Preparation of Specific Polyclonal Antibody Against the Recombinant Mutacin Produced by sfGFP Fusion Protein Technology
Lamis Al-Homsi1, Souad Al-Okla2, Abdul Q. Abbady3, *
1 Department of Biotechnology, Faculty of Agriculture, Damascus University, Syria
2 Department of Animal Biology, Faculty of Sciences, Damascus University, Syria
3 Department of Molecular Biology and Biotechnology, Atomic Energy Commission of Syria (AECS), Damascus, Syria
Mutacin I, a bacteriocin produced by streptococcus mutans, displays an antimicrobial activity against many
gram positive and some gram negative bacteria. Because of its medical importance, production of this short peptide in
large scale for future applications is a significant challenge. This work described the improvement of a novel system to
produce the recombinant mutacin using fusion protein technology.
The short peptide was expressed directly as a fusion protein with a superfolder form of the green florescent protein
(sfGFP), resulting in a high yield expression of soluble sfGFP-mutacin fusion protein (30 kDa) in the cytoplasm of
E. coli. Mutacin was released from the fusion by enzymatic cleavage at the tobacco etch virus (TEV) protease recognition
site and separated from the carrier sfGFP by nickel affinity and gel filtration chromatography. An additional advantage of
this fusion system was tested in the generation of mutacin-specific polyclonal antibodies. Specific anti-mutacin IgGs
were affinity purified, and were able to recognize the mutacin-sfGFP fusion protein or the cleaved forms of mutacin.
Even though it was efficiently produced (25 mg/L) by this method, pure mutacin was devoid of antibiotic activity. Fourier
transform infrared spectroscopy (FTIR) analysis revealed the absence of thioether bonds in the purified mutacin, which
are critical for final structure and function of this antibiotic. Determining whether the activity of pure mutacin could be
recovered by the reformation of such structures by chemical reaction needs more investigations. The development of this
system will provide large quantities of mutacin for future studies and applications as broad spectrum antibacterial peptide.
open-access license: This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited.
* Address correspondence to this author at the Department of Molecular
Biology and Biotechnology, Atomic Energy Commission of Syria (AECS),
Damascus, Syria; Tel: +963 11 213580; Fax: +963 11 6112289;