ATCC11924 as a tester and S.lavendulae JCM4055 as a driver was carried out using a PCR-select bacterial genome subtrac-tionkit(Clontech)accordingtothemanufacturer’sinstruction manual, except that the hybridization temperature was set to 75 °C. The secondary PCR products, which were enriched for the S.lavendulae ATCC11924-specific DNA. The morphology of citric acid production strains of Aspergillus niger is sensitive to a variety of factors, including the concentration of manganese (Mn2+). Upon increasing the Mn2+ concentration in A. Niger (ATCC 11414) cultures to 14 ppb or higher, the morphology switches from pelleted to filamentous, accompanied by a rapid decline in citric acid production. The molecular mechanisms through.

Amersham Biosciences Parts. Showing all 5 results. Amersham GE IV-908 AKTA Valve $ 1,600.00 Add to cart; Amersham Pharmacia, AKTA, HPLC, FPLC, M-925 mixer. Vickie has 30 years’ experience in drug development, including research, discovery and non-clinical support for 2 NDAs. She was a co-founder of Rx3 Pharmaceuticals in 2004, which became Trius Therapeutics in 2007. Vickie is leading research development efforts for Zim Biosciences.

Many biologists see far-reaching potential for proteomics. This field emerges from several decades of discoveries from protein chemists exploring polypeptides, and today's technology supplies scientists with a growing array of tools for attacking even more complicated questions about proteins. Edward T. Maggio, chairman and chief executive officer at Structural Bioinformatics, said, 'Proteins are fundamental to most biological processes, so they are a focus in life science and drug discovery.'

Proteomics describes the study of all the proteins in a cell or tissue. This science presents some interesting challenges, even when compared to genomics. Proteins are more complex than DNA in several ways. DNA consists of just four different bases, but 20 different amino acids make up proteins. Moreover, the linear sequence of DNA bases determines the information in genes, but a protein's activity relies on its amino acid sequence plus its three-dimensional conformation.

The collective activities of proteins in a cell account for how a cell behaves in its environment, because proteins perform several jobs. Some proteins make cells motile. Others function as receptors for signals from outside a cell. Proteins also send signals from one location in a cell to another. In addition, other chemical moieties, including sugars, can attach to proteins and change their properties.

Just as proteins can change quickly, so does the field of proteomics. Rudolf Aebersold, cofounder of the Institute for Systems Biology, sees two ongoing changes in proteomics. First, he said this field started by trying to simply identify all of the proteins—enough of a challenge in itself—but now scientists hope to determine the differential expression of proteins under varying circumstances, interactions between proteins, and the three-dimensional structure for all proteins. In addition, Aebersold said that two-dimensional (2-D) gel electrophoresis dominated proteomics research at first—and remains an integral technology to this field—but today's experiments also employ chip based measurements and high throughput mass spectrometry (MS).

AN OLD FRIEND

In the mid 1970s, protein chemists started separating polypeptides with 2-D gel electrophoresis. These systems consist of two stages. The first dimension uses isoelectric focusing, and the second dimension utilizes a denaturing polyacrylamide gel matrix.

The first gel, or dimension, traditionally incorporated ampholytes into the gel. These free ampholytes migrate in the gel to form a pH gradient, which helps separate the proteins based on their isoelectric points. Preparing gels that produce consistent results is not easy, but immobilized pH-gradient strips from suppliers solve the drifting pH gradient that resulted from ampholytes.

After the first dimension, the separated proteins go to a vertical gel electrophoresis unit. This stage includes a uniform molecular sieve that can separate proteins based on their molecular weight. These gels denature the proteins. That is, the gel unwinds proteins—turning their three-dimensional conformation into more or less linear molecules, which can then migrate through the matrix. Smaller molecules move faster than the larger molecules through the gel, which further isolates different polypeptides.

A number of companies—including Amersham Biosciences, Bio-Rad Laboratories, and Invitrogen Corporation—offer complete systems to perform protein separations. Pieter Noordeloos, vice president of marketing for proteomics at Amersham Biosciences, said, 'We have been involved with 2-D electrophoresis for more than 25 years. Our innovations make the results stable and reproducible.' Amersham Bioscience offers essentially everything needed to apply 2-D electrophoresis to proteomics, including precast gels, instruments, reagents, and more.

According to Noordeloos, Amersham Bioscience's product line includes the Ettan DIGE which 'provides quantification and increased speed by allowing a researcher to compare normal samples with diseased or treated ones at the same time, in the same gel.' Although few would call 2-D electrophoresis simple or completely straightforward, Joakim Rodin, vice president of product development at Amersham Biosciences said, 'We use innovative chemistry and smart software together with experimental design in our Ettan DIGE system to do true 2-D differential protein expression analysis, helping the customer generate more accurate data—actually making it possible to analyze subtle changes in the biology and not the experimental variation'

OTHER FAVORITES

Other favorite tools used in protein studies include antibodies, liquid chromatography, mass spectrometry, and X-ray crystallography. Over time, manufacturers continually improved the performance and capabilities of these products, with special attention to how they can be used to study proteins. Antibodies, for example, provide ideal tools for identifying and isolating proteins. BD Biosciences, Chemicon, R&D Systems, and others provide antibodies and related products for a broad range of applications.

QED Bioscience provides customized antibodies—from DNA sequences alone—including chicken antibodies. These antibodies are increasingly being used in large-scale proteomics studies because they are relatively inexpensive, nonmammalian in origin, which makes them more immunoreactive and specific, and can be generated quickly.

Antibodies generated to a specific protein can be used to find it in a cellular extract. Traditionally, chromatography media in columns secured antibodies to a solid support to capture a protein of interest. Then, antigen-antibody complexes pulled out the protein. These products are offered by Amersham Biosciences, Pierce Biotechnology, Tosoh Biosep, and others.

Some old favorites in protein studies can now be run and analyzed using multiplexed assays. Based on Luminex Corporation's xMAP technology, MiraiBio's (formerly a division of Hitachi Software Engineering America) Luminex 100 and Qiagen's LiquiChip system run multiple tests simultaneously. Steve Lee, director of research and development at Hitachi, explained that this product includes several basic parts. It starts with an assay, which can be an immunoassay, such as ELISAs, receptor-ligand analyses, or nucleic acid assays, including single nucleotide polymorphisms. This system uses polystyrene beads—5.6 micrometers across—and two internal dyes in varying ratios that create up to 100 unique colors that can be simultaneously associated with 100 different assays. The beads flow single file past two lasers, where the first identifies the bead and the second excites a reporter dye. A digital signal processor captures the data, and MiraiBio's MasterPlex QT analyzes the data. This system has already been used on studies of cytokines and viral antibodies, including HIV.

MiraiBio's FMBIO III Fluorescence Imaging System, with both phosphor and fluorescence capabilities, provides multitasking. For 2-D gels, for example, this system can scan and analyze two different lysates labeled with two different dyes that go through electrophoresis simultaneously. Lee said, 'This way you can quantify your results without any gel-to-gel variability.' Making this work depends in part on this machine's multiple excitation capabilities with 488, 532, and 635 nanometer lasers resulting in multicolor fluorescence detection and high sensitivity. The very large scanning area, which is 55 by 40 centimeters, facilitates simultaneous scanning of multiple gels, microtitre plates, or up to 84 complete microarrays. The software program Image Analysis 3.0 is packaged with the scanning system.

PUTTING PRESSURE ON PROTEINS

High performance liquid chromatography, or HPLC, separates molecules under high pressure in a stainless steel column filled with a solid matrix. HPLC pulls out molecules of interest by using the attractive forces between molecules carrying charged groups of opposite signs. Agilent Technologies, Alltech Associates, and Phenomenex specialize in HPLC.

Emmet Welch, manager of product development and commercialization at Phenomenex, said, 'The most exciting thing in today's world of HPLC for proteomics is multidimensional chromatography.' He and his colleagues are working to improve this technology by increasing the so-called peak capacity, essentially increasing the number of data peaks resolved per unit time. To do that, this group of scientists looks at one dimension of HPLC at a time. They started with the reverse phase dimension, and the Jupiter Proteo HPLC Column increased peak capacity by 20 percent to 60 percent over previous technology. Welch said, 'we're on the right track and must focus on each dimension.' Now this company's scientists are working on improving the ion exchange dimension as well.

A WEIGHTY OPTION

Mass spectrometry can identify individual proteins. For example, scientists analyze 2-D gel profiles with mass spectrometry, such as the matrix-assisted laser desorption ionization time-of-flight (MALDI-ToF) technique. Proteins get digested, and mass spectrometry can analyze the resulting peptides. The data gathered from the digestion of a single protein spot can then be compared to properties of known proteins and an exact match, or identification, can be made. Amersham Biosciences, Applied Biosystems, Micromass, PerkinElmer Life Sciences, Thermo Finnigan, and others manufacture these instruments.

As the workload increases, single MS instruments such as Amersham Biosciences' Ettan MALDI-ToF Pro could help investigators screen proteins of interest. Noordeloos said, 'In the first screening, a majority of proteins can be identified, and thus free up time on your high-end MS-MS systems for more difficult samples. In other words, we are seeing a shift from studying proteins with pure high-end sequencing applications to single mass spectrometry screening methods.'

'To get more scientists to use mass spectrometry, though,' Rodin said, 'we have to bring it down to earth.' That is, from a scientist's perspective, mass spectrometry must be easier to use and the data must be easier to interpret. Rodin and his colleagues hope to improve mass spectrometry along those very lines. He said, 'We are pursuing ease of use, user friendliness, and getting answers instead of just spikes.' In collaboration with Thomas Keough and R. Scott Youngquist at Procter & Gamble, Amersham Biosciences developed the Ettan CAF-MALDI Sequencing Kit—the first of its kind to use chemically assisted fragmentation (CAF) chemistry—and it significantly increases the rate of protein identification.

One of the widest lines of mass spectroscopy equipment for proteomics and drug discovery comes from Applied Biosystems. Steve Martin, director of the Proteomics Research Center, said, 'Tandem mass spectroscopy, or MS-MS, is the real driver in proteomics.' For example, scientists can identify and further characterize proteins with the API QSTAR, a hybrid quadrupole time-of-flight instrument. This product can also be used in drug metabolism studies.

Scientists can use Applied Biosystems' ICAT Reagent technology—developed by scientists at the Institute for Systems Biology—to compare levels of protein expression in complex samples, including human serum or diseased versus normal tissue homogenates. The technology works in concert with MS-MS based systems to analyze these complex mixtures and automatically quantify and identify the hundreds of proteins present. Martin said, 'Scientists can use QSTAR with ProICAT software to automatically identify and quantify the proteins of interest.'

For low-abundant proteins, Martin said, 'A common approach is using multidimensional liquid chromatography for separation and then high resolution MS-MS systems to identify the proteins.' Applied Biosystems' 4700 Proteomics Analyzer can analyze a complex protein sample that has been separated into many fractions and then rapidly do MS and MS-MS on the various fractions to identify and characterize the large number of peptides and proteins present. Martin said, 'Low-abundant proteins are hidden by very abundant ones, so separation lets you pull apart different proteins.' Getting enough sample fractionation depends on sample preparation. Applied Biosystems' VISION Workstation provides a wide variety of separation techniques, including ion exchange, reverse phase, affinity, and more.

In addition to the MALDI based 4700 system, Applied Biosystems also introduced the QTRAP, an electrospray-ionization system for protein and metabolite identification. It is the first commercial quadrupole linear ion trap-based system and expands the choices for selecting an appropriate mass spectrometry system. The QTRAP's higher performance capabilities allow for greater numbers of protein identifications from complex samples than traditional ion trap technology.

Scientists at Eppendorf also see the value in improved sample preparation. Joern Kirchhuebel, product manager of Eppendorf's molecular technology group, said, 'There is a great demand for enhanced sample preparation for electrophoresis and chromatographic methods.' Consequently, Eppendorf is developing products that make sample handling easier and more standardized. In addition, Kirchhuebel said, 'We are trying to make products with a broad range, that can be used for 2-D electrophoresis, liquid chromatography, and mass spectrometry. We are planning one technology platform with different formats to fit the appropriate application.'

NEWER ARRIVALS

Proteins usually function in families or pathways and interact with other related proteins. Researchers often begin studying proteins by attempting to understand how a protein with unknown function relates to other known proteins. A technique called the yeast two-hybrid method helps researchers identify related proteins.

In this technique, a transcription factor binds to a gene and causes it to produce RNA and then protein. This transcription factor consists of two parts: one that binds to a gene is called the binding domain, and the one that turns on RNA production is called the activation domain. The two domains can separate and each fuses to a different protein of interest. The two hybrid proteins can then be tested to see if they interact with each other. If they are related, the two domains will bind to each other and cause RNA production. This system is now offered by several companies like BD Biosciences, Invitrogen, and Stratagene.

New approaches to purifying proteins and studying expression also push proteomics ahead. Some expression systems insert small peptide sequences into a specific protein to identify and purify it. Scientists can find these systems at BD_Biosciences, Qiagen, and Sigma-Aldrich Corporation. Kerstin Steinert, Qiagen's associate director of research and development in protein expression and proteomics, said, 'As the number of proteins being studied increases, automation becomes essential. Using automated Ni-NTA [nickel-nitrilotriacetic acid] technology, 96 different recombinant proteins can be easily purified without a need to optimize individual purification conditions.'

CASHING IN ON CHIPS

Protein arrays consist of large numbers of regularly arranged spots of discreet elements— antibodies, enzymes, substrates, and so on—that recognize a protein or protein binder of interest. This technique can be used to monitor a cell's metabolism and response to external stimuli. Moreover, any biological protein assay that uses a specific ligand-receptor interaction can be miniaturized into a protein chip.

Despite some years of experience with DNA microarrays, similar chips for proteins create new challenges. Karin Hughes, vice president of research and development at Prolinx, said, 'A lot of the chip chemistries that work for DNA do not produce good results for proteins. Proteins are more sticky and fragile, so surface chemistry is an issue.' Prolinx battles that problem by making a slide that incorporates a three-dimensional polymer to help capture the desired proteins. Hughes said, 'We designed this surface for proteins, and it works quite well. It gives a very high signal-to-noise ratio and there is little to no nonspecific protein binding.'

Beyond this advance in surface chemistry, Prolinx added other new features to its protein microarray technology. Leslie Linkkila, director of sales and marketing, said, 'Proteins vary in size, how fragile they are, and in other characteristics, so our system allows a user to select how to bind proteins to the array surface on a protein-by-protein basis. This makes the Prolinx method a diverse and robust screening technology.' Despite these advanced features, Prolinx made sure to keep this product compatible with standard microarray equipment. Customers simply buy the slide and then spot, process, and detect the results with instrumentation originally designed for use with nucleic acid arrays.

Scientists at Prolinx hope that their technology helps biologists answer broader questions than they could address in the past. Robert J. Kaiser, Prolinx's chief technology officer, said 'To this point much of biological science has been carried out in a rather isolated fashion. You'd find a particular characteristic of interest and dissect it one piece at a time, largely in vitro. Now we're trying to go from there to simultaneously investigating a huge number of interactions that must be understood in vivo to quantify a stimulus-response.' He paused before adding: 'These are very complex questions. Once such questions start to be answered, we'll have come a long way toward describing biology as biology, and not as a test-tube model.'

Chasing such questions, though, produces megatons of information, such as protein sequence data. A number of companies—including Compugen and Incyte Genomics—offer analysis software for this situation. For example, MiraiBio's MasterPlex QT, mentioned above, takes multiple files and simultaneously analyzes them. The user gets a report that includes the ability to search all of the data.

DISCOVERING NEW DRUGS

Alex Vodenlich, vice president of contract services at PanVera Corporation, said, 'Proteomics is already accelerating the process of developing therapeutic agents by identifying and validating new drug targets.' Once new proteins get identified and validated, PanVera's scientists can create them—accurately, quickly, and in quantity. Vodenlich said, 'The need for proteins is increasing dramatically, and we have a capability of taking validated gene sequences and turning them into proteins with an industrialized process.' Currently, PanVera offers more than 100 recombinant proteins with off-the-shelf availability. Vodenlich said, 'We generally focus on certain gene families that are of broad therapeutic interest, such as the protein kinases, which are involved in cell signaling and regulation. We've produced over 30 different kinases that can be used for high throughput screening for people who are looking for inhibitors of these kinases.' This company also manufactures custom proteins, and it has created more than a thousand specialized polypeptides.

Manufactured proteins from PanVera can also be used in structural proteomics. Investigators can more easily study a protein's structure if they have large quantities of it, and it must be the right protein.

Using proteomics to make better drugs often involves structure. Ming Li, president of Bioinformatics Solutions, said, 'Software helps biologists speed up their study of proteins and cuts the costs. Instead of going to wet labs, they can use dry labs to find the structure of proteins.' Before working on a protein's structure though, a scientist must know just what protein it is. Li and his colleagues provide their PEAKS software, which determines a peptide sequence by using data from mass spectroscopy. Li said, 'PEAKS consists of an automated de novo sequencing component and a semi-manual sequencing component. PEAKS significantly improves the accuracy of all existing commercial de novo sequencing software with a new algorithm.' Li adds, 'It also handles the troublesome posttranslational modifications including phosphorylation.'

To determine an identified protein's structure, Li's company owns an exclusive license for PROSPECT, a package developed at Oak Ridge National Laboratory. This program uses the known structures of proteins with similar sequences—plus a variety of mathematical techniques—to predict a new protein's structure. Li said this program can determine the structure of about 50 percent of the proteins tested. 'Researchers working in drug discovery can use this program as a prescreening step,' Li said, 'before they work on potential drug targets in the wet lab.' As databases of protein structures improve, PROSPECT will find the structures of even more proteins.

SEEKING THE RIGHT SHAPE

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Finding the best pharmaceutical often depends on how it fits its target, which is generally a protein. Maggio said, 'Protein structure has been hard to come by in the past, because it depended on X-ray crystallography or nuclear magnetic resonance.' The scientists at Structural Bioinformatics make it easier to determine a protein's shape through augmented homology modeling. In essence, this technique takes a protein with an unknown structure and uses its sequence to find a similar protein with a known structure.

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Structural Bioinformatics' proprietary algorithms use the relationship between the known sequence and structure to build an accurate internal core structure for a protein of interest. Then the software completes the structure using proprietary and patented algorithms to generate accurate surface loops, which overcomes the inaccurate surface structures that plagued earlier homology methods. After additional mathematical refinement, the resulting structures are comparable in quality to X-ray structures, but at a fraction of the time and cost. Structural Bioinformatics also created StructureBank, a system for large-scale management, data sharing, and comparative analysis of three-dimensional protein structures now in use by a growing number of drug discovery companies. According to Maggio, 'StructureBank's simple but powerful, intuitive user interface provides access to protein structure at the touch of a few keystrokes. This makes rational experimental design practical across nearly all life science disciplines, increasing speed and efficiency in many aspects of drug discovery.'

Kal Ramnarayan, Structural Bioinformatics' chief scientific officer, said, 'To make this process work, you need an understanding of the three-dimensional structure of proteins and how they react with targets.' Ramnarayan indicated that the crucial aspect of this process involves how the program represents the protein loops in the secondary structure that lead to the protein's tertiary, or three-dimensional, structure. This group's software computes the structures of short sequences of amino acids—usually six to 30 that compare well with known structures—to get the loops right. In addition, an expert protein modeling team goes over the results to be sure that loops align properly.

This technology works very well for proteins that come from families with well-known structures. For 98 percent of proteins examined in benchmark studies, Structural Bioinformatics' approach generated a structure in which amino acids lie within no more than two angstroms of where X-ray crystallography places them. For 96 percent of the proteins, this technique places the residues within 1.5 angstroms of the exact location.

In the past, this type of modeling failed in drug discovery. Past protein structure modeling provided reasonable results for a protein's core structure, but poor results for its surface, where drug interactions take place. Augmented homology, however, creates a very accurate model of the core and the surface. Consequently, scientists at Structural Bioinformatics take a potential drug target, determine its three-dimensional structure, and then look for active small molecules as potential drugs. David D. Muth, president and chief operating officer, said, 'Beyond the target's structure, you also need the structure of closely related proteins in patients, because they cause the side effects. This is yet another powerful use of StructureBank.'

So far, scientists at Structural Bioinformatics are batting a thousand. With 10 new targets, they found promising drug candidates. They are working on drugs against anthrax, asthma, cancer, diabetes, inflammation, and more.

BENEFITS FROM BREAKDOWNS

Experiments in drug discovery go beyond making new proteins. Scientists also study the destruction of proteins. Every cell contains proteases, or enzymes, that break down proteins. Proteases play a fundamental role in viral infections. In the infection process, viral RNA gets translated to polypeptides, and the assembled sequence of amino acids includes several proteins connected end-to-end. Proteases cut these proteins apart, which turns them on so that the virus can infect more cells. Protease inhibitors block the protease enzyme, and that can fight the spread of a virus. Investigators at ICN Pharmaceuticals, for example, focus on protease inhibitors.

Drugs can also attack viruses in other ways. Steven Dowdy and his colleagues at Washington University School of Medicine attacked HIV with another protein, called caspase-3. When this protein turns on, it causes a cell to commit suicide. Dowdy's team makes HIV cells take up this protein. When the virus tried to reproduce, it turned on caspase-3, which killed the cells. Dowdy pointed out that this approach could also attack other viral diseases, including hepatitis C and herpes.

Investigators at ICN Pharmaceuticals also see the potential of caspase and related proteins. Ron Mischak, director of research and development, indicated that his company provides fluorescent probes for caspases. He said, 'Our novel probe design provides direct visualization of intercellular caspase activity. I think this is an excellent model system for reagents that might target other activated enzymes in a cell.'

UPCOMING OBSTACLES AND ADVANCES

Aebersold pointed to a couple challenges in current proteomics research. First he said, 'it's difficult to find the right technology to do certain types of protein measurements on a global scale, which is of particular biological importance.' Posttranslational modifications, for instance, often change a protein's function, such as phosphorylation turning a protein on or off. Aebersold said, 'There are no good tools to measure these modifications systematically.' He also pointed out the ongoing challenge of informatics. He asked: 'What do you do with all of the data? We measure multiple types of data from one type of protein, and all of these measurements create huge amounts of data and each must be processed. Eventually, all of the data must also be integrated.'

Aebersold also wants to bring high throughput techniques for proteomics to academic laboratories. Academic scientists have little access to high throughput proteomic facilities. Aebersold hopes to change that by creating centralized facilities where academic scientists can go to use equipment as well as data gathering and analysis tools. He said, 'It is not a question of money and buying lots of mass spectrometers. It is more complex, because you need equipment and personnel and a computational infrastructure.' With such facilities academic researchers might also enjoy the high throughput capabilities, which are largely limited to today's industrial scientists.

Mike May is a freelance writer based in Madison, Indiana, U.S.A. Gary Heebner is a marketing consultant serving the scientific industry, based in Foristell, Missouri, U.S.A.

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