Antimicrobial peptides are active against bacteria, fungi and viruses as part of the innate immune system in animals and insects. Such peptides are currently produced by extracting them from the host organism or by solid phase peptide synthesis; both techniques are expensive and produce low yields. Recombinant DNA technology opens a window to produce these peptides inexpensively and in large quantities utilizing E. coli expression systems. Two antimicrobial peptides, indolicidin and PGQ, were the focus of this work. They are short amphipathic alpha helical antimicrobial peptides that target a broad range of microorganisms. Genes encoding multimers of indolicidin, PGQ and a hybrid of indolicidin:PGQ were placed into protein expression vectors pET32a+ and pET43.1a+, for peptide production in E. coli. A combination of multimerization and the use of a fusion protein were utilized to mask the toxicity of these peptides in E. coli. The multimerized peptide fusion construct was purified using Ni/NTA affinity chromatography. Methionine residues flanking each monomeric unit were utilized to enable cleavage of the multimerized protein and liberating a biologically active peptide. A Trx:indolicidin trimer fusion was produced in the greatest yield of all constructs investigated. Upon cyanogen bromide cleavage, a band corresponding to the theoretical molecular weight of an indolicidin monomer was observed with SDS-PAGE. Antimicrobial activity of monomeric recombinant indolicidin was tested resulting in zones of clearing. Overall the results indicate that multimerizing antimicrobial peptide genes can potentially produce a larger quantity of peptide per bacterial cell. These studies suggest that multimerization of antimicrobial peptide genes represents a means to control in vivo toxicity of the recombinant peptides and increase production relative to single gene fusions.
| Identifer | oai:union.ndltd.org:wpi.edu/oai:digitalcommons.wpi.edu:etd-theses-1279 |
| Date | 25 April 2003 |
| Creators | Morin, Kimberly M |
| Contributors | David S. Adams, Committee Member, Samuel M. Politz, Committee Member, , Charlene Mello |
| Publisher | Digital WPI |
| Source Sets | Worcester Polytechnic Institute |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Masters Theses (All Theses, All Years) |
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