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

• Main Authors: Deguang Sun, Xiaonan Zang, Yalin Guo, Dongfang Xiao, Xuexue Cao, Zhu Liu, Feng Zhang, Yuming Jin, Jiawei Shi, Zhendong Wang, Rui Li, Zhaxi Yangzong
• Format: Article
• Language: English
• Published: MDPI AG 2019-04-01
• Series: Genes
• Subjects: <i>Gracilariopsis lemaneiformis</i>; phycocyanin; fluorescence; cloning; expression
• Online Access: https://www.mdpi.com/2073-4425/10/5/322

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 &#946;-subunit (<i>pc</i>B) contained 519 nucleotides encoding a protein of 172 amino acids, and the full length of phycocyanin &#945;-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 &#946; subunit and the Cys-84 of the &#945; 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.