Efficient purification protocol for bioengineering allophycocyanin trimer with N-terminus Histag

Allophycocyanin plays a key role for the photon energy transfer from the phycobilisome to reaction center chlorophylls with high efficiency in cyanobacteria. Previously, the high soluble self-assembled bioengineering allophycocyanin trimer with N-terminus polyhistidine from Synechocystis sp. PCC 6803 had been successfully recombined and expressed in Escherichia coli strain. The standard protocol with immobilized metal-ion affinity chromatography with chelating transition metal ion (Ni(2+)) was used to purify the recombinant protein. Extensive optimization works were performed to obtain the desired protocol for high efficiency, low disassociation, simplicity and fitting for large-scale purification. In this study, a 3(3) full factorial response surface methodology was employed to optimize the varied factors such as pH of potassium phosphate (X(1)), NaCl concentration (X(2)), and imidazole concentration (X(3)). A maximum trimerization ratio (Y(1)) of approximate A(650 nm)/A(620 nm) at 1.024 was obtained at these optimum parameters. Further examinations, with absorbance spectra, fluorescence spectra and SDS-PAGE, confirmed the presence of bioengineering allophycocyanin trimer with highly trimeric form. All these results demonstrate that optimized protocol is efficient in purification of bioengineering allophycocyanin trimer with Histag.