Part:BBa_K2114998

pelB_aGFPnano

This part contains the anti-GFP nanobody described by Kubala et al.¹ 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^2,3 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: Expression analysis of the anti-GFP nanobody. (A) Purified pelB anti-GFP nanobody. The pelB leader sequence facilitates an easy and reliable purification at high concentrations. (B) Lysate and pellet of E. coli BL21 after over-expression of anti-GFP nanobody without pelB leader sequence. Overexpression of anti-GFP nanobody without the pelB leader sequence results in the formation of inclusion bodies that remain in the bacterial pellet and therefore decreases the purification efficiency.

The anti-GFP nanobody was 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). In comparison, the bacterial over-expression of the anti-GFP nanobody without pelB leader sequence results in low yields since a considerable amount of expressed proteins remains in inclusion bodies in the bacterial pellet and therefore it cannot be purified from the bacterial lysate (figure 2 B).

Binding kinetics

Figure 3. Binding kinetics of the anti-GFP nanobody. The interaction of the anti-GFP nanobody to His-tagged GFP was determined by biolayer interferometry. The resulting sensogram represents the complex formation and is devided into 4 sections. The section depicts the binding of the His-tagged GFP to the biosensor followed by the adjustment of the baseline in section 2. As shown in section 3 the incubation with the purified anti-GFP nanobody resulted in its association to GFP. Subsequently, the dissociation of the complex was determined in binding buffer without purified nanobody.

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. Kubala, M. H., Kovtun, O., Alexandrov, K. & Collins, B. M. Structural and thermodynamic analysis of the GFP:GFP-nanobody complex. Protein Sci. 19, 2389–2401 (2010).
2. Hamers-Casterman, C. et al. Naturally occurring antibodies devoid of light chains. Nature 363, 446–8 (1993).
3. 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).

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]