title: Efficient purification protocol for bioengineering allophycocyanin trimer with N-terminus Histag.
abstract: 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 fromSynechocystissp. PCC 6803 had been successfully recombined and expressed inEscherichia colistrain. The standard protocol with immobilized metal-ion affinity chromatography with chelating transition metal ion (Ni2+) 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 33full factorial response surface methodology was employed to optimize the varied factors such as pH of potassium phosphate (X1), NaCl concentration (X2), and imidazole concentration (X3). A maximum trimerization ratio (Y1) of approximate A650 nm/A620 nmat 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.
authors.affiliation: Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003, China; University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Wenjun
authors.initials: W
authors.lastname: Li
authors.affiliation: School of Agriculture, Ludong University, Yantai 264025, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Pu
authors.affiliation: South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China.
authors.forename: Na
authors.initials: N
authors.lastname: Gao
authors.affiliation: Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003, China.
authors.forename: Zhihong
authors.initials: Z
authors.lastname: Tang
authors.affiliation: Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003, China.
authors.forename: Lufei
authors.initials: L
authors.lastname: Song
authors.affiliation: Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003, China.
authors.forename: Song
authors.initials: S
authors.lastname: Qin
keywords: Bioengineering allophycocyanin trimer; Efficient purification; Histag; Optimization