Figure 1 | Scientific Reports

Figure 1

From: Structural basis of nanobody-mediated blocking of BtuF, the cognate substrate-binding protein of the Escherichia coli vitamin B12 transporter BtuCD

Figure 1

Selection of BtuF-specific nanobodies and sequence alignment. (A) SDS-PAGE analysis of pull-down assays. His-tagged nanobodies (Nb) were immobilized on Ni-NTA beads and mixed with a 1.5 - fold molar excess of tag-less BtuF. Controls were performed by omission of nanobody (no Nb) to detect unspecific BtuF binding and a fraction of unspecifically bound BtuF was observed. Gel lanes correspond to 1 = initial mix, 2 = flow through, 3 = wash and 4 = elution. MW, marker proteins with masses indicated on the left. (B) Amino acid sequence alignment of the six nanobodies selected in pull-down assays, and the non-binder Nb12, which was a member of the second-largest family identified in panning. The three CDR regions are labeled. The secondary structure elements of Nb9 are indicated above the alignment. Cysteine positions for disulfide bond formation are numbered in green. Note the additional disulfide bond between C72 (CDR2) and C128 (CDR3) in Nb9, Nb15 and Nb10. The ESPript server 3.0 available online was used to generate the alignment48.

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