Targeting quantum dots to surface proteins in living cells with biotin ligase - PubMed (original) (raw)

Comparative Study

. 2005 May 24;102(21):7583-8.

doi: 10.1073/pnas.0503125102. Epub 2005 May 16.

Affiliations

Comparative Study

Targeting quantum dots to surface proteins in living cells with biotin ligase

Mark Howarth et al. Proc Natl Acad Sci U S A. 2005.

Abstract

Escherichia coli biotin ligase site-specifically biotinylates a lysine side chain within a 15-amino acid acceptor peptide (AP) sequence. We show that mammalian cell surface proteins tagged with AP can be biotinylated by biotin ligase added to the medium, while endogenous proteins remain unmodified. The biotin group then serves as a handle for targeting streptavidin-conjugated quantum dots (QDs). This labeling method helps to address the two major deficiencies of antibody-based labeling, which is currently the most common method for targeting QDs to cells: the size of the QD conjugate after antibody attachment and the instability of many antibody-antigen interactions. To demonstrate the versatility of our method, we targeted QDs to cell surface cyan fluorescent protein and epidermal growth factor receptor in HeLa cells and to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors in neurons. Labeling requires only 2 min, is extremely specific for the AP-tagged protein, and is highly sensitive. We performed time-lapse imaging of single QDs bound to AMPA receptors in neurons, and we compared the trafficking of different AMPA receptor subunits by using two-color pulse-chase labeling.

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Figures

Fig. 1.

Fig. 1.

General strategy for targeting QDs to cell surface proteins. (A) The size of GFP is compared with unconjugated QD605 and QD605-streptavidin conjugated to biotinylated secondary antibody and primary antibody, as used to label a cell surface protein. (B) The AP, GLNDIFEAQKIEWHE (shown in pink), is genetically encoded at the N terminus or C terminus of the protein of interest. Recombinantly expressed biotin ligase (BirA) is added to the cell medium with ATP and biotin, biotinylating AP. Excess biotin is removed by washing. Streptavidin-conjugated QDs are added to bind the biotinylated surface proteins. B, biotin.

Fig. 2.

Fig. 2.

Specific labeling of a cell surface protein by BirA shown by imaging. HeLa cells expressing AP-CFP-TM were biotinylated with BirA for 10 min. Biotinylated proteins at the cell surface were detected with streptavidin-Alexa Fluor 568. Controls are shown with a point mutation in AP (Ala-CFP-TM) or with BirA or biotin omitted from the labeling reaction.

Fig. 3.

Fig. 3.

Specific labeling of cell surface proteins by BirA shown by immunoblot. HeLa cells transfected with AP-CFP-TM were biotinylated with BirA for 5 min. Total cell lysates were blotted with streptavidin-horseradish peroxidase to detect biotinylated proteins. Results are shown for: lane 1, a point mutation in AP (Ala-CFP-TM); lane 2, without BirA; lane 3, without biotin; lane 4, with all components present. Endogenous biotinylated proteins are labeled with arrows and serve as a control for equivalent loading in each lane.

Fig. 4.

Fig. 4.

QD labeling of cell surface proteins in HeLa cells. HeLa cells expressing AP-CFP-TM were biotinylated with BirA for 5 min. Biotinylation was detected with streptavidin-QD605. A control is shown with a point mutation in AP (Ala-CFP-TM). The QD605 signal is shown with 3-s and 0.2-s exposure.

Fig. 5.

Fig. 5.

Targeting QDs to AP-tagged AMPA receptors in neurons. Neurons transfected with AP-GluR2 were labeled with BirA for 5 min and then incubated with streptavidin-QD605. A control is shown with a point mutation in AP (Ala-GluR2). CFP was used as a cotransfection marker.

Fig. 6.

Fig. 6.

The effect of QD size on surface labeling in neurons. (A) Neurons expressing AP-GluR2 and the synaptic marker PSD-95-YFP were labeled with BirA for 10 min at 37°C. After biotinylation, neurons were stained with streptavidin-QD605. (Middle) The staining at higher zoom. (Bottom) The same experiment, except the biotinylation was detected with streptavidin-Alexa Fluor 568 instead of streptavidin-QD605. (B) Neurons expressing AP-GluR2 were labeled with BirA for 10 min at 37°C. They were then incubated simultaneously with streptavidin-Alexa Fluor 488 and streptavidin-QD605.

Fig. 7.

Fig. 7.

Pulse–chase labeling of AMPA receptor subunits. Neurons were transfected with AP-GluR1 or AP-GluR2 and labeled with BirA for 10 min at 37°C followed by streptavidin-Alexa Fluor 488 for 10 min at room temperature. After 3 min at room temperature with 200 μM glycine, the neurons were again incubated with BirA for 10 min at 37°C and streptavidin-QD605 for 10 min at 4°C before imaging.

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