Biosynthesis of fluorescent cyanobacterial allophycocyanin trimer in Escherichia coli

Abstract

Allophycocyanin (APC), a cyanobacterial photosynthetic phycobiliprotein, functions in energy transfer as a light-harvesting protein. One of the prominent spectroscopic characteristics of APC is a strong red-shift in the absorption and emission maxima when monomers are assembled into a trimer. Previously, holo-APC a and b subunits (holo-ApcA and ApcB) were successfully synthesized in Escherichia coli. In this study, both holo-subunits from Synechocystis sp. PCC 6803 were co-expressed in E. coli, and found to self-assemble into trimers. The recombinant APC trimer was purified by metal affinity and size-exclusion chromatography, and had a native structure identical to native APC, as determined by characteristic spectroscopic measurements, fluorescence quantum yield, tryptic digestion analysis, and molecular weight measurements. Combined with results from a study in which only the monomer was formed, our results indicate that bilin synthesis and the subsequent attachment to apo-subunits are important for the successful assembly of APC trimers. This is the first study to report on the assembly of recombinant ApcA and ApcB into a trimer with native structure. Our study provides a promising method for producing better fluorescent tags, as well as a method to facilitate the genetic analysis of APC trimer assembly and biological function.

Photocurrent generation by recombinant allophycocyanin trimer multilayer on TiO2 electrode

“…S1) [9]. The native structure, stability, and fluorescent characteristics of the recombinant APC trimer were determined using fluorescence and absorption spectrometry, and the results supported a previous report that the longer His tag near the N-terminus did not significantly interfere with the trimer assembly [9,10]. Large-scale purification of the recombinant APC trimer has also been achieved.…”

“…Moreover, the very similar fluorescence emission spectra from the APC trimer complexes in solution and on TiO 2 both exhibited a fluorescence emission maximum at 660 nm with a shoulder at around 640 nm (Fig. 2a); this demonstrated that the APC trimer complexes were not disassembled following the formation of the multilayer structure [9]. It is possible that electrostatic interactions, especially hydrogen bonds, which are strong noncovalent forces in biological systems through functional groups such as carbonyl, amide, or imidazole, especially in macromolecules, are responsible for the multilayer adsorption, as noted previously [5,15,18].…”

“…1 shows a set of images (CLSM, AFM, and FE-SEM), demonstrating the deposition of an APC trimer complex multilayer on the sensitized surface of the photoanode. CLSM is a sensitive and convenient method for detecting signals from the recombinant APC trimer because of its maximum fluorescence emission at 661 nm under 600 nm excitation and fluorescence quantum yield at 0.72 [9,11]. The bright fluorescence image revealed the presence of adsorbed APC trimer complexes with non-uniform patterns, compared with the faint visual field of the blank control (unsensitized photoanode) under the same conditions (Fig.…”

“…In the preparation of the bioengineered APC trimer, the strains and purification methods reported previously were used [9]. Briefly, the recombinant APC trimer was purified through nickel affinity chromatography.…”

Biosynthesis of a stable allophycocyanin beta subunit in metabolically engineered Escherichia coli

“…Several phycobiliproteins subunits including holo-apcA, holoapcB and holo-cpcA have been biosynthesized in E. coli (13,16,21). The recombinant proteins are much smaller than their corresponding native proteins (1/6 or 1/12 of that of native proteins), and their spectroscopic properties are similar to those of corresponding native proteins.…”