Cloning And Expression Of Phycoerythrin Gene From Porphyridium Cruentum

As a natural pigment, phycoerythrin can be used in food and cosmetics industries as well as widely applyed in the pharmaceutical industry for photosensitizer and fluorescent probes. In this paper, we cloned phycoerythrin (PE) subunit gene and then expressed in Pichia pastoris and Escherichia coli BL21 for heterologous expression. The results were as follows:Group member Wu Yicheng has clonedβsubunits of phycoerythrin, base on his results, amplified phycoerythrinβ,αsubunit and intergenic region by LA-PCR, then sequenced(GenBank Accession Number:HM535635), cDNA sequence has 1134 bp bases, including the P subunit coding region of 534 bp (encoding 177 amino acids),105 bp spacer region and 495 bp a subunit (encoding 164 amino acids). cDNA sequences arranged in order of 5'UTR-peB-spacer region-peA. The analysis of genomic sequence showed that Porphyridium cruentum gene of PE has no introns. The primary structure, the secondary structure, and the three-dimensional structure of the PE a subunit in Porphyridium cruentum were predicted through the homologous peA with known structure in different data bases. The results suggested that the main structure of the a subunit of PE in Porphyridium cruentum was suited for the feature of light-harvesting protein.The peA, peB cDNA were subcloned into pPIC9K vector and the recombinant plasmid was transformed into Pichia pastoris GS115. The peA, peB was overexpressed in Pichia pastoris GS115 after induction under the control of methanol, but the expression of peA, peB were low. Then, the encoding gene of peA, peB were inserted into the plasmid pPET28a and was successfully expressed in E. coli BL21 by IPTG.Codon usage bias analysis shows that peA, peB have significant preference of the A or T ending codons. While compare to the E.coli and Pichia pastoris, the preference of peA, peB codon usage were significant different. If we want to increase the significantly efficiency of peA, peB gene expression, the partial codon in gene's open reading frame should be reconstituted.