Profluorescent protein fragments for fast bimolecular fluorescence complementation in vitro.
Here, we present a protocol for isolating the large N-terminal fragment of enhanced green fluorescent protein (EGFP) with a preformed chromophore. By itself, the chromophore-containing EGFP fragment exhibits very weak fluorescence, but it rapidly becomes brightly fluorescent upon complementation with the corresponding small, C-terminal EGFP fragment. Each EGFP fragment is cloned and overexpressed in E. coli as a fusion with self-splitting intein. After solubilizing and refolding these fusions from inclusion bodies, both EGFP fragments are cleaved from intein and purified using chitin columns. When these EGFP fragments are linked with the two complementary oligonucleotides and combined in equimolar amounts, fluorescence develops within a few minutes. The isolation of profluorescent protein fragments from recombinant E. coli cells requires approximately 3 d, and their conjugation to oligonucleotides requires 1-4 h.
Demidov
Vadim V
VV
Center for Advanced Biotechnology, Boston University, 36 Cummington Street, Boston, Massachusetts 02215, USA. vvd@bu.edu
Broude
Natalia E
NE
D004926:Escherichia coli; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D047668:Inteins / Q000235:genetics; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry
D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins