Part:BBa_K1590009

Spermidine/putrescine-binding periplasmic protein

Escherichia coli PotD sequence, encoding Spermidine/putrescine-binding periplasmic protein.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 429
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 148
  
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

For semen detection our main target ligand is the polyamine spermidine which is found in relatively high concentrations in seminal fluid (5-15 mM). Spermidine is made from another polyamine called putrescine and is the precursor of spermine. Regulation of polyamine synthesis, degradation and transport is tightly controlled in bacteria. In E. coli, two of three identified transport systems are ABC transporters composed of a periplasmic binding protein, a pair of transmembrane proteins and a membrane protein possessing ATPase catalytic activity. Out of these three components, we were interested in investigating the periplasmic binding protein PotD which specifically binds spermidine. The fact that this protein is responsible for transportation of spermidine and lacking in enzymatic activity meant that it was an ideal candidate for use in FluID for semen detection. S

6-His - PotD was purified by Ni-affinity chromatography and subsequently blotted against.

Figure 1: Chromatogram showing the purification profile of the his-tagged bacterial protein, PotD. The fractions corresponding to the two peaks observed on the chromatograph were further analysed on SDS page gel. B) Coomassie Stain of the purified fractions (A12 + B12). 10 µl of each fraction was mixed with 10 µl of 2x Laemmli buffer and loaded onto an SDS gel which was ran for 1 hour at 100V and then stained. These were then western blotted to confirm the presence of our target protein PotD-His.

Figure 2: Samples from the SEC were ran on a 12.5% SDS-PAGE gel and then transferred to a nitrocellulose membrane and probed with an anti-His antibody. The band detected in the blot corresponds to the expected size of PotD of 37kDa.