Human beta Defensin-3 Recombinant

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Human beta Defensin-3 Recombinant

$70.00$2,700.00


accession P81534


Source Optimized DNA sequence encoding Humanbeta Defensin-3 mature chain was expressed in Escherichia Coli.
Molecular weight Native humanBD-3 is generated by the proteolytic removal of the signal peptide and propeptide, the molecule has a calculated molecular mass of approximately kDa. Recombinant beta Defensin-3 is a monomer protein consisting of46 amino acid residue subunits, and migrates as an approximately kDa protein under non-reducing and reducing conditions in SDS-PAGE.
Purity >98%, as determined by SDS-PAGE and HPLC
Biological Activity The ED(50) wasDetermined by its antimicrobial resistance toE.Coli and was determined to be <3 ug/ml.

Protein Sequence MRIHYLLFAL LFLFLVPVPG HGGIINTLQK YYCRVRGGRC AVLSCLPKEE QIGKCSTRGR KCCRRKK
Endotoxin Endotoxin content was assayed using a LAL gel clot method. Endotoxin level was found to be less than.1 ng/µg(1EU/µg).
Presentation RecombinantHuman betaDefensin-3 was lyophilized from.2 μm filtered100mM NaCl,mM PB, pH.4.
Reconstitution A quick spin of the vial followed by reconstitution in distilled water to a concentration not less than.1 mg/mL. This solution can then be diluted into other buffers.
Storage The lyophilized protein is stable for at least years from date of receipt at -20° C. Upon reconstitution, this cytokine can be stored in working aliquots at° -° C for one month, or at -20° C for six months, with a carrier protein without detectable loss of activity. Avoid repeated freeze/thaw cycles.
Usage This cytokine product is for research purposes only.It may not be used for therapeutics or diagnostic purposes.


Molecular function Antimicrobial

Methods

Cryo Immunoelectron microscopy

  • Parental strains NTHI 86-028NP::rpsL and the isogenic sapBC mutant strain were grown to mid-log phase in sBHI, normalized for cell number, and pelleted.
  • Pellets were resuspended in 10 mM sodium phosphate buffer pH 7.4, supplemented with 2% sBHI.
  • Human LL-37 cathelicidin or human beta defensin 3 were added to cultures at a final concentration of 0.25 µg LL-37/ml or 1 µg hBD3/ml.
  • Cultures containing AMPs or cells alone were incubated at 37°C, 5% CO2, static for 30 minutes.
  • Samples were transfered to ice and cells were pelleted by centrifugation, washed in 100 mM PIPES pH 7.0, and then resuspended in 100 mM PIPES.
  • For immunolocalization of AMPs at the ultrastructural level, bacteria were fixed in 4% paraformaldehyde/0.05% glutaraldehyde in 100 mM PIPES/0.5 mM MgCl2, pH 7.2 (or PBS) for 1 hr at 4°C.
  • Samples were then embedded in 10% gelatin and infiltrated overnight with 2.3 M sucrose/20% polyvinyl…
  • Parental strains NTHI 86-028NP::rpsL and the isogenic sapBC mutant strain were grown to mid-log phase in sBHI, normalized for cell number, and pelleted.
  • Pellets were resuspended in 10 mM sodium phosphate buffer pH 7.4, supplemented with 2% sBHI.
  • Human LL-37 cathelicidin or human beta defensin 3 were added to cultures at a final concentration of 0.25 µg LL-37/ml or 1 µg hBD3/ml.
  • Cultures containing AMPs or cells alone were incubated at 37°C, 5% CO2, static for 30 minutes.
  • Samples were transfered to ice and cells were pelleted by centrifugation, washed in 100 mM PIPES pH 7.0, and then resuspended in 100 mM PIPES.
  • For immunolocalization of AMPs at the ultrastructural level, bacteria were fixed in 4% paraformaldehyde/0.05% glutaraldehyde in 100 mM PIPES/0.5 mM MgCl2, pH 7.2 (or PBS) for 1 hr at 4°C.
  • Samples were then embedded in 10% gelatin and infiltrated overnight with 2.3 M sucrose/20% polyvinyl pyrrolidone in PIPES/MgCl2 at 4°C.
  • Samples were trimmed, frozen in liquid nitrogen, and sectioned with a UCT cryo-ultramicrotome .
  • 50 nm sections were blocked with 5% FBS/5% NGS for 30 min and subsequently incubated with rabbit anti-hBD3 or rabbit anti-CAP-18 (LL-37) antibody for 1 hr at room temperature.
  • Sections were then washed in block buffer and probed with 18 nm colloidal gold-conjugated anti-rabbit IgG (H+L) for 1 hr at room temperature.
  • Sections were washed in PIPES buffer followed by a water rinse, and stained with 0.3% uranyl acetate/2% methyl cellulose.
  • Samples were viewed with a JEOL 1200EX transmission electron microscope (JEOL USA Inc., Peabody, MA).
  • All labeling experiments were conducted in parallel with controls omitting the primary antibody.
  • These controls were consistently negative at the concentration of colloidal gold conjugated secondary antibodies used in these studies.

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The SapF ATPase is dispensable for resistance to the AMPs hBD-3 and LL-37.

hBD-3
  • The parent strain and the SapF ATPase-deficient strain (A and C) or the Wild Type and SapD-ATPase deficient strain (B and D) were incubated with increasing concentrations of hBD-3 (A and B) or LL-37 (C and D) for 1 h and then plated on chocolate agar for enumeration of viable bacteria.