Isailovic Dragan, Sultana Ishrat, Phillips Gregory J, Yeung Edward S
Ames Laboratory-U.S. Department of Energy and Department of Chemistry, Iowa State University, Ames, IA 50011, USA.
Anal Biochem. 2006 Nov 1;358(1):38-50. doi: 10.1016/j.ab.2006.08.011. Epub 2006 Aug 23.
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.
在将藻胆蛋白R-藻红蛋白的重组脱辅基亚基与藻红胆素发色团孵育后，对荧光蛋白的形成进行了探索。从红藻多管藻中克隆出R-藻红蛋白的α和β脱辅基亚基基因，并将其克隆到质粒pET-21d(+)中。带有六组氨酸标签的α和β脱辅基亚基在大肠杆菌中表达。尽管表达的脱辅基亚基形成了包涵体，但在用藻红胆素孵育大肠杆菌细胞后，形成了荧光全蛋白亚基。全蛋白亚基同时含有藻红胆素和尿胆素发色团。荧光和微分干涉相差显微镜显示全蛋白亚基包涵体在细菌细胞中的极性定位。通过荧光显微镜和流式细胞术发现，含有荧光全蛋白亚基的细胞比对照细胞亮几倍。与包涵体中蛋白质的表达相比，向细胞中添加藻红胆素未显示出细胞毒性作用。为了提高溶解性，将R-藻红蛋白脱辅基亚基与麦芽糖结合蛋白融合，并在体外和体内与藻红胆素一起孵育。形成了以藻红胆素为唯一发色团的高荧光可溶性融合蛋白。通过荧光显微镜观察，融合蛋白定位于整个大肠杆菌细胞或细胞两极。流式细胞术显示，含有荧光融合蛋白的细胞比对照细胞亮达10倍。结果表明，藻红胆素与藻胆蛋白表达的脱辅基亚基结合形成的荧光蛋白可作为荧光探针，用于通过显微镜和流式细胞术分析细胞。这些荧光报告分子的一个独特特性是它们在正确折叠的（可溶性）亚基和聚集在包涵体中的亚基中都有用。
