High-throughput purification of recombinant proteins using self-cleaving intein tags.

High throughput methods for recombinant protein production using E. coli typically involve the use of affinity tags for simple purification of the protein of interest. One drawback of these techniques is the occasional need for tag removal before study, which can be hard to predict. In this work, we demonstrate two high throughput purification methods for untagged protein targets based on simple and cost-effective self-cleaving intein tags. Two model proteins, E. coli beta-galactosidase (ßGal) and superfolder green fluorescent protein (sfGFP), were purified using self-cleaving versions of the conventional chitin-binding domain (CBD) affinity tag and the nonchromatographic elastin-like-polypeptide (ELP) precipitation tag in a 96-well filter plate format. Initial tests with shake flask cultures confirmed that the intein purification scheme could be scaled down, with >90% pure product generated in a single step using both methods. The scheme was then validated in a high throughput expression platform using 24-well plate cultures followed by purification in 96-well plates. For both tags and with both target proteins, the purified product was consistently obtained in a single-step, with low well-to-well and plate-to-plate variability. This simple method thus allows the reproducible production of highly pure untagged recombinant proteins in a convenient microtiter plate format.

The genome of the natural genetic engineer Agrobacterium tumefaciens C58.

The 5.67-megabase genome of the plant pathogen Agrobacterium tumefaciens C58 consists of a circular chromosome, a linear chromosome, and two plasmids. Extensive orthology and nucleotide colinearity between the genomes of A. tumefaciens and the plant symbiont Sinorhizobium meliloti suggest a recent evolutionary divergence. Their similarities include metabolic, transport, and regulatory systems that promote survival in the highly competitive rhizosphere; differences are apparent in their genome structure and virulence gene complement. Availability of the A. tumefaciens sequence will facilitate investigations into the molecular basis of pathogenesis and the evolutionary divergence of pathogenic and symbiotic lifestyles.

Conquering the proteome: complexity, cooperation and commerce.

The Protein Expression meeting was held in McLean, Virginia, USA, 5-6 April 2001.

Optimized single-step affinity purification with a self-cleaving intein applied to human acidic fibroblast growth factor.

To reduce the number of recovery steps during downstream processing and to overcome the limitations of present fusion-based affinity separations, a controllable self-splicing protein element in the form of a mini-intein was used to optimize the recovery of proteins for both batch and flow purification strategies. The ability to recover purified proteins was demonstrated using a tripartite fusion consisting of a maltose binding domain, a truncated intein as a controllable linker molecule, and a protein of interest. To characterize expression level, solubility, cleavage rates, pH and temperature controllability, and protein activity, recombinant human acidic fibroblast growth factor (aFGF) was used as a model protein. A simple mass transport model, based on cleavage reaction-limited mass transfer and constant dispersion, was successfully used to predict product concentration and peak shape in relation to critical process parameters (with no fitting parameters). Insight into the nature of the cleavage reaction and its regulation was obtained via temperature- and pH-dependent kinetic data.

A genetic system yields self-cleaving inteins for bioseparations.

A self-cleaving element for use in bioseparations has been derived from a naturally occurring, 43 kDa protein splicing element (intein) through a combination of protein engineering and random mutagenesis. A mini-intein (18 kDa) previously engineered for reduced size had compromised activity and was therefore subjected to random mutagenesis and genetic selection. In one selection a mini-intein was isolated with restored splicing activity, while in another, a mutant was isolated with enhanced, pH-sensitive C-terminal cleavage activity. The enhanced-cleavage mutant has utility in affinity fusion-based protein purification. These mutants also provide new insights into the structural and functional roles of some conserved residues in protein splicing.

Two-component transcriptional regulation of N-acyl-homoserine lactone production in Pseudomonas aureofaciens.

Production of phenazine antibiotics by the biological control bacterium Pseudomonas aureofaciens 30-84 is regulated in part by the PhzI/PhzR N-acyl-homoserine lactone (AHL) response system (L. S. Pierson III, V. D. Keppenne, and D. W. Wood, J. Bacteriol. 176:3966-3974, 1994; D. W. Wood and L. S. Pierson III, Gene 168:49-53, 1996). Two mutants, 30-84W and 30-84.A2, were isolated and were found to be deficient in the production of phenazine, protease, hydrogen cyanide (HCN), and the AHL signal N-hexanoyl-homoserine lactone. These mutants were not complemented by phzI, phzR, or the phenazine biosynthetic genes (phzFABCD) (L. S. Pierson III, T. Gaffney, S. Lam, and F. Gong, FEMS Microbiol. Lett. 134:299-307, 1995). A 2.2-kb region of the 30-84 chromosome which fully restored production of all of these compounds in strain 30-84W was identified. Nucleotide sequence analysis of this region revealed a single open reading frame encoding a predicted 213-amino-acid protein which is very similar to the global response regulator GacA. Strain 30-84.A2 was not complemented by gacA or any cosmid from a genomic library of strain 30-84 but was complemented by gacS (formerly lemA) homologs from Pseudomonas fluorescens Pf-5 (N. Corbel and J. E. Loper, J. Bacteriol. 177:6230-6236, 1995) and Pseudomonas syringae pv. syringae B728a (E. M. Hrabek and D. K. Willis, J. Bacteriol. 174:3011-3020, 1992). Transcription of phzR was not altered in either mutant; however, phzI transcription was eliminated in strains 30-84W and 30-84.A2. These results indicated that the GacS/GacA two-component signal transduction system of P. aureofaciens 30-84 controls the production of AHL required for phenazine production by mediating the transcription of phzI. Addition of exogenous AHL did not complement either mutant for phenazine production, indicating that the GacS/GacA global regulatory system controls phenazine production at multiple levels. Our results reveal for the first time a mechanism by which a two-component regulatory system and an AHL-mediated regulatory system interact.

Deep pockets or blueprint for change: traumatic brain injury (TBI) proactive strategy.

The Pacific Conference scheduled for October 1-3, 1988, is a critical event in the development of an integrated community-based plan for a comprehensive continuum of services to address the "silent epidemic," Traumatic Brain Injured (TBI). This paper provides insights of the complex nature and the special problems faced by the TBI survivors; their families, natural supports and caregivers, as well as the health, social and educational care providers in Hawaii. Process for the development of the community plan is presented.

Agrobacterium VirD2 protein interacts with plant host cyclophilins.

Agrobacterium tumefaciens induces crown gall tumors on plants by transferring a nucleoprotein complex, the T-complex, from the bacterium to the plant cell. The T-complex consists of T-DNA, a single-stranded DNA segment of the tumor-inducing plasmid, VirD2, an endonuclease covalently bound to the 5' end of the T-DNA, and perhaps VirE2, a single-stranded DNA binding protein. The yeast two-hybrid system was used to screen for proteins interacting with VirD2 and VirE2 to identify components in Arabidopsis thaliana that interact with the T-complex. Three VirD2- and two VirE2-interacting proteins were identified. Here we characterize the interactions of VirD2 with two isoforms of Arabidopsis cyclophilins identified by using this analysis. The VirD2 domain interacting with the cyclophilins is distinct from the endonuclease, omega, and the nuclear localization signal domains. The VirD2-cyclophilin interaction is disrupted in vitro by cyclosporin A, which also inhibits Agrobacterium-mediated transformation of Arabidopsis and tobacco. These data strongly suggest that host cyclophilins play a role in T-DNA transfer.

Helicobacter pylori: a review.

Helicobacter pylori is a gram-negative bacterium that was first isolated in 1982. In the years following its discovery, H. pylori infection in humans has been shown to be associated with gastritis, peptic ulcer disease, and gastric carcinoma, as well as other, nongastrointestinal disorders. The epidemiology, transmission, and virulence factors of this bacteria have been an area of intense study. Successful treatment improves cure rates of gastritis and ulceration of the stomach and duodenum. Treatment with antimicrobials also decreases the recurrence rates of these diseases. Clinicians have numerous diagnostic tools and treatment options at their disposal. Vaccination in high-endemic areas may be available in the near future. Here, we review the pharmalogical basis of these treatment options, including their efficacy and economic considerations.

Homoserine lactone-mediated gene regulation in plant-associated bacteria.

Many plant-associated bacteria produce and utilize diffusible N-acyl-homoserine lactones (AHLs) to regulate the expression of specific bacterial genes and operons. AHL-mediated regulation utilizes two genes that encode proteins similar to the LuxI/LuxR system originally studied in the marine symbiont Vibrio fischeri. The LuxI-type proteins are AHL synthases that assemble the diffusible AHL signal. The LuxR-type proteins are AHL-responsive transcriptional regulatory proteins. LuxR proteins control the transcription of specific bacterial genes in response to the levels of AHL signal. To date, AHL-mediated gene regulation has been identified in a broad range of gram-negative bacteria, most of which are host-associated. However, it seems unlikely that such a widely conserved regulatory mechanism would be limited only to host-microbe interactions. These signals probably play central roles in ecological interactions among organisms in microbial communities by affecting communication among bacterial populations as well as between bacterial populations and their eukaryotic hosts.

N-acyl-homoserine lactone-mediated regulation of phenazine gene expression by Pseudomonas aureofaciens 30-84 in the wheat rhizosphere.

Pseudomonas aureofaciens 30-84 is a soilborne bacterium that colonizes the wheat rhizosphere. This strain produces three phenazine antibiotics which suppress take-all disease of wheat by inhibition of the causative agent Gaeumannomyces graminis var. tritici. Phenazines also enhance survival of 30-84 within the wheat rhizosphere in competition with other organisms. Expression of the phenazine biosynthetic operon is controlled by the phzR/phzI N-acyl-homoserine lactone (AHL) response system (L. S. Pierson III et al., J. Bacterial 176:3966-3974, 1994; D. W. Wood and L. S. Pierson III, Gene 168:49-53, 1996). By using high-pressure liquid chromatography coupled with high-resolution mass spectrometry, the AHL produced by PhzI has now been identified as N-hexanoyl-homoserine lactone (HHL). In addition, the ability of HHL to serve as an interpopulation signal molecule in the wheat rhizosphere has been examined by using isogenic reporter strains. Disruption of phzI reduced expression of the phenazine biosynthetic operon 1,000-fold in the wheat rhizosphere. Coinoculation of an isogenic strain which produced the endogenous HHL signal restored phenazine gene expression in the phzI mutant to wild-type levels in situ. These results demonstrate that HHL is required for phenazine expression in situ and is an effective interpopulation signal molecule in the wheat rhizosphere.

Genetic definition of a protein-splicing domain: functional mini-inteins support structure predictions and a model for intein evolution.

Inteins are protein-splicing elements, most of which contain conserved sequence blocks that define a family of homing endonucleases. Like group I introns that encode such endonucleases, inteins are mobile genetic elements. Recent crystallography and computer modeling studies suggest that inteins consist of two structural domains that correspond to the endonuclease and the protein-splicing elements. To determine whether the bipartite structure of inteins is mirrored by the functional independence of the protein-splicing domain, the entire endonuclease component was deleted from the Mycobacterium tuberculosis recA intein. Guided by computer modeling studies, and taking advantage of genetic systems designed to monitor intein function, the 440-aa Mtu recA intein was reduced to a functional mini-intein of 137 aa. The accuracy of splicing of several mini-inteins was verified. This work not only substantiates structure predictions for intein function but also supports the hypothesis that, like group I introns, mobile inteins arose by an endonuclease gene invading a sequence encoding a small, functional splicing element.

The phzI gene of Pseudomonas aureofaciens 30-84 is responsible for the production of a diffusible signal required for phenazine antibiotic production.

The production of phenazine (Ph) antibiotics in Pseudomonas aureofaciens (Pau) 30-84 is positively regulated by PhzR, a protein belonging to the LuxR family of transcriptional activators. We have now identified phzI, a second gene required for PH production. The product of phzI is a member of the LuxI family of N-acyl-homoserine lactone (N-acyl-HSL) synthases. Inactivation of phzI results in the loss of Ph production in Pau 30-84. The presence of phzI in Escherichia coli is sufficient for the production of a diffusible signal which activates phzB expression in Pau 30-84 and traA expression in a N-acyl-HSL-dependent reporter strain of Agrobacterium tumefaciens. In addition, synthetic N-(3-oxohexanoyl)-L-HSL induces phzB expression in Pau 30-84. These results suggest that Pau 30-84 produces a N-acyl-HSL signal that regulates Ph production, and that phzI plays a central role in this signaling pathway.

Phenazine antibiotic biosynthesis in Pseudomonas aureofaciens 30-84 is regulated by PhzR in response to cell density.

We have identified a gene that acts in trans to activate the expression of the phenazine biosynthetic genes in the biological control organism Pseudomonas aureofaciens 30-84. This gene, phzR (phenazine regulator), is located upstream of and divergently transcribed from the phenazine biosynthetic genes. Thus, the phenazine biosynthetic locus consists of at least two divergently transcribed operons. A functional phzR gene is required for phenazine production. The nucleotide sequence of phzR revealed an open reading frame of 723 nucleotides encoding a protein of ca. 27 kDa. The predicted amino acid sequence of PhzR has homology with other bacterial positive transcriptional activators, including LasR of Pseudomonas aeruginosa, LuxR of Vibrio fischerii, and TraR of Agrobacterium tumefaciens. The addition of cell-free supernatants from late-exponential-phase cultures of strain 30-84 resulted in expression of a genomic phzB:lacZ reporter strain at a lower cell density than normal, indicating the possible presence of an autoinducer. These results indicate that PhzR is a member of a two-component sensor-regulator family with known or predicted carboxy-terminal DNA-binding domains which regulates gene expression in response to environmental and cell density signals.

Trends in prevalences of behavioral risk factors: recent Hawaiian experience.

Recent time trends were studied for the prevalences of behavioral risk factors in Hawaii during the 5-year period from 1986 through 1990. The presence of linear time trend was analyzed by the multiple logistic regression method on weighted data, adjusting for confounding factors. The risk factors studied were seatbelt nonuse, lack of exercise, obesity, hypertension, smoking, acute drinking, chronic drinking, and driving while intoxicated. Seatbelt nonuse showed a significant decline, from 8.6% to 4.8%, with a mean annual decrease of 0.9 percentage point. Lack of exercise and obesity increased steadily, from 48.0% to 62.4% and from 16.7% to 21.6%, respectively, with respective annual mean increases of 3.3 and 1.4 percentage points.

Asia-Pacific prevention research. CHallenges, opportunities, and implementation.

An effort is underway in the Asia-Pacific region to carry out multidisciplinary prevention research, with an emphasis on understanding health-related behaviors. In partnerships with the U.S. Centers for Disease Control, the U.S. Public Health Service, international health agencies, governments, and universities in the region, researchers at the University of Hawaii are pursuing a paradigm for international, multicultural prevention research in the field of health promotion and disease prevention. An integrated framework for guiding a program of research is discussed along with several factors that present challenges to the conduct of prevention research in the Asia-Pacific region.

Psychology doctoral training in work and health.

Psychology as a profession has an opportunity and obligation to advocate for and develop healthy work environments. This will require the design and conduct of doctoral-level training in occupational health psychology. A model for training might well be based on the assumptions that there is a viable role for occupational health psychologists trained at the doctoral level for both academic and applied work settings, and that doctoral training would be based on the integration of health psychology and public health. Issues remaining to be addressed in the development of doctoral training programs include appropriate predoctoral training, academic standards, the interdisciplinary nature of faculties, and appropriate settings for training. Future directions in establishing doctoral training in occupational health psychology will best be taken in dialogue with several other professions and institutions that share a common interest in reducing leading work-related diseases and injuries and promoting public health in the workplace.

Gram-negative identification card for identification of Salmonella, Escherichia coli, and other Enterobacteriaceae isolated from foods: collaborative study.

Twelve laboratories evaluated the Gram-Negative Identification (GNI) Card to identify members of the Enterobacteriaceae. Eighty-four isolates, previously isolated from foods, were used in the collaborative study; the isolates represented 12 genera within the Enterobacteriaceae group. Each collaborator streaked each isolate on tryptic soy agar plates for purity. In the method, plates are incubated 18-24 h at 35 degrees C. Isolated colonies are then subcultured to tryptic soy agar slants and incubated 18-24 h at 35 degrees C. An emulsion is made from the growth on the slant in 1.8 mL 0.45% sodium chloride solution. The GNI Card is filled and placed in a reader/incubator. Isolates are identified and an identification is printed. The Vitek System correctly identified 96.7% of Salmonella sp., 97.0% of Escherichia coli, and an average of 93.8% of the other enteric genera. The method using the Vitek System and GNI Card has been approved interim official first action by AOAC as a screening method for the presumptive identification of Salmonella sp., E. coli, and other Enterobacteriaceae isolated from foods.

Environmental education: a fast-growing field in Latin America.

PIP: Private conservation organizations have become more prevalent throughout Latin America in the last decade. They range from well organized and financed, internationally-recognized organizations, to small volunteer groups. They are committed to preserving the natural resources and cover national park management, the protection of wildlife, reforestation, environmental contamination and other issues. Also in Latin America environmental education has become an important part of the resource conservation programs. One of the most advanced and successful programs is in Ecuador and is called Natura. It has produced many educational materials including slide programs, television spots and programs, booklets, a complete primary school curriculum, posters, radio programs and a profile of Ecuador's environment. Their programs have been successful because they analyze their target audience and tailor the program to their needs and desires. They also follow up with evaluation questionnaires and testing for each program. It has been difficult for organizations such as these to implement programs in rural areas. There are some groups developing environmental education programs in rural areas through alliances with development assistance organizations. There are essential for the protection of wild areas where human needs must be balanced with longterm ecological priorities.^ieng