Abstract Five genes (cpcA, hox1, pcyA, cpcE and cpcF), involved in the biosynthesis of phycocyanin holo-α-subunit, were cloned from the genomic DNA from Spirulina maxima. These genes were digested by the corresponding restriction enzymes and sequentially ligated into the expression plasmid pETDuet-1 with two multiple cloning sites to construct the expression vector pETDuet-6. The recombinant vector pETDuet-6 was then transformed into E. coli BL21 (DE3) and ampicillin resistant transformant ZJGSU02 was selected. The fidelity of the recombinant vector was confirmed by DNA sequencing. After induction with isopropyl-β-D-thiogalactopyranoside (IPTG), the cells in the culture of the engineered strain exhibited a pronounced blue. The color change was not seen in the cells in the culture of the control strain. Visualization of the proteins on coomassie-stained SDS-PAGE gel showed about 21kDa distinct band. After the SDS-PAGE gel being soaked in zinc ion solution, a bright fluorescence band was visualized by UV illumination. Western blotting indicated the specific binding of the recombinant protein to 6×His-tag monoclonal antibody. The absorption spectrum of the recombinant phycocyanin holo-α-subunit had a λmax at 623nm, and the maximum fluorescence emission wavelength of it was at 645.8nm, which were in agreement with those of the natural phycocyanin holo-α-subunit from S. maxima. These results demonstrated that phycocyanin holo-α-subunit of S. maxima was successfully expressed in the heterologous E. coli. This study provided not only a new strategy for the expression of pigment-binding protein in the heterologous host, but also a reference for exploring the construction and expression of five or more heterologous protein genes using one vector and the study of the interaction among the genes.
