Use of Fluorescent Protein in Studying Protein Half-Life

How long a protein remains in cell and at what equilibrium level depends on several factors: 1) how fast it is translated; 2) how fast it is degraded; 3) how much dilution by cell division affects its balance. A good method for tracking protein degradation requires live cell measurement methods that show high resolution because the changes may be small and gradual; and that do not interfere with cellular processes. One simple method was recently described in Science by Eden et al. that relies on bleaching fluorescent protein (FP) tagged to cellular protein of interest.

To track protein half-life, only a small fraction of FP is bleached with a pulse of light that would irreversibly damage the chromophore of the FP. This treatment, called bleach chase, would produce a population of proteins that are non-fluorescent and cannot be replenished. By comparing the fluorescence of this population and the control, unbleached population, it is possible to determine the half-life of the fused proteins using equation T1/2=ln(2)/a, where a is the slope of decay of the difference between bleached and unbleached protein fluorescence on a semilogarithmic plot. (This part is recited from AlleleNews)

Conversely, instead of photobleaching a FP to create a protein population, a fluorescent signal can be created and chased by photoactivating a photoactivable FP that is fused to a cellular protein under study. Plachta et al. published in a recent issue of Nature Cell Biology that by following the half-life, or kinetics of pluripotency-related transcription factor Oct4, cell fates are predicted in early embryo development.

In fact, there is a third method, perhaps soon to be published, that a photoconvertible FP can be used for tracking fusion protein half life. By using a photoconvertible FP, such as mClavGR (already offered by Allele), a fluorescent protein population can be created as in the aforementioned studies; but unlike bleaching or photoactivating, photoconversion keeps both populations (converted and unconverted, green or red in the case of mClavGR) present. This way all readings can be internally controlled to compensate for factors not directly related to protein metabolism per se, such as cell death, equipment variation, etc.

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Tags: bleach chase, cell fate, mClavGR, Oct4 kinetics, photoactivable FP, photoconvertible FP, protein half life, pulse chase, YFP

State of the Biomedical Research–Not So Good for Pharma R&D

Exerpt from From Science News

Pfizer’s R&D budget, $9.3 billion in 2010, will drop to less than $8.5 billion this year and to between $6.5 billion and $7 billion in 2012, and the company will stop funding research in internal medicine, allergy and respiratory diseases, urology, and tissue repair.

In fact, the pharmaceutical industry as a whole faces financial pressures, as companies are producing fewer new drugs than in the past. In these conditions, even highly promising research has gotten the ax; in November, Roche cut its RNA interference research unit after spending $400 million over 3 years.

Drug companies also seem less wary nowadays about outsourcing. Among other examples, Eli Lilly began outsourcing animal toxicology studies in 2008, and Wyeth (purchased by Pfizer in 2009) began out sourcing data management for its clinical trials in 2003. In 2007, AstraZeneca even decided to move the production of many active pharmaceutical ingredients—perhaps the core activity of a drug company—to China.

These downsizing events are not particularly caused by still depressed economy, they have more to do with industry-specific patent expiration and productivity issues with large pharmas. What does it all mean to current graduate students and postdocs? Perhaps an even tighter job market than now for starting researchers for some years to come until the next round of sea change comes around. Be aware of what’s going on in smaller, more productive, and focused biotech companies.

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Tags: budget, drug market, patent expiration, Pfizer, R&D, RNAi, Roche

Happy Chinese New Year!

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Tags: Chinese New Year