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Preparation of Specific Polyclonal Antibody Against the Recombinant Mutacin Produced by sfGFP Fusion Protein
Lamis Al-Homsi1, Souad Al-Okla2, Abdul Q Abbady3
1Department of Biotechnology, Faculty of Agriculture, Damascus University, Syria.
Abstract:
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.
Insights
Researchers developed a novel system for producing recombinant mutacin (a bacteriocin) using fusion protein technology. This method yields large quantities of mutacin, but the purified peptide lacks antimicrobial activity due to missing thioether bonds.
Area of Science:
- Microbiology
- Biotechnology
- Protein Engineering
Background:
- Mutacin I, a bacteriocin from Streptococcus mutans, has significant medical potential due to its antimicrobial activity.
- Large-scale production of mutacin for therapeutic applications presents a considerable challenge.
Purpose of the Study:
- To develop and optimize a novel recombinant protein expression system for producing mutacin.
- To investigate the feasibility of using fusion protein technology for high-yield mutacin production.
- To assess the functional activity of the purified mutacin and identify limitations.
Main Methods:
- Engineered a fusion protein system expressing mutacin with superfolder green fluorescent protein (sfGFP) in E. coli.
- Utilized enzymatic cleavage (TEV protease) to release mutacin from the sfGFP carrier.
- Employed nickel affinity and gel filtration chromatography for purification.
- Generated and purified mutacin-specific polyclonal antibodies.
- Analyzed purified mutacin using Fourier transform infrared spectroscopy (FTIR).
Main Results:
- Achieved high-yield expression (25 mg/L) of soluble sfGFP-mutacin fusion protein.
- Successfully purified mutacin using chromatographic techniques.
- Demonstrated the utility of the system for generating mutacin-specific antibodies.
- Found that purified mutacin lacked antimicrobial activity.
- FTIR analysis indicated the absence of critical thioether bonds in the purified mutacin.
Conclusions:
- The developed fusion protein system enables efficient, large-scale production of recombinant mutacin.
- The lack of antimicrobial activity in purified mutacin is attributed to the absence of essential thioether bonds.
- Further research is needed to chemically reform these bonds and restore mutacin's antibiotic function.
- This system provides a valuable platform for future studies and applications of mutacin as a broad-spectrum antibacterial peptide.
