Methods for Manipulation of Transferrin-Binding Proteins

DeWinter, Leanne M.; Schryvers, Anthony B.

doi:10.1385/1-59259-148-5:109

Cited by 1 publication

(1 citation statement)

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“…The verified recombinant plasmids were transformed into E. coli strain C41 (DE3) [31] and the resulting colonies were tested for expression by the solid-phase binding assay and affinity-capture assays [32]. The inserts were identified by colony PCR and confirmed by sequencing.…”

Section: Production Of Recombinant Tbpa Proteinsmentioning

confidence: 99%

Steric and allosteric factors prevent simultaneous binding of transferrin-binding proteins A and B to transferrin

Silva

Calmettes

et al. 2012

Biochemical Journal

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The ability to acquire iron directly from host Tf (transferrin) is an adaptation common to important bacterial pathogens belonging to the Pasteurellaceae, Moraxellaceae and Neisseriaceae families. A surface receptor comprising an integral outer membrane protein, TbpA (Tf-binding protein A), and a surface-exposed lipoprotein, TbpB (Tf-binding protein B), mediates the iron acquisition process. TbpB is thought to extend from the cell surface for capture of Tf to initiate the process and deliver Tf to TbpA. TbpA functions as a gated channel for the passage of iron into the periplasm. In the present study we have mapped the effect of TbpA from Actinobacillus pleuropneumoniae on pTf (porcine Tf) using H/DX-MS (hydrogen/deuterium exchange coupled to MS) and compare it with a previously determined binding site for TbpB. The proposed TbpA footprint is adjacent to and potentially overlapping the TbpB-binding site, and induces a structural instability in the TbpB site. This suggests that simultaneous binding to pTf by both receptors would be hindered. We demonstrate that a recombinant TbpB lacking a portion of its anchor peptide is unable to form a stable ternary TbpA–pTf–TbpB complex. This truncated TbpB does not bind to a preformed Tf–TbpA complex, and TbpA removes pTf from a preformed Tf–TbpB complex. Thus the results of the present study support a model whereby TbpB ‘hands-off’ pTf to TbpA, which completes the iron removal and transport process.

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Section: Production Of Recombinant Tbpa Proteinsmentioning

confidence: 99%