Recombinant phycobiliprotein can be used as fluorescent label in immunofluorescence assay. In this study, pathway for phycocyanin beta subunit (CpcB) carrying noncognate chromophore phycoerythrobilin (PEB) and phycourobilin (PUB) was constructed in Escherichia coli. Lyase CpcS and CpcT could catalyze attachment of PEB to Cys84-CpcB and Cys155-CpcB, respectively. However, PEB was attached only to Cys84-CpcB when both CpcS and CpcT were present in E. coli. A dual plasmid expression system was used to control the expression of lyases and the attachment order of PEB to CpcB. The production of PEB-Cys155-CpcB was achieved by L-arabinose-induced expression of CpcS, CpcB, Ho1, and PebS, and then the attachment of PEB to Cys84-CpcB was achieved by IPTG-induced expression of CpcS. The doubly chromophorylated CpcB absorbed light maximally at 497.5 nm and 557.0 nm and fluoresced maximally at 507.5 nm and 566.5 nm. An amount of light energy absorbed by PUB-Cys155-CpcB is transferred to PEB-Cys84-CpcB in doubly chromophorylated CpcB, conferring a large stokes shift of 69 nm for this fluorescent protein. There are interactions between chromophores of CpcB which possibly together with the help of lyases lead to isomerization of PEB-Cys155-CpcB to PUB-Cys155-CpcB.

中文翻译：

---

大肠杆菌中带有两个非同源生色团的藻蓝蛋白β亚基的生物合成。

重组藻胆蛋白可以用作免疫荧光测定中的荧光标记。在这项研究中，在大肠杆菌中构建了携带非同源生色团藻红蛋白（PEB）和藻尿素（PUB）的藻蓝蛋白β亚基（CpcB）的途径。裂解酶CpcS和CpcT可以分别催化PEB与Cys84-CpcB和Cys155-CpcB的结合。但是，当大肠杆菌中同时存在CpcS和CpcT时，PEB仅与Cys84-CpcB连接。使用双重质粒表达系统来控制裂解酶的表达和PEB与CpcB的连接顺序。PEB-Cys155-CpcB的生产是通过L-阿拉伯糖诱导的CpcS，CpcB，Ho1和PebS的表达来实现的，然后通过IPTG诱导的CpcS的表达来实现PEB与Cys84-CpcB的连接。双发色的CpcB在497处最大吸收光。5 nm和557.0 nm并在507.5 nm和566.5 nm处发出最大荧光。PUB-Cys155-CpcB吸收的光能被转移到双发色的CpcB中的PEB-Cys84-CpcB中，从而为该荧光蛋白带来了69 nm的大斯托克斯位移。CpcB的发色团之间存在相互作用，这可能与裂解酶一起导致PEB-Cys155-CpcB异构化为PUB-Cys155-CpcB。