Purifying HIS-Tagged Proteins from Insect and Mammalian Cells

Posted on by Gary Kobs

MSextractcroppedMany different polypeptide fusion partners or affinity tags have been developed to facilitate purification of target proteins. The most commonly used tag for the purification and detection of recombinant expressed proteins is the His tag. Cloning vectors designed to generate His-tagged proteins contain 5–10 histidine residues at either the C- or N terminus of the expressed protein. The His tag adds only 0.84kDa to the mass of the protein and is nonimmunogenic. Also, because the tertiary structure of the tag is not important for purification, His-tagged proteins can be purified using native or denaturing conditions. The affinity of histidine residues for immobilized nickel allows selective purification of His-tagged proteins. The MagneHis™ Ni-Particles can bind up to 1mg of His-tagged protein per milliliter of particles providing a fast, efficient method for purifying His-tagged proteins with high yield and low background in a highly scalable format.

Bacterial expression of recombinant His-tagged proteins is a common technique. However, use of other systems, such as Sf9 insect cells,or HeLa or CHO mammalian cells for expression of recombinant proteins either intracellularly or secreted into the culture medium is increasing. These eukaryotic expression systems may allow more natural processing and modification of recombinant His-tagged proteins.
The following article:  illustrates the use of FastBreak™ Cell Lysis Reagent and the MagneHis™ Protein Purification System with insect and mammalian cell lysates. Proteins are purified from culture medium in the presence or absence of serum with only minior modifications to the standard protocol for bacterial cultures are required for purification from these diverse sources.
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How a Magazine Ad Helped Convict a Rapist

Posted on by Terri Sundquist
Trial

In May of 1986, a woman in Orange County, Florida, was surprised by a man who entered her apartment and raped her at knifepoint. Despite the fact that she got a glimpse of his face, the chances of identifying and convicting her rapist were slim. Although law enforcement officers did their best to identify the perpetrator, their investigative techniques in the case were limited compared to our current set of forensic tools. That changed when Jeffrey Ashton, an assistant attorney for the state of Florida, saw an advertisement for DNA-based paternity testing in a magazine and began to wonder if DNA testing could also be used to identify the man responsible for the attack.

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Monitoring Mass Spec Instrument Performance and Sample Preparation

Posted on by Michele Arduengo, PhD

Proteomics, the analysis of the entire protein content of a living system, has become a vital part of life science research, and mass spectrometry (MS) is the method for analyzing proteins.  MS analysis of protein content allows researchers to identify proteins, sequence them and determine the nature of post translational modifications.

LC/MS performance monitoring. Each run used 1μg of human predigested protein extract injected into the instrument (Waters NanoAquity HPLC System interfaced to a Thermo Fisher Q Exactive™ Hybrid Quadrupole-Orbitrap Mass Spectrometer). Peptides were resolved with a 2-hour gradient. Weekly monitoring with the human extract ensured consistent analytical performance of the instrument.
LC/MS performance monitoring. Each run used 1μg of human predigested protein extract injected into the instrument (Waters NanoAquity HPLC System interfaced to a Thermo Fisher Q Exactive™ Hybrid Quadrupole-Orbitrap Mass Spectrometer). Peptides were resolved with a 2-hour gradient. Weekly monitoring with the human extract ensured consistent analytical performance of the instrument.

Mass spectrometry allows characterization of molecules by converting them to ions so that they can be manipulated in electrical and magnetic fields. Basically a small sample (analyte) is ionized, usually to cations by loss of an electron. After ionization, the charged particles (ions) are separated by mass and charge;  the separated particles are measured and data displayed as a mass spectrum. The mass spectrum is typically presented as a bar graph where each peak represents a single charged particle having a specific mass-to-charge (m/z) ratio. The height of the peak represents the relative abundance of the particle. The number and relative abundance of the ions reveal how different parts of the molecule relate to each other.

For the study of large, organic macromolecules, matrix associated laser desorption/ionization (MALDI) or tandem mass spec/collision induced dissociation (MS/MS) techniques are often used to generate the charged particles from the analyte. MS analysis brings sensitivity and specificity to proteome analysis. The technique has excellent resolution and is able to distinguish one ion from another, even when their m/z ratios are similar. Macromolecules are present in extremely different concentrations in the cells, and MS analysis can detect biomolecules across five logs of concentration.

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Hope for Treatment of Carbapenem-Resistant Bacteria

Posted on by Isobel Maciver

Structure of the antibiotic meropenem
Structure of the antibiotic meropenem
Last month brought some hopeful news on the subject of antibiotic resistance. A paper published in Nature on June 26 described the isolation of a fungal compound that restored the antibiotic sensitivity of carbapenem-resistant enterobacteria. An editorial accompanying the paper took encouragement from the article–considering it a sign that the well of potential sources of new antimicrobial agents, and agents that inhibit resistance mechanisms, is not yet dry:

But the reservoir of natural products with the potential to act as antibacterial drugs has not yet been exhausted. In contrast to general thinking by drug companies, screening for such products may well still have a bright future” Nature News and Views: “Antibiotic resistance: To the rescue of old drugs” Meziane-Cherif & Courvalin, Nature 510, 477–478.

The emergence of bacteria that are resistant to antibiotics has been an object lesson in the relentlessness of natural selection; the moment a new antibiotic is developed and introduced, the countdown to the emergence of resistance begins. The race to keep the one step ahead of emerging resistance mechanisms has been going on since antibiotics were first introduced.

The history of the development of penicillin and related antibiotics is both an illustration of the ingenuity of scientists and of the never-ending nature of this battle with emerging resistance. The Nature paper is the latest installment in that story. Continue reading “Hope for Treatment of Carbapenem-Resistant Bacteria”

Shedding Light on Protein:Protein Interactions with NanoBRET™ Technique

Posted on by Michele Arduengo, PhD

NanoBRET™ TechnologyIf you are trying to investigate protein:protein interactions inside cells, you know how important physiologically relevant results are. If you overload your cells with fusion constructs, your protein interactions may not actually reflect what is going on in the cell, and if your BRET energy donor and acceptor do not have sufficiently separated spectra, you can pick up a fair amount of noise in your experiment. Using the new superbright NanoLuc® Luciferase, and the HaloTag® Technology, we have developed a sensitive BRET system to help you take a better look specific protein interactions that interest you. Promega research scientist, Danette Daniels, describes the system in the Chalk Talk below:

Optimize Your Western Blot

Posted on by Ben Schmidt
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Western Blot Detection.

You’ve probably been there. You’ve got a new antibody or you’re testing out one you’ve made yourself. After weeks or months of work, your antibody is going to help move your research project forward. As you excitedly head to the dark room to develop your film, your mood is crushed when you see…bands, more bands, and smears. Alas, science has played one more cruel joke on you as you experience what so many of your fellow scientists have before. Despite such a dismal beginning, you often can still get good western blots by changing steps in your protocol.

Several steps in the western blot protocol can be optimized.

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Mass Spec-Compatible Proteome Reference Material

Posted on by Gary Kobs
MSextractcropped

The complexity of biological samples places high demand on mass spec analytical capability. Adequate monitoring of instrument performance for proteomics studies requires equally complex reference material such as whole-cell extracts. However, whole-cell extracts available commercially are developed for general research (e.g., enzymatic or Western blot analysis) and contain detergents and salts that interfere with reverse phase liquid chromatography and mass spectrometry. Even after clean up, the extracts need to be digested, requiring time, labor and experience to generate samples for use in mass spectrometry. To address the need for complex protein material, we have developed whole-cell protein extracts from yeast and human cells. The yeast extract offers the convenience of a relatively small and well annotated proteome, whereas the human extract provides a complex proteome with large dynamic range. The human extract also serves as reference material for studies targeting the human proteome.

The extracts are free of compounds that interfere with reverse phase liquid chromatography-mass spectrometry (LC-MS), and have been reduced with DTT and alkylated with iodoacetamide then digested with Trypsin/Lys-C Mix and lyophilized. These digested extracts (tryptic peptides) can be readily reconstituted in trifluoroacetic acid (TFA) or formic acid and injected into an instrument. The same human and yeast whole-cell extracts also are provided in an intact (undigested) form for users who would like to develop an independent method for preparing protein mass spectrometry samples. For convenience, the intact extracts are provided as a frozen solution.

Consistent extract protein composition is ensured by tight control over cell culture conditions and manufacturing process. Lot-to-lot consistency of extracts is monitored by various protein and peptide qualitative and quantitation methods, including LC-MS. (Quality control results are provided upon request.) Our manufacturing process assures compatibility with reverse phase liquid chromatography and mass spectrometry, minimal nonspecific protein fragmentation, nonbiological post-translational modifi cations and,for digested extracts, minimal undigested peptides. The extracts are optimized for a high number of peptide and protein identifications in mass spectrometry analysis.

Genetically Modified Mosquitoes Fight Malaria

Posted on by Terri Sundquist
Image courtesy of James Gathany and the CDC
Image courtesy of James Gathany and the CDC

Mosquitos: They are the scourge of summer activities—the annoying buzzing noise as they fly around our ears and the pain, itching and swelling associated with their bites. Worst of all, certain species of mosquitoes can transmit diseases such as West Nile virus, Dengue fever and malaria. Defense mechanisms such as mosquito repellent, covering my head with netting and wearing heavy clothing are often insufficient against the swarm of hungry insects. It’s enough to make me want to stay indoors.

Those people who cannot escape these pests have a higher risk of being bitten and contracting a disease such as malaria, which killed an estimated 627,000 people in 2012, mostly in Africa and southeast Asia (1). A common step in malaria reduction programs in high-risk areas is reducing the number of Anopheles gambiae mosquitoes, which act as the host for malaria-causing parasites. This often involves massive amounts of insecticides, including limited amounts of the much maligned but very effective insecticide dichlorodiphenyltrichloroethane (DDT). Due to these programs, the World Health Organization (WHO) estimates that between 2000 and 2012, malaria mortality rates decreased by 42% worldwide, including a 48% decrease in children under 5 years of age. Clearly these programs are saving lives, but wouldn’t it be nice to achieve the same thing with fewer pesticides?

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Screening for Antiviral Compounds under Level 4 Containment Conditions

Posted on by Isobel Maciver

Working with bacteria and viruses that cause life-threatening diseases with no currently available treatment options takes guts. Most scientists are familiar with the routine requirements of good aseptic technique, are highly aware of laboratory safety requirements, and are more than familiar with autoclaves and sterilization issues, but if we make a mistake the consequences are usually only lost time or a spoiled experiment—not a lost life.

Scientists working with highly virulent organisms deal with a whole other level of risk that requires adherence to the strictest of safety regulations, and these containment regulations can sometimes place constraints on the type of experiment that can be performed with dangerous pathogens. A paper published in the April 2014 issue of Assay and Drug Development Technologies brought this to my attention and reminded me of the serious issues some scientists face on a daily basis as they research ways to combat infectious diseases.

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