Journal: Scientific Reports
Article Title: Clumping factor A of Staphylococcus aureus interacts with AnnexinA2 on mammary epithelial cells
Figure Lengend Snippet: Recombinant AnnexinA2 interacted with ClfA35-559. ( A ) Purification of recombinant AnnexinA2. lane1: purified GST-AnnexinA2 (arrow), lane2–3: cell lysate of bacteria BL21-RIL harbouring the plasmid GST-AnnexinA2, lane4: Protein marker P7702S (BioLabs), kDa unit, lane5: The flow-through after overnight cleavage by TEV, two bands showed AnnexinA2 (36 Kda) and TEV (24 Kda) (arrow), lane6–7: Purified AnnexinA2 (arrow) after removal of the GST tag by TEV. ( B ) Purification of recombinant GST-ClfA35-559 and the complex GST-ClfA35-559/fibrinogen. Lane 1: protein marker P7702S (BioLabs), kDa unit, 2–3: cell lysate of bacteria BL21-RIL harbouring the plasmid GST-ClfA35-559, 4: purified GST-ClfA35-559 (arrow), 5: purified GST-ClfA35-559 mixed with bovine blood plasma, 6: flow-through of 5, 7: the last wash of 5, 8: the complex of GST-ClfA35-559/fibrinogen. Arrows pointing the three weak bands are fibrinogen α (63.5 Kda), β (56 Kda) and γ chain (47 Kda) and the arrow pointing to the strong band is GST-ClfA35-559. ( C ) In vitro binding assay confirmed ClfA35-559 interacted with AnnexinA2. The interaction was not dependent on whether ClfA was free or forming the complex with fibrinogen. Similarly the presence or absence of 0.1 mM Ca2+ did not influence the interaction. Lane 1: protein marker P7702S (BioLabs), kDa unit, 2: the control GST (26 Kda) (arrow) mixed with AnnexinA2 (36 Kda) (arrow), 3–4: GST-ClfA35-559 (arrow) mixed with AnnexinA2, without and with 0.1 mM Ca2+ respectively, 5: the complex GST-ClfA35-559/fibrinogen mixed with AnnexinA2, 6, 7, 8, 9: the flow-through from 2, 3, 4, 5 respectively, 10, 11, 12, 13: the first wash from 2, 3, 4, 5 respectively, 14, 15, 16, 17: the second wash from 2, 3, 4, 5 respectively, 18, 19, 20: the third wash from 2, 3, 5 respectively, 21, 22, 23, 24 (arrows): the elute from 2, 3, 4, 5 respectively. The elute in lane 21 contains GST alone whereas lane 22, 23 and 24 contain GST-ClfA35-559 and AnnexinA2 indicative of an interaction.
Article Snippet: Protein expression and purification The constructs were transformed into E. coli BL21(DE3)-RIL (Novagen).
Techniques: Recombinant, Purification, Plasmid Preparation, Marker, Flow Cytometry, In Vitro, Binding Assay