Recombinant production of rhesus θ-defensin-1 (RTD-1) using a bacterial expression system (original) (raw)
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Efficient one-pot cyclization/folding of Rhesus θ-defensin-1 (RTD-1)
Bioorganic & medicinal chemistry letters, 2012
We report an efficient approach for the chemical synthesis of Rhesus θ-defensin-1 (RTD-1) using Fmoc-based solid-phase peptide synthesis in combination with an intramolecular version of native chemical ligation. The corresponding linear thioester precursor was cyclized and folded in a onepot reaction using reduced glutathione. The reaction was extremely efficiently yielding natively folded RTD-1 with minimal or no purification at all. This approach is fully compatible with the high throughput production of chemical libraries using this peptide scaffold.
Bioorganic & Medicinal Chemistry, 2017
We report the high-yield heterologous expression of bioactive θ-defensin RTD-1 inside Escherichia coli cells by making use of intracellular protein trans-splicing in combination with a high efficient split-intein. RTD-1 is a small backbone-cyclized polypeptide with three disulfide bridges and a natural inhibitor of anthrax lethal factor protease. Recombinant RTD-1 was natively folded and able to inhibit anthrax lethal factor protease. In-cell expression of RTD-1 was very efficient and yielded ≈ 0.7 mg of folded RTD-1 per gram of wet E. coli cells. This approach was used to generate of a genetically-encoded RTD-1-based peptide library in live E. coli cells. These results clearly demonstrate the possibility of using genetically-encoded RTD-1-based peptide libraries in live E. coli cells, which is a critical first step for developing in-cell screening and directed evolution technologies using the cyclic peptide RTD-1 as a molecular scaffold.
Biosynthesis and Antimicrobial Evaluation of Backbone-Cyclized α-Defensins
Biochemistry, 2011
Defensins are antimicrobial peptides that are important in the innate immune defense of mammals. Upon stimulation by bacterial antigens, enteric α-defensins are secreted into the intestinal lumen where they have potent microbicidal activities. Cryptdin-4 (Crp4) is an α-defensin expressed in Paneth cells of the mouse small intestine and the most bactericidal of the known cryptdin isoforms. The structure of Crp4 consists of a triple-stranded antiparallel β-sheet, but lacks three amino acids between the fourth and fifth cysteine residues, making them distinct from other αdefensins. The structure also reveals that the α-amino and C-terminal carboxylic groups are in proximity (d ≈ 3 Å) in the folded structure. We present here the biosynthesis of backbone-cyclized Crp4 using a modified protein splicing unit or intein. Our data show that cyclized Crp4 can be biosynthesized by using this approach both in vitro and in vivo, although the expression yield was significantly lower when produced inside the cell. The resulting cyclic defensins retained the native α-defensin fold and showed equivalent or better microbicidal activities against several Gram-positive and Gram-negative bacteria when compared to native Crp4. No detectable hemolytic activity against human red blood cells was observed for either the native or cyclized variants of Crp4. Moreover, both forms of Crp4 also showed high stability to degradation when incubated with human serum. Altogether, these results indicate the potential for backbone cyclized defensins in the development of novel peptide-based antimicrobial compounds.
Efficient Production of Human β-Defensin 2 (HBD2) in Escherichia coli
Protein & Peptide Letters, 2009
Human -defensin 2 (HBD2) has been shown to interact with pathogenic bacteria and components of the mammalian innate and adaptive immune response. We describe a quick and reliable method for the production of HBD2 in Escherichia coli. HBD2 was expressed as an insoluble fusion, chemically cleaved and oxidised to give a single, folded HBD2 -isoform. The purified peptide was analysed by high resolution mass spectrometry, displayed a well-dispersed 1 H NMR spectrum, was a chemoattractant to HEK293 cells expressing CCR6 and acted as an antimicrobial agent against E. coli, P. aeruginosa and C. albicans.
Chemical synthesis, characterization and activity of RK-1, a novel α-defensin-related peptide
Journal of Peptide Science, 2000
The 32-residue peptide, RK-1, a novel kidney-derived three disulfide-bonded member of the antimicrobial h-defensin family, was synthesized by the continuous flow Fmoc-solid phase method. The crude, cleaved and S-reduced linear peptide was both efficiently folded and oxidized in an acidic solution of aqueous dimethyl sulfoxide. Following purification of the resulting product, it was shown by a variety of analytical techniques, including matrix assisted laser desorption time of flight mass spectrometry, to possess a very high degree of purity. The disulfide bond pairing of the synthetic peptide was determined by 1 H-NMR spectroscopy and confirmed to be a Cys 1 -Cys 6 , Cys 2 -Cys 4 , Cys 3 -Cys 5 arrangement similar to other mammalian h-defensin peptides. The synthetic RK-1 was also shown to inhibit the growth of Escherichia coli type strain NCTC 10418.
Proceedings of The National Academy of Sciences, 2003
Human defensins form a family of small, cationic, and Cys-rich antimicrobial proteins that play important roles in innate immunity against invading microbes. They also function as effective immune modulators in adaptive immunity by selectively chemoattracting T lymphocytes and immature dendritic cells. On the basis of sequence homology and the connectivity of six conserved Cys residues, human defensins are classified into ␣ and  families. Structures of several -defensins have recently been characterized, confirming the disulfide connectivity conserved within the family, i.e., Cys 1 -Cys 5 , Cys 2 -Cys 4 , and Cys 3 -Cys 6 . We found that human -defensin 3 (hBD3), a recently described member of the growing  family, did not fold preferentially into a native conformation in vitro under various oxidative conditions. Using the orthogonal protection of Cys 1 -Cys 5 and of Cys 1 -Cys 6 , we chemically synthesized six topological analogs of hBD3 with predefined disulfide connectivities, including the (presumably) native  pairing. Unexpectedly, all differently folded hBD3 species exhibited similar antimicrobial activity against Escherichia coli, whereas a wide range of chemotactic activities was observed with these analogs for monocytes and cells transfected by the chemokine receptor CCR6. Furthermore, whereas substitution of all Cys residues by ␣-aminobutyric acid completely abolished the chemotactic activity of hBD3, the bactericidal activity remained unaffected in the absence of any disulfide bridge. Our findings demonstrate that disulfide bonding in hBD3, although required for binding and activation of receptors for chemotaxis, is fully dispensable for its antimicrobial function, thus shedding light on the mechanisms of action for human -defensins and the design of novel peptide antibiotics.
Mammalian defensins (R as well as forms) have a-hairpin structural motif spanning approximately 20 residues at the carboxy-terminal end. We have investigated the antibacterial activity and biophysical properties of synthetic peptides corresponding to the carboxy-terminal segment of bovine-defensin-2 (BNBD-2): VRNHVTC 1 RINRGFC 2 VPIRC 3 PGRTRQIGTC 4 FGPRIKC 5 C 6 RSW (positions of disulfide bonds are C 1-C 5 , C 2-C 4 , and C 3-C 6). The parent sequence chosen was RCPGRTRQIGTIF-GPRIKCRSW (P1), which spans the carboxy-terminal region of BNBD-2. Since the dipeptide sequence D-Pro-Gly favors nucleation of-hairpin structures even in acyclic peptides, analogues of P1 with one D-Pro-Gly at the central portion and two D-Pro-Gly segments near the N-and C-terminal ends were generated. An analogue in which GP (residues 14 and 15) in P1 was switched to PG was also synthesized. It was observed that the cyclic form as well as their linear forms exhibited antibacterial activity. Circular dichroism and theoretical studies indicated that while the-hairpin conformation is populated, there is conformational plasticity in the cyclic and linear peptides. The mode of bacterial killing was by membrane permeabilization. The entire mammalian defensin sequence does not appear to be essential for manifestation of antibacterial activity. Hence, short peptides corresponding to the C-terminal segments of mammalian defensins could have potential as therapeutic agents.
Antimicrobial Agents and Chemotherapy, 2007
Beta defensins comprise a family of cationic, cysteine-rich antimicrobial peptides, predominantly expressed at epithelial surfaces. Previously we identified a unique five-cysteine defensin-related peptide (Defr1) that, when synthesized, is a mixture of dimeric isoforms and exhibits potent antimicrobial activity against Escherichia coli and Pseudomonas aeruginosa. Here we report that Defr1 displays antimicrobial activity against an extended panel of multidrug-resistant nosocomial pathogens for which antimicrobial treatment is limited or nonexistent. Defr1 fractions were collected by high-pressure liquid chromatography and analyzed by gel electrophoresis and mass spectrometry. Antimicrobial activity was initially investigated with the type strain Pseudomonas aeruginosa PAO1. All fractions tested displayed equivalent, potent antimicrobial activity levels comparable with that of the unfractionated Defr1. However, use of an oxidized, monomeric six-cysteine analogue (Defr1 Y5C), or of reduced Defr1, gave diminished antimicrobial activity. These results suggest that the covalent dimer structure of Defr1 is crucial to antimicrobial activity; this hypothesis was confirmed by investigation of a synthetic one-cysteine variant (Defr1-1cys). This gave an activity profile similar to that of synthetic Defr1 but only in an oxidized, dimeric form. Thus, we have shown that covalent, dimeric molecules based on the Defr1 -defensin sequence demonstrate antimicrobial activity even in the absence of the canonical cysteine motif.
Defensins: Antimicrobial peptides for therapeutic development
Biotechnology Journal, 2007
A novel class of endogenous antimicrobial peptides called defensins has shown great versatility in their activity against a diverse range of microorganisms including bacteria, viruses and fungi. Their mode of action of bacterial cell lysis seems largely nonspecific and so promises to avert the development of resistance. These two features have made them an area of intense research activity and growing commercial interest. A successful multidisciplinary effort to investigate and develop novel defensins analogues has been established in Singapore that involves computer modeling, biochemistry, proteomics, chemical synthesis, molecular biology and clinical sciences.