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

Working on the Wnt signaling pathway

Wnt proteins form a family of highly conserved, secreted glycolipoproteins that regulate cell proliferation, cell polarity and cell fate determination during embryonic development and tissue homeostasis. Mutations in Wnt genes or the Wnt signaling pathway components are often linked to human birth defects, cancer and other diseases.

Since the discovery of the Wnt-1 gene 27 years ago, a complex Wnt signaling network with many components having multiple distinct roles and acting in different cellular compartments has been established. The studies of Wnt signaling in human diseases, in stem cell biology and tissue regeneration holds promise for translational medicine.

A few commonly asked questions regarding studies on the Wnt pathways will be touched upon in this blog series:

How to activate the Wnt Signaling Pathway?

1) Recombinant Wnt Proteins: there are several commercial sources for recombinant Wnt proteins. As most of them are produced in bacteria, endotoxin, purity and active concentration are of primary concern. From our own experience using commonly used commercial Wnt proteins, the activities vary from batch-to-batch, and sometimes the products are simply inadequate.

2) Conditioned Medium: this approach is convenient and of low cost. ATCC provides two mouse fibroblast cell lines, which over-express mouse Wnt3A (CRL-2647) and mouse Wnt5A (CRL-2814). Researchers can maintain the cell lines and collect conditioned medium whenever they need Wnt proteins. However, these are the only two Wnt over-expressing cell lines available on the market, yet there are 19 members in the Wnt gene family. If you are planning on working with Wnt proteins other than 3A and 5A, you would have to develop relevant cell lines on your own. On the other hand, because over-expression of Wnt proteins will also activate the secretion of some growth factors, such as FGFs, the conditioned medium should be a mixture of several kinds of secreted factors.

3) Over-expression Wnt proteins directly in your cells of interests: Over-expression is a basic strategy for functional biology research. One of the barriers for introducing cDNAs into cells is the transfection efficiency. The advent of retroviral transfection (MMLV based or Lenti based) technically resolves this problem, as the transduction efficiency can reach almost 100%. However, highly efficient retrovirus packaging remains a difficult process for most research labs.

Allele Biotech plans to introduce all the human Wnt cDNAs on its HiTiter™ Expression Lentivirus platform as shelf products. The package size will be 5 vials, each vial 100 µl, the titer is 10e8 TU/ml at a price of $799. This product line will be under “Product-on-Demand”, with clones ready but packaged only upon order for the first time. First-time orders will qualify for a 10% discount, if the first-time customer provides the cDNA, there will be an additional 20% discount. These lentiviruses can be used directly in target cells or to build over-expressing cell lines for conditioned medium. Next topic is How to inhibit Wnt signaling pathway?

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Tags: cell fate, cell polarity, cell proliferation, conditioned medium, growth factors, lentivirus, signaling pathways, Wnt