Cloning of pcB and pcA Gene from Gracilariopsis lemaneiformis and Expression of a Fluorescent Phycocyanin in Heterologous Host.

Sun Sun,Guo Guo,Wang Wang,Li Li,Cao Cao,Jin Jin,Zhang Zhang,Zang Zang,Xiao Xiao,Yangzong Yangzong,Shi Shi,Liu Liu

doi:10.3390/genes10050322

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.

Highlights

Phycobilisomes (PBSs) are a complex light-harvesting protein mainly found in cyanobacteria and red algae, which capture light energy in a spectrum range of 480 to 650 nm and transmit it to the photosystem II [1,2]
The open reading frame (ORF) started from ATG and ended at TAA, which encoded a protein of 172 amino acids
A number of studies have achieved heterologous expression contributes to many important biological activities, the such as anti-oxidation, antiof optically active phycocyanin

Summary

Introduction

Phycobilisomes (PBSs) are a complex light-harvesting protein mainly found in cyanobacteria and red algae, which capture light energy in a spectrum range of 480 to 650 nm and transmit it to the photosystem II [1,2]. PBSs consist of two parts, including brightly colored phycobiliproteins (PBPs) and non-pigmented linker proteins. The colored phycobiliproteins (PBPs) are responsible for the absorption and transmission of light energy. The function of the non-pigmented linker protein is to guide the assembly of PBSs [2,3]. PBP can be used in many fields, as a fluorescent probe or food additive, and as an anti-oxidation, anti-tumor, and anti-inflammatory agent [4,5,6]. PBP is normally composed of phycoerythrin (PE), phycocyanin (PC), and allophycocyanin (APC)

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Conclusion