Fluorescent protein pair emit intracellular FRET signal suitable for FACS screening.
The fluorescent proteins ECFP and HcRed were shown to give an easily resolved FRET-signal when expressed as a fusion inside mammalian cells. HeLa-tat cells expressing ECFP, pHcRed, or the fusion protein pHcRed-ECFP were analyzed by flow cytometry after excitation of ECFP. Cells expressing HcRed-ECFP, or ECFP and HcRed, were mixed and FACS-sorted for FRET positive cells: HcRed-ECFP cells were greatly enriched (72 times). Next, cloned human antibodies were fused with ECFP and expressed anchored to the ER membrane. Their cognate antigens (HIV-1 gp120 or gp41) were fused to HcRed and co-expressed in the ER. An increase of 13.5+/-1.5% (mean+/-SEM) and 8.0+/-0.7% in ECFP fluorescence for the specific antibodies reacting with gp120 or gp41, respectively, was noted after photobleaching. A positive control (HcRed-ECFP) gave a 14.8+/-2.6% increase. Surprisingly, the unspecific antibody (anti-TT) showed 12.1+/-1.1% increase, possibly because overexpression in the limited ER compartment gave false FRET signals.
Johansson
Daniel X
DX
Karolinska Institutet, Department of Medicine, Karolinska Hospital, 171 76 Stockholm, Sweden.
Brismar
Hjalmar
H
Persson
Mats A A
MA
D002469:Cell Separation / Q000379:methods*; D004721:Endoplasmic Reticulum / Q000378:metabolism* / Q000648:ultrastructure; D005434:Flow Cytometry / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D000097573:Red Fluorescent Protein
D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein; C485182:Cyan Fluorescent Protein