GFP fusion

When Great is not Good Enough—VHH Antibodies Engineered for 10 Fold Affinity Increase

Single Domain antibodies (VHH fragments, nanobodies, or as we call them, nAbs) have been generated by injecting llamas with ligand-bound GPCR for the purpose of obtaining crystals of active-state structures. Such structural information could be critical in understanding drug functions and screening for new drugs. The unique ability of VHH fragments to fit into protein-protein complex crevices and hold proteins together was demonstrated by two Nature publications from Brian Kobilka’s group at Stanford ([1, 2], also see Allele Newsletter of Sep 4th, 2013). The nano antibody used in those studies, Nb80, showed affinity towards only the active state of the target GPCR.

However, even with an antibody as great as Nb80, the authors were only able to co-crystal GPCR beta2-adrenoceptor (b2AR) with high affinity agonists, not its natural agonists such as adrenaline. In yet another Nature paper published just now, the Kobilka lab showed that Nb80 could be further improved by 10 times in affinity, through in vitro evolution [3]. They presented Nb80 on the surface of yeast using an existing yeast display system, then applied standard limited mutagenesis and magnetic separation technologies for screening. After about 5 rounds of selection, a new version of VHH Nb6B9 was isolated that bound to ligand-loaded GPCR with a kD of 6.4 nM. For the first time, a co-crystal of b2AR-adrenoline was made.

Rasmussen et al. Nature, 2011 Structure of a nanobody-stabilized active state of the b2 adrenoceptor
Rasmussen et al. Nature, 2011 Crystal structure of the b2 adrenergic receptor–Gs protein complex
Ring et al. Nature, 2013 Adrenaline-activated structure of b2-adrenoceptor stabilized by an engineered nanobody

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mTFP1 is an excellent FRET donor

Because of its excitation and emission wavelength, sharp excitation and emission peaks, high quantum yield, and exceptional photostability, mTFP1 has always been considered a very good Forster resonance energy transfer (FRET) donor (1). More recently, several groups have investigated the use of mTFP1 in various FRET experiments and imaging modalities and have shown that mTFP1 is indeed one of the best choices (2, 3, 4).

In one recent publication, Padilla-Parra et al (2) tested a number of different FRET couples to determine which was the best for fluorescence lifetime imaging (FLIM)-FRET experiments, and found that the mTFP1-EYFP pair was by far the best pair for FLIM-FRET. This group also confirmed that the fluorescence lifetime decay of mTFP1 fits well to a single exponential, and that the time constant for this decay is unaffected by photobleaching, making mTFP1 an excellent choice for any kind of fluorescence lifetime imaging applications, including FLIM-FRET. This group also notes that it is likely that the use of Venus or mCitrine variants in place of EYFP would improve the performance of this FRET pair even further.

In a mathematical analysis of the potential FRET efficiency of mTFP1 with Venus YFP, Day et al. (3) showed that compared with Cerulean (currently the brightest cyan Aequorea GFP variant), one can expect up to 17% better FRET efficiency using mTFP1. This group went on to characterize the mTFP1-Venus pair in live-cell FRET and FLIM-FRET experiments and showed that it worked as predicted in both cases. They also note that mTFP1 has superior brightness and photostability when compared to Cerulean in live cells, which is consistent with all in vitro data reported previously (1). In a related paper, Sun et al. (4) demonstrated that mTFP1 is also an excellent FRET donor for the orange fluorescent protein mKO2.

Together, these recent independent studies confirm that mTFP1 among the best options when choosing a fluorescent protein as a FRET donor. With its proven track record of successful fusions, mTFP1 is also an excellent all-around performer that will enhance almost any live-cell imaging experiment.

(1) Ai et al., (2006) Biochem. J. 400:531-540.
(2) Padilla-Parra et al., (2009) Biophys J. 97(8):2368-76.
(3) Day et al., (2008) J Biomed Opt. 13(3):031203.
(4) Sun et al., (2009) J Biomed Opt. 14(5):054009.

AlleleBlog Admin, by Nathan Shaner

Video of the month (NEW!): Protein Expression Systems on youtube ( and at our protein expression page.

Discount of the week (Dec 14-20): 15% off Phoenix Retrovirus Expression System 2.0 (with selection medium provided)

New product(s) of the week: 48 fluorescent protein fusions on ready-to-infect virus that get into primary mammalian cells as subcellular markers (, 20 infections, only $249 for a limited introduction time.

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Tuesday, December 15th, 2009 Allele Mail Bag, Fluorescent proteins No Comments

Camelid Antibodies

When it was discovered that animals in the camel family produce antibodies with no light chains, the idea that a single-domain fragment can bind as well as a full 4-chain antibody formed a breakthrough. So far it has been a relatively less known one.

Smaller antibody fragments have been tested for therapeutic uses because classical IgG antibodies are too bulky to penetrate tissues well, and very expensive to produce. Different combinations of antigen-binding variable regions are used, e.g. scFv, Fab, diabody, all to some degree of success. In comparison, the N-terminal domain of camelid antibodies, termed VHH domain (nanobody, VHH antibody), represents a naturally evolved, only 13-15 kD in size, fully functional target binding fragment with many advantages.

The only other known species outside camelidae family that has heavy chain antibodies is particular cartilaginous fish, nurse shark. Although the arrangement of CDRs is somewhat different between the camel and shark heavy chain variable regions, they share many characteristics such as extremely high stability (maintaining functions after100 C heat and extreme pH treatment).

Accumulating reports have demonstrated the therapeutic potentials of camelid antibody-based fragments in treating cancer, neural diseases, even use in hair dandruff preventing shampoo. For basic research, the tiny antigen binders can be used as tools for quantitative pull down with unmatched efficiency, recognizing previously inaccessible enzyme cleft as antigens, and providing libraries for binding partner selection.

Allele Biotech has been working on display antibody selection from its early days through an NIH grant, and recently carried out an NIH/NCI contract for scFv yeast display.

Check out Allele’s current Camelid antibody products:

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