Recombinant Bacillus subtilis Putative aminopeptidase ysdC (ysdC)

Basic Research Questions

• What is Bacillus subtilis ysdC and what is its known function?

  Bacillus subtilis ysdC (UniProt: P94521) is a putative aminopeptidase classified under EC 3.4.11.- enzymatic group. As an aminopeptidase, it likely catalyzes the removal of amino acids from the N-terminus of proteins or peptides. While its exact biological function remains to be fully characterized, it belongs to the broader family of aminopeptidases in B. subtilis, which play important roles in protein turnover, nutrient acquisition, and potentially in sporulation processes. Based on sequence analysis, it contains conserved metal-binding motifs characteristic of metallopeptidases .

  The full-length protein consists of 361 amino acids with a molecular weight of approximately 40 kDa. Its predicted secondary structure suggests a typical aminopeptidase fold with metal-binding residues that likely coordinate zinc or another divalent cation for catalytic activity .

• How does ysdC compare to other aminopeptidases in B. subtilis?

  B. subtilis possesses several distinct aminopeptidases with different substrate specificities, cellular localizations, and functions:

  Aminopeptidase	Characteristics	Physiological Role
  ysdC (P94521)	Putative aminopeptidase; metal-binding motifs present	Not fully characterized
  ywaD (BSAP168)	Extracellular aminopeptidase; double-zinc aminopeptidase	Plays important role in supplying amino acids during growth in protein-rich media
  dppA	D-aminopeptidase; octamer of 30 kDa subunits; pH optimum 9-11; Zn²⁺-dependent	Highest activity with D-Ala-D-Ala and D-Ala-Gly-Gly; likely adaptation to nutrient deficiency
  TmPep1050/CelM	M42 family aminopeptidases; prefer nonpolar aliphatic substrates	Oligomeric structure (dodecameric or dimeric)

  Unlike dppA, which specifically targets D-amino acid substrates, ysdC likely processes L-amino acid peptides, as is typical for most aminopeptidases. Unlike ywaD, which is secreted extracellularly, the subcellular localization of ysdC has not been definitively established .

• What expression systems are available for recombinant ysdC production?

  Recombinant ysdC can be produced in multiple expression systems, each with distinct advantages for different research applications:

  Expression System	Product Code	Advantages	Considerations
  E. coli	CSB-EP308668BRJ	High yield; cost-effective; rapid production	May have challenges with proper folding
  Yeast	CSB-YP308668BRJ	Post-translational modifications; improved folding	Longer production time than E. coli
  Mammalian cell	CSB-MP308668BRJ	Native-like post-translational modifications	Higher cost; lower yields
  Baculovirus	CSB-BP308668BRJ	Suitable for large or complex proteins	Specialized expression system required

  Additionally, a biotinylated version (CSB-EP308668BRJ-B) is available with an Avi-tag for applications requiring immobilization or detection via streptavidin .

• What is the optimal method for storing and reconstituting recombinant ysdC?

  For optimal stability and activity retention of recombinant ysdC:

  Storage recommendations:

  • Store at -20°C for standard storage

  • For extended storage, maintain at -80°C

  • Avoid repeated freeze-thaw cycles

  • Working aliquots can be kept at 4°C for up to one week

  Reconstitution protocol:

  1. Briefly centrifuge the vial before opening to collect all material at the bottom

  2. Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

  3. Add glycerol to a final concentration of 5-50% (optimally 50%)

  4. Prepare small aliquots to avoid repeated freeze-thaw cycles

  5. Flash freeze aliquots in liquid nitrogen before transferring to -80°C for long-term storage

  The shelf life is approximately 6 months for liquid preparations at -20°C/-80°C and approximately 12 months for lyophilized preparations .