Recombinant production of rhesus -defensin-1 (RTD-1) using a bacterial expression system

Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90033, USA.
Molecular BioSystems (Impact Factor: 3.21). 04/2012; 8(4):1359-65. DOI: 10.1039/c2mb05451e
Source: PubMed


Defensins are antimicrobial peptides that are important in the innate immune defense of mammals. In contrast to mammalian α- and β-defensins, rhesus θ-defensin-1 (RTD-1) comprises only 18 amino acids stabilized by three disulfide bonds and an unusual backbone cyclic topology. In this work we report for the first time the recombinant expression of the fully folded θ-defensin RTD-1 using a bacterial expression system. This was accomplished using an intramolecular native chemical ligation in combination with a modified protein-splicing unit. RTD-1 was produced either in vitro or in vivo. In-cell production of RTD-1 was estimated to reach an intracellular concentration of ~4 μM. Recombinant RTD-1 was shown to be correctly folded as characterized by heteronuclear-NMR and by its ability to specifically inhibit lethal factor protease. The recombinant production of folded θ-defensins opens the possibility to produce peptide libraries based on this peptide scaffold that could be used to develop in-cell screening and directed evolution technologies.

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    • "The resulting thioester bond is resolved by nucleophilic attack of the N-terminal amino group of the former cysteine (intramolecular aminolysis). Via an S→N acyl shift, this leads to the irreversible formation of a backbone-cyclized peptide (Austin et al., 2010; Gould et al., 2012). In PTS, inactive N-and C-intein fragments are recombinantly fused to the C-and N-terminus of the target polypeptide chain, which has been equipped with an extra N-terminal glycine (Figure 5B). "
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