Biosynthesis and characterization of a recombinant eukaryotic allophycocyanin using prokaryotic accessory enzymes

• Published in: MicrobiologyOpen (Weinheim) Vol. 9; no. 3; pp. e989 - n/a
• Main Authors: Dagnino‐Leone, Jorge, Figueroa, Maximiliano, Uribe, Elena, Hinrichs, María Victoria, Ortiz‐López, Diego, Martínez‐Oyanedel, José, Bunster, Marta
• Format: Journal Article
• Language: English
• Published: England John Wiley & Sons, Inc 01-03-2020; John Wiley and Sons Inc; Wiley
• Subjects: Agarophyton chilensis; Algae; Biosynthesis; Biotechnology; Cloning; Cyanobacteria; Deoxyribonucleic acid; DNA; E coli; Electrophoresis, Polyacrylamide Gel; Enzymes; Eukaryota - genetics; Fluorescence; Food dyes; functional characterization; Gene Expression; Genes; Genetic Vectors - genetics; Genomes; Glycerol; Heme; Molecular Weight; Oligomerization; Original; Oxidoreductase; Oxygenase; Phycobiliproteins; Phycocyanin - biosynthesis; Phycocyanin - genetics; Phycocyanin - isolation & purification; Phycocyanobilin; Prokaryotic Cells - enzymology; Proteins; recombinant allophycocyanin; Recombinant Proteins; Seaweeds; Spectroscopy; Spectrum Analysis; structural characterization; Trimers; Agarophyton chilensis; structural characterization; recombinant allophycocyanin; functional characterization
• ISSN: 2045-8827; 2045-8827
• DOI: 10.1002/mbo3.989

Phycobiliproteins (PBPs) are colored fluorescent proteins present in cyanobacteria, red alga, and cryptophyta. These proteins have many potential uses in biotechnology going from food colorants to medical applications. Allophycocyanin, the simplest PBP, is a heterodimer of αβ subunits that oligomerizes as a trimer (αβ)3. Each subunit contains a phycocyanobilin, bound to a cysteine residue, which is responsible for its spectroscopic properties. In this article, we are reporting the expression of recombinant allophycocyanin (rAPC) from the eukaryotic red algae Agarophyton chilensis in Escherichia coli, using prokaryotic accessory enzymes to obtain a fully functional rAPC. Three duet vectors were used to include coding sequences of α and β subunits from A. chilensis and accessorial enzymes (heterodimeric lyase cpc S/U, heme oxygenase 1, phycocyanobilin oxidoreductase) from cyanobacteria Arthrospira maxima. rAPC was purified using several chromatographic steps. The characterization of the pure rAPC indicates very similar spectroscopic properties, λmaxAbs, λmaxEm, fluorescence lifetime, and chromophorylation degree, with native allophycocyanin (nAPC) from A. chilensis. This method, to produce high‐quality recombinant allophycocyanin, can be used to express and characterize other macroalga phycobiliproteins, to be used for biotechnological or biomedical purposes.
A heterologous expression of Allophycocyanin from A.chilensis in E.coli was successfully accomplished. The enzymes for the synthesis of the holo‐APC were obtained from Arthrospira maxima, and they were active to introduce phycocyanobilin to Allophycocyanin. The spectroscopic and biochemical characterization of the protein confirmed a trimeric recombinant Allophycocyanin. The trimeric recombinant APC produced was completely functional, with high quality and it showed similar properties with native APC.