Part:BBa_K2114998

pelB_aGFPnano

This part contains the anti-GFP nanobody described by Kubala et al. and the pelB leader sequence in the N-terminal section.

Usage and Biology

The discovery of camelid heavy-chain antibodies and their subsequent modification to single-domain antibodies also called nanobodies[1,2] provide researchers with a wide range of tools including labeling methods for imaging, receptor modulation or therapeutic agents. They exhibit a small size of only 15 kDa and their easy structure enables a high-efficiency production in bacterial strains such as E. coli. The anti-GFP nanobody represents an established and well characterized variant of those proteins. In order to directly utilize their functionality, the purification of the over-expressed nanobody provides the advantage of acquiring a highly concentrated protein solution. The BioBrick in the iGEM registry BBa_K929104 contains the full sequence of the anti-GFP nanobody. However, the purification of the nanobody from a bacterial lysate requires its export into the periplasmatic space in order to avoid inclusion bodies and provide an oxidative environment which promotes the formation of the characteristic disulfide bond. To avoid the formation of inclusion bodies and increase the yield of purified nanobodies the pelB leader sequence was included at the N-terminal section of the nanobody. This sequence facilitates the export of the protein into the periplasmatic space. The nanobody can be expressed in standard expression vectors such as pGEX or pET in an appropriate bacterial strain.

Figure 1. Construction of the improved pelB anti-GFP nanobody. The anti-GFP nanobody was amplified with primers carrying extensions for the pelB leader sequence. The resulting fragments were assembled by Gibson assembly.

Characterization

Bacterial expression

Figure 2. Analysis of the overexpressed anti-GFP nanobodies by SDS-PAGE.(A) Purified pelB anti-GFP nanobody. (B) Lysate and pellet of E.coli BL21 after overexpression of anti-GFP nanobody without pelB leader sequence.

The anti-GFP nanobody can be expressed in pET303 including a C-terminal 10xHis-tag in the E. coli strain BL21 after induction with IPTG for 3 hours at 37 °C. The lysate was purified using nickel columns and analysed by SDS-PAGE and coomassie staining to verifiy the expected size of 17 kDa (figure 2 A).

Binding kinetics

Binding kinetic of purified anti-GFP nanobody to 10xHisGFP

The evaluation of the binding properties of the anti-GFP nanobody was performed with the Octet RED96 system based on biolayer interferometry. The biosensors were loaded with His-tagged GFP at a concentration of 1.25 µg/mL. After baseline adjustment the association rate ka and the dissociation rate kd of the anti-GFP nanobody at a concentration of 4.8 µg/mL were measured. The resulting sensogram contained intereference patterns over time and was analyzed by the supplemented software. The dissociation constant KD was determined at 1.57 nM.

References

1. Hamers-Casterman, C. et al. Naturally occurring antibodies devoid of light chains. Nature 363, 446–8 (1993).
2. Muyldermans, S. & Lauwereys, M. Unique single-domain antigen binding fragments derived from naturally occurring camel heavy-chain antibodies. J. Mol. Recognit. 12, 131–140 (1999).
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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 87
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 54
  
• 1000
  COMPATIBLE WITH RFC[1000]