Dataset Information

Cloning and Expression of Beta Subunit Gene of Phycocyanin From Spirulina platensis in Escherichia coli.

ABSTRACT: BACKGROUND:C-Phycocyanin (C-PC) from blue-green algae such as Spirulina has been reported to have various pharmacological characteristics, including anti-in?ammatory and anti-tumor activities. Recombinant ?-subunit of C-PC (C-PC/?) is an inhibitor of cell proliferation and an inducer of cancer cell apoptosis. OBJECTIVES:Since C-PC/? has a big potential to be used as a promising cancer prevention or therapy agent, the purpose of this study was to clone and express Spirulina platensis cpcB gene in a bacterial expression system. This is a significant step for the production of this compound. MATERIALS AND METHODS:The cpcB gene was amplified using specific primers and cloned in a bacterial expression vector, namely pET43.1a+. Gene expression of cpcB was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the dot blotting technique. RESULTS:The SDS-PAGE analysis and dot blotting confirmed the production of recombinant C-PC/? in the bacterial expression system. Over-expression of cpcB gene was optimized in induction by 1 mM Isopropyl-?-D-Thiogalactoside (IPTG), after four hours of inoculation at 30°C. CONCLUSIONS:Over-expression of the synthetic CPC/? protein in the bacterial system (Escherichia coli BL-21) showed that E. coli can be used as a basis for further research to produce this desired protein in large quantities.

SUBMITTER: Shoja Z

PROVIDER: S-EPMC4600200 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

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Cloning and Expression of Beta Subunit Gene of Phycocyanin From Spirulina platensis in Escherichia coli.

Shoja Zahra Z Rajabi Memari Hamid H Roayaei Ardakani Mohammd M

Jundishapur journal of microbiology 20150829 8

<h4>Background</h4>C-Phycocyanin (C-PC) from blue-green algae such as Spirulina has been reported to have various pharmacological characteristics, including anti-inﬂammatory and anti-tumor activities. Recombinant β-subunit of C-PC (C-PC/β) is an inhibitor of cell proliferation and an inducer of cancer cell apoptosis.<h4>Objectives</h4>Since C-PC/β has a big potential to be used as a promising cancer prevention or therapy agent, the purpose of this study was to clone and express Spirulina platens ...[more]

PMID: 26464761

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Project description:Brachyspira (Serpulina) hyodysenteriae induces a mucohemorrhagic diarrheal disease in pigs. The production of a beta-hemolysin has been considered a major virulence attribute of this organism. Previous reports have failed to correlate a specific cloned gene sequence with a purified beta-hemolytic protein sequence. Thus, questions still remain concerning the structural gene sequence of the hemolysin. To answer this question unequivocally, the beta-hemolytic toxin was purified from extracts of log-phase spirochetes, and the N-terminal amino acid sequence was determined (K-D-V-V-A-N-Q-L-N-I-S-D-K) and compared with the translated sequences of previously cloned genes, tlyA to tlyC. The lack of homology between tlyA to tlyC translated sequences and the purified beta-hemolytic toxin sequence resulted in the study that is reported here. A degenerate probe was designed based on the N-terminal amino acid sequence of the purified beta-hemolysin and used to screen a B. hyodysenteriae genomic library. Three overlapping clones were identified, and one was sequenced to reveal an open reading frame coding for a putative 8.93-kDa polypeptide containing the N-terminal sequence of the purified beta-hemolysin. To distinguish this gene from the tlyA to tlyC genes, it has been designated hlyA. A hemolysis-negative Escherichia coli strains containing hlyA was beta-hemolytic on blood agar media. Also, the hemolytic activity of the recombinant protein had identical protease and lipase sensitivities and electrophoretic mobility to those of native B. hyodysenteriae beta-hemolysin. Based on sequence analysis, the translated protein had a pI of 4.3, an alpha-helical structure, and a phosphopantetheine binding motif. Hybridization analysis of genomic DNA indicated that the hlyA gene was present in B. hyodysenteriae and B. intermedia but was not detected in B. innocens, B. pilosicoli, or B. murdochii under high-stringency conditions. The location of hlyA on the chromosomal map was distinct from the locations of tlyA, tlyB, and tlyC.

Dataset Information

Cloning and Expression of Beta Subunit Gene of Phycocyanin From Spirulina platensis in Escherichia coli.

Publications

Cloning and Expression of Beta Subunit Gene of Phycocyanin From Spirulina platensis in Escherichia coli.

Similar Datasets

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