Fluorescent Protein-Based Assay Development II

FPs as pH and redox sensors:

The uses of FPs extend well beyond simple expression and fusion reporters.  While pH sensitivity (usually quenching of fluorescence by acidic pH) is generally considered a drawback for fusion tagging, it becomes a useful property for constructing pH sensors.  FPs specifically engineered to take advantage of pH sensitivity (“pHluorins”) report pH as either a change in fluorescent intensity or a change in the ratio of excitation at two different wavelengths, and may be used to monitor processes such as endocytosis or other pH-variable processes.  In such an application, the pH-sensitive FP is fused to a localization tag for the compartment of interest which experiences variable pH.  This technique can be used, for example, to visualize release of neurotransmitter-containing vesicles.  In addition to pH-sensitive FPs, redox-sensitive Aequorea GFP variants have been produced (roGFPs and others) which produce similar changes in fluorescence intensity or excitation ratio when exposed to differing redox conditions or reactive oxygen species.

Sensors based on circularly permuted FPs:

Because FPs have such a compact and stable beta-barrel fold with N and C termini close together, it is possible to engineer circularly permuted variants which retain their fluorescent properties.  Studies on circular permutation of FPs have led to the development of several different sensors which take advantage of domains inserted into sensitive areas of the fluorescent protein backbone.  The most famous of these are the GCaMP calcium sensors, in which a calmodulin domain has been inserted into a loop in GFP, yielding a sensor that reports calcium concentration as a change in fluorescent intensity.  Other circularly permuted FP variants, such as cpVenus (a yellow Aequorea GFP variant), have found usefulness in improving FRET sensor dynamic range (see next section).

FRET sensors:

Fluorescence resonance energy transfer (FRET) is a quantum mechanical process that allows the transfer of excited state energy between two fluorophores when they are in close physical proximity.  Because this process operates with a strong distance (1/r^6) and orientation dependence (strongest when chromophore dipoles are parallel or antiparallel), it lends itself to the construction of highly sensitive reporters of biochemical activity.  In FP FRET, excited state energy from a higher-energy (shorter wavelength) “donor” fluorescent protein is transferred to a lower-energy (longer wavelength) “acceptor” FP, leading to sensitized fluorescent emission from the acceptor and reduced emission (quenching) from the donor.  By linking donor and acceptor FPs with a domain which changes conformation in response to a biochemical activity of interest, this activity is reported as a change in the ratio of sensitized emission to direct-excitation emission of the acceptor (or a simple ratio of donor and acceptor emission).  FRET sensors have been engineered to specifically sense a wide variety of activities, including many protein kinases, as well as small molecules such as Ca2+ and neurotransmitters.  While design of a new FRET sensor generally requires a great deal of optimization and trail-and-error, this class of probe is among the most powerful tools currently available for investigating live-cell biochemistry.

New Product of the Week 03-15-10 to 03-21-10: Oct4-Sox2 2-in-1 lentivirus ABP-SC-LVI2in1 for effective iPS generation link:
Promotion of the Week 03-15-10 to 03-21-10: 5% off plate oligos at all scales! We are doing our “window promotion” again, during a hour-long window, get any Allele’s High efficiency competent cells at 30% regular price, the time will be announced tomorrow on our Facebook page.

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Wednesday, March 17th, 2010 Uncategorized No Comments

GPCR: problems and resolutions in high content screening, Part II

2) Without a mark (continued from Part I by Niels Yuhui Ni, MD Ph.D. of Allele Biotech)

In this section, the author focused on the “Label-Free” system. “A newer alternative that has given hope” because these systems are closer to the real cellular conditions most GPCR studies are meant to address. However, label-free systems must depend on complicated detection system for high content analysis. As commented, “For some scientist though, this technology is simply still too new and for now, too expensive for many categorical assessments…” While I am a believer in label-free detection, however, here is my question, for this group of scientists, are there any good alternatives now, before the label-free systems become more accessible? Using the traditional over-expression cell lines seems less and less attractive (see Part I under the same blog topic here)? A system that combines immortalized primary cells and a non-integrated expression system could be a nice system that does not require high end equipment or heavy commitment in technology development especially if the components were commercially available. The third, critical component of this system could be the application of newer, brighter and monomeric and thus less toxic fluorescent proteins or FRET pairs as sensors. Obviously part of the reason that I thought about such a system was because our research team at Allele Biotech has the background in all 3 components, whereas others might have their own preferred methods. To me, it seems that immortalized primary cells present a renewable cell source, and for non-integration delivery, we prefer Baculo viral delivery vehicles for mammalian infection (called BacMam by some). Both platforms are being offered as products or services already or in the pipeline teed up for launching.

Baculo2Mam non-intergrated viral delivery system:
Many people may know about Baculovirus such as in the Bac-to-Bac system from Invotrogen or the Sapphire baculovirus system from Orbigen (acquired by Allele Biotech). But how many of us know that even though mammalian cells are not the nature host of baculovirus, they still can be infected with modest modifications on the virus. Both the safety and efficiency of Baculovirus for mammalian use are superb. Based on the data from our customers’ projects, most protein expression require baculoviral protein expression in insect cells, only about 10% require Baculo2Mam. We actually feel a sense of responsibility for introducing this technology to as many as researchers as possible.

The following list shows some of the advantages of Baculo2Mam I can list right now, for more details check back on our blog articles in coming days or contact us at any time for discussion.
1) Baculoviruses are Risk Group 1 or biosafety 1 agents.
They are produced in insect cells and can not replicate in mammalian cells. They express genes in human or mouse cells in non-integrated state for about 2 weeks (varies in different cells).
2) Baculoviruses can be easily generated in high titer and production rapidly scaled-up.
That is when compared with other viral systems. For example, baculovirus is a budding virus that is released into cell medium, unlike adenovirus that requires lysing cells during productions. Allele Biotech now provides Baculo2Mam viral packaging service at an affordable price for routine use. Your viral clones can be stored in Allele Biotech’s Baculo2Mam virus bank; if you need the virus again, you can just order a production service at an even lower price.
3) Broad host cell range including many primary cells.
Many terminally differentiated primary cells such as neuron, adipocytes have been tested in Allele Biotech’s lab as target for modified Boculovirus. To assess the infection efficiency, you can order a pre-made Baculo2Mam-mWasabi GFP or Baculo2Mam-LanRFP control for a test run. Once you order custom or regular Baculo2Mam products, the cost of the control will be credited back.
4) Up to now, little or no cytopathic effects were observed of using baculovirus in mammalian cell cultures.
5) Other points that may be related to GPCR assays in relevance to mimicking natural cellular environment:
a) Delivery of biosensor to cells just prior to assay without establishing cell lines
b) Large insert capacity for expressing long cDNAs.
c) Multiple virus transductions, simultaneous delivery of multiple genes
d) Expression level can be adjusted by viral titer
e) Finally, Baculo2Mam Viruses can be stably stored at 4oC for up to 3 months, and even longer as seed stocks (i.e. titer will drop but still amplifiable).

    Promotion of the first week of 2010:

in the spirit of celebrating Allele’s 10th anniversary and in line with the ongoing “get oligos free for a month” program, we offer $20 off for oligos on 3’ TAMRA or FAM modifications.

    New product of the week:

iPS specific gene promoter-fluorescent protein reporter lentiviruses.

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