CLONING OF MUTACIN GENE FROM Streptococcus mutans AND ITS PROTEIN EXPRESSION USING pT7-HIS PLASMID

Al-Homsi, L.; Al-Okla, S.; Abbady, A. Q.

doi:10.21608/jacb.2012.54716

CLONING OF MUTACIN GENE FROM Streptococcus mutans AND ITS PROTEIN EXPRESSION USING pT7-HIS PLASMID

Document Type : Original Article

Authors

¹ Biotechnology Dept., Fac. Agric., Damascus University, Syria.

² Animal Biology Dept., Fac. of Science, Damascus University, Syria.

³ Department of Molecular Biology and Biotechnology, Atomic Energy Commission of Syria (AECS), Damascus, Syria.

10.21608/jacb.2012.54716

Abstract

Streptococcus mutans is gram positive, facultative anaerobic bacteria and commonly found in the human oral cavity. The most important feature is its ability to produce an antimicrobial short peptide (Bacteriocin) known as Mutacin which is a short peptide chain of amino acids. Mutacin can inhibit the growth of other mutans streptococci, many gram positive and some gram negative bacteria. In this work, we aimed to design a novel system to produce the Mutacin using recombinant methods. These include the construction of the coding sequence of the peptide by special PCR followed by gene cloning and expression in the plasmid pT₇-His. Mutacin was produced as a part of a high solubility and productivity fusion protein which is able to be purified and detected by its colorimetric properties.
The whole gene of Mutacin was constructed using PCR with long overlapping primers which cover the whole gene and with two additional short primers to amplify it. Digested Mutacin fragment, as well as the pT₇-His plasmid, were ligated and then used to transform E. coli strain. The positive colonies expressed a soluble fusion protein (30 kDa) of the Mutacin with the Green Florescent Protein (GFP) in the cytoplasm. This fusion protein was purified by metal affinity chromatography, as shown after SDS-PAGE separation and gel staining. In the next work, we will test the best conditions to release the Mutacin from the fusion protein in order to study its antibiotic activity against many bacterial strains. The development of this system will provide large quantities of the Mutacin for future studies and applications as broad spectrum antibacterial peptide.

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