Formation of fluorescent proteins by the attachment of phycoerythrobilin to R-phycoerythrin alpha and beta apo-subunits

Abstract

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 plasmids pET-21d (+). Hexa-histidine tagged apo-alpha and apo-beta subunits were expressed in Escherichia coli. Although expressed apo-subunits formed inclusion bodies, fluorescent holo-subunits were constituted after incubation of Escherichia coli cells with phycoerythrobilin. 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. 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, Rphycoerythrin apo-subunits were fused to maltose binding protein and incubated with phycoerythrobilin both in vitro and in vivo. Highly-fluorescent soluble fusion proteins were formed containing phycoerythrobilin as the sole chromophore. Fusion proteins were localized by fluorescence microscopy either throughout Escherichia coli cells or at cell poles. Flow cytometry showed that cells containing fluorescent fusion proteins were up to ten 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.

Phycobilin:cystein-84 biliprotein lyase, a near-universal lyase for cysteine-84-binding sites in cyanobacterial phycobiliproteins

“…¶ An increasing number of lyases has recently been identified that catalyze the chromophore addition and are specific not only for the chromophore but also for the apoprotein and the binding site (12)(13)(14)(15)(16). Based on the capacity of several of the respective apoproteins to also bind the chromophores autocatalytically (17)(18)(19)(20)(21), a chaperone-like function has been suggested (12). It enhances and guides the autocatalytic binding, which is generally of low fidelity, possibly by conformational control of the chromophore (18).…”

Progress in biosynthesis of phycobiliprotein

“…20世纪90年代, Kahn等人 [56] 发现裂合酶CpeY和 CpeZ可能会催化PE的α及β亚基与PEB共价结合. 2006 年, Isailovic等人 [57] [59] . 2002年, 美 国加州大学的Glazer研究组 [60] 进行了holo-PecA在大 [62,63] .…”

Advances in Cyanobacterial Phycobilisome Assembling in vitro

“…在藻红蛋白方面, 针对脱辅基 PE 和亚基结合 PEB 的研究还只是处于初步阶段 [29,30] , 色基 PUB 或 PVB 与脱辅基蛋白如何结合的研究还没有进展. 结合 PCB 的 holo-PC 亚基, 是 Glazer 研究组 [31] 在 E. coli 体内通过 PAT 和 PCB 两个载体, 成功表达 了包含 cpcE/F 裂合酶在内的 5 个基因, 并通过 His 6 标签纯化得到的.…”