| Summary: | In order to study the assembly mechanism of phycocyanin in red algae, the apo-phycocyanin genes (<i>pcB</i> and <i>pcA</i>) were cloned from <i>Gracilariopsis lemaneiformis</i>. The full length of phycocyanin β-subunit (<i>pc</i>B) contained 519 nucleotides encoding a protein of 172 amino acids, and the full length of phycocyanin α-subunit(<i>pc</i>A) contained 489 nucleotides encoding a protein of 162 amino acids. Expression vector pACYCDuet-<i>pcB</i>-<i>pcA</i> was constructed and transformed into <i>E. coli</i> BL21 with pET-<i>ho</i>-<i>pcyA</i> (containing <i>ho</i> and <i>pcyA</i> gene to synthesize phycocyanobilin). The recombinant strain showed fluorescence activity, indicating the expression of optically active phycocyanin in <i>E. coli</i>. 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.
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