Formation of fluorescent proteins by the attachment of phycoerythrobilin to R-phycoerythrin alpha and beta apo-subunits.
Formation of fluorescent proteins was explored after incubation of recombinant apo-subunits of phycobiliprotein R-phycoerythrin with phycoerythrobilin chromophore. Alpha and beta apo-subunit genes of R-phycoerythrin from red algae Polisiphonia boldii were cloned in plasmid pET-21d(+). Hexahistidine-tagged alpha and beta apo-subunits were expressed in Escherichia coli. Although expressed apo-subunits formed inclusion bodies, fluorescent holo-subunits were constituted after incubation of E. coli cells with phycoerythrobilin. Holo-subunits contained both phycoerythrobilin and urobilin chromophores. Fluorescence and differential interference contrast microscopy showed polar location of holo-subunit inclusion bodies in bacterial cells. Cells containing fluorescent holo-subunits were several times brighter than control cells as found by fluorescence microscopy and flow cytometry. The addition of phycoerythrobilin to cells did not show cytotoxic effects, in contrast to expression of proteins in inclusion bodies. In an attempt to improve solubility, R-phycoerythrin apo-subunits were fused to maltose-binding protein and incubated with phycoerythrobilin both in vitro and in vivo. Highly fluorescent soluble fusion proteins containing phycoerythrobilin as the sole chromophore were formed. Fusion proteins were localized by fluorescence microscopy either throughout E. coli cells or at cell poles. Flow cytometry showed that cells containing fluorescent fusion proteins were up to 10 times brighter than control cells. Results indicate that fluorescent proteins formed by attachment of phycoerythrobilin to expressed apo-subunits of phycobiliproteins can be used as fluorescent probes for analysis of cells by microscopy and flow cytometry. A unique property of these fluorescent reporters is their utility in both properly folded (soluble) subunits and subunits aggregated in inclusion bodies.
Isailovic
Dragan
D
Ames Laboratory-U.S. Department of Energy and Department of Chemistry, Iowa State University, Ames, IA 50011, USA.
Sultana
Ishrat
I
Phillips
Gregory J
GJ
Yeung
Edward S
ES
D001059:Apoproteins / Q000096:biosynthesis / Q000737:chemistry*; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics; D003001:Cloning, Molecular; D029968:Escherichia coli Proteins / Q000737:chemistry; D008164:Luminescent Proteins / Q000138:chemical synthesis*; D058619:Maltose-Binding Proteins; D052980:Phycobilins / Q000737:chemistry*; D010799:Phycoerythrin / Q000096:biosynthesis / Q000737:chemistry*; D021122:Protein Subunits / Q000096:biosynthesis / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D000461:Rhodophyta / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D001059:Apoproteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D008164:Luminescent Proteins; D058619:Maltose-Binding Proteins; D052980:Phycobilins; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D010799:Phycoerythrin; C011070:phycoerythrobilin