Date of Award

1996

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

William J. Todd

Abstract

Cecropins are basic, amphipathic peptides with antibacterial activity against many Gram-positive and Gram-negative bacteria. By retaining the positively charged, amphipathic characteristic of cecropin B, a cecropin analog S1, in which 54% of amino acid residues were substituted, was synthesized and shown to possess a broad spectrum of antimicrobial activity. To determine expression of the cecropin analog S1 in Sf9 cells, two gene constructs were designed, and inserted into Autographa californica nuclear polyhedrosis virus under the control of the polyhedrin promoter. One construct, pBS1, consisted of a gene encoding the analog peptide, so that the gene was intended for expression in the cytoplasm. The other construct, pBCES1, was designed to be expressed as a secretory peptide, consisting of a hybrid gene between a preprosequence-encoding region of cecropin B and the analog peptide-encoding gene. Expression was observed in the pBCES1 construct but not in the pBS1, indicating that this peptide is not stable in the cytoplasm. While the expressed peptide was observed to be maintained stably, the antibacterial activity of analog peptides was inhibited by fetal bovine serum without observation of peptide degradation. This indicates that fetal bovine serum binds to the expressed peptides and may help its stable expression. A gene encoding preprocecropin B was observed to be expressed as procecropin B in Sf9 cells, indicating that the cells do not have an enzyme, dipeptidyl aminopeptidase, to process the prosequence. While Sf9 cell lysates degraded cecropin B, the expressed procecropin B was not degraded in the medium after lysis of Sf9 cells. Unlike the analog peptide, the antibacterial activity of cecropin B was not inhibited by fetal bovine serum. Both peptides were not stably expressed in bacterial expression systems, where mature peptide-encoding genes were used for making fusion proteins with protein A or maltose-binding protein (MBP). In the protein A fusion system, both peptides were observed to be expressed in Staphylococcus aureus but not in Escherichia coli. The analog peptide showed more degradation than cecropin B in the MBP fusion system. However, none of peptides could be purified in functional form due to precipitate formation after a chemical or enzymatic cleavage.

ISBN

9780591289022

Pages

111

DOI

10.31390/gradschool_disstheses.6327

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