of sequence databases has allowed the genome-scale search and identification of members of different gene families by using sequence information as well as structural or functional annotations (2, 3, 13-15). However, a systematic cloning, sequence analysis, and functional validation effort for any of these gene sets has been chal...

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