Part:BBa_K4760007

Lpp-OmpA-SpyTag003

This is counterpart of Lpp-OmpA-SpyCatcher003(BBa_K4760005). The two parts, expressed on two kinds of OMV surfaces, would bind with high affinity to each other, resulting in OMV crosslinking. The SpyTag we used is an optimized version improved by Keeble et al.¹

Uses and Biology

To realize the in situ recovery of heavy metal ions, we chose the intermediate of outer membrane vesicle(OMV)s. To faciliate this goal, we were in demand of a means to slow OMVs' flow speed and increase equivalent surface area of the OMVs, to maximize the chance of the surface diplayed His-Tags to encounter the nickel surface. Therefore, we introduced the surface displayed SpyTag and SpyCather, to cross-link the OMVs. SpyTag and SpyCatcher are classic peptide pairs that react with each other to form an isopeptide bond. Keeble et al. ¹ improved this pair into SpyTag003 and SpyCatcher003, dramatically increasing the reacion efficiency. Here, we linked a SpyCatcher003 to a modified basic part we designed this year, Lpp-OmpA-2xEAAAAK, in which we added a rigid linker to the C-terminus of Lpp'OmpA. We believe this can help display the small-size peptides better. Our results also proved this scaffold to be fully useful in displaying this pair.

Design

The SpyTag and SpyCatcher, though unsuitable for OMV recovery, are considered quite useful for the cross-linking challenge, given their infinite affinity feature. We utilized the SpyTag003 and SpyCatcher003 pair, for their reportedly higher efficiency.¹ The part Lpp’OmpA-2xEAAAAK we introduced is expected to further function here, where we worry that the short peptide SpyTag003 might be hard to meet SpyCatcher003 displayed on the other type of OMV. The rigid linker EAAAAKEAAAAK is expected to stabilize the SpyTag003 peptide’s orientation.

Build

We built the two parts similarly as how we built Lpp’OmpA-2xEAAAAK-His10, with RE cleavage and ligation. We consider the 12 bp scar, translated into RGSA, should be of minimal influence to both peptides’ function. In order that we can detect the binding of the proteins, we added two antibody detection tags behind both peptides.

Fig. 11 Composition of Lpp’OmpA-2xEAAAAK-SpyTag003-3xFlag and Lpp’OmpA-2xEAAAAK-SpyCatcher003-HA

The two parts were separately used to transform two E.coli. BL21 strains, and the expression was verified via Western Blot.

Fig. 2 Expression verification of SpyTag003(ST) and SpyCatcher003(SC). (A) SDS-PAGE clearly showing the expression of SpyTag. (B)(C) Western blotting of SpyTag003 and SpyCatcher003.

Test

We then tested whether the displayed SpyTag003 and SpyCatcher003 can still react and help cross-link the OMVs. We prepared the protein-of-interest-containing OMVs through ultracentrifugation, and mixed them at different dilution multiples. Since the production of SpyCatcher003 was more unstable and overall short in supply, we decided to reduce the influence of expression instability through another group of mixing with higher concentration of SpyTag003 OMVs used. The reaction continued overnight, until we tested the OMVs’ diameter distribution with NTA and the protein reaction with Western blotting.

Fig. 13 Verification of SpyTag003/SpyCatcher003 containing OMVs’ binding affinity.

Reference

1.Keeble AH, Turkki P, Stokes S, et al. Approaching infinite affinity through engineering of peptide-protein interaction. Proc Natl Acad Sci U S A. 2019;116(52):26523-26533.

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 463
  Illegal BamHI site found at 475
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 55
  
• 1000
  COMPATIBLE WITH RFC[1000]