Cloning of <i>pcB</i> and <i>pcA</i> Gene from <i>Gracilariopsis lemaneiformis</i> and Expression of a Fluorescent Phycocyanin in Heterologous Host

Abstract

In order to study the assembly mechanism of phycocyanin in red algae, the apo-phycocyanin genes (pcB and pcA) were cloned from Gracilariopsis lemaneiformis. The full length of phycocyanin β-subunit (pcB) contained 519 nucleotides encoding a protein of 172 amino acids, and the full length of phycocyanin α-subunit(pcA) contained 489 nucleotides encoding a protein of 162 amino acids. Expression vector pACYCDuet-pcB-pcA was constructed and transformed into E. coli BL21 with pET-ho-pcyA (containing ho and pcyA gene to synthesize phycocyanobilin). The recombinant strain showed fluorescence activity, indicating the expression of optically active phycocyanin in E. coli. To further investigate the possible binding sites between phycocyanobilin and apo-phycocyanin, Cys-82 and Cys-153 of the β subunit and the Cys-84 of the α subunit were respectively mutated, and four mutants were obtained. All mutant strains had lower fluorescence intensity than the non-mutant strains, which indicated that these mutation sites could be the active binding sites between apo-phycocyanin and phycocyanobilin (PCB). This research provides a supplement for the comprehensive understanding of the assembly mechanism of optically active phycocyanin in red algae.

Keywords

• <i>Gracilariopsis lemaneiformis</i>
• phycocyanin
• fluorescence
• cloning
• expression