CLONING AND EXPRESSION OF C-PHYCOCYANIN Β SUBUNIT OF Spirulina platensis IN Escherichia coli

AC07

Porangaba, TM (1) tmporanga@yahoo.com.br , Piazza RMF (1), Sircili MP (1).

(1) Laboratório de Bacteriologia, Instituto Butantan, Brazil

Introduction: C-phycocyanin (C-PC) is a pigment soluble in water. It’s found in some blue-green microalgae such as Spirulina platensis, which are used in many countries as dietary supplements. It is consisted of two subunits, α and β, with molecular masses of 16 and 17kDa, respectively. This protein has shown hepato-protective, anti-oxidant, radical scavenger, anti-arthritic, anti-mutagenic, anti-viral, and anti-tumoral properties. One recent study demonstrated that β subunit of Anabaena C-PC (βC-PC) has anti-tumoral activity, since the βC-PC inhibits cell proliferation and promotes apoptosis on cancer cells. Objective: The aim of this study was cloning and expression the β subunit of S. platensis C-PC in Escherichia coli. Methods: The β subunit was cloned in pGEMT- easy plasmid vector originating pTMP-01 plasmid and subcloned in pET28a plasmid vector originating pTMP-02 plasmid. When the cells transformed with the plasmid pTMP-02 reached growth D.O.₆₀₀ between 0.5 to 0.6, it was induced with 1mM of IPTG during 4 h. Results: The β subunit was cloned and expressed in E. coli BL21.The presence of the βC-PC gene in the pTMP-01 plasmid was confirmed by sequencing. The gene expression was confirmed by RT-PCR and immunoblotting. Since the construction has one Histag, we use anti - his antibody to detect the recombinant protein. Discussion: The production of recombinant proteins in E. coli has been very useful in search of new biotechnology products. In this study was obtained a genetically modified microorganism capable of expressing a recombinant protein (βC-PC), which according to the literature, is capable of causing apoptosis in tumor cells. Experiments will be performed to determine the effects of this recombinant protein on cancer cells in vitro.  Because the number of cases of cancer increase each year and the treatments existing for this disease, are not specific, it is essential to search for new therapies against cancer.