Method: The N-terminal region of PadA binds to platelets, and, therefore, several N-terminal fragments were cloned and expressed in E. coli BL21 (DE3) cells harboring the appropriate vectors for each fragment. These N-terminal fragments were purified using a three-step protocol. First, the histidine-tagged proteins were purified over a nickel affinity column, then purified by anionic exchange, and finally polished over size exclusion chromatography. Highly purified (>99%) proteins were concentrated under nitrogen gas and then screened for crystallization conditions using the hanging drop method. Several hits were obtained, and these crystallization conditions were further optimized to grow diffraction quality crystals.
Result: Among several fragments of PadA that were highly purified, only one fragment of PadA readily crystallized. X-ray diffraction data was collected on an RU-H3R generator operating at 50 V and 100 A, fitted with an RAXIS IV image plate detector. Data collected and scaled indicated that the PadA crystals adopted a trigonal space group (either P321, P3121, or P3221) with the following cell parameters: a = 154.46, b = 154.46, c = 235.62, and α=β= 90 with γ= 120.
Conclusion: The functional region of Streptococcus gordonii PadA implicated in binding to the platelet integrin GPIIbIIIa has been crystallized, and structure solution is in progress.
Support: NIDCR-5T32DE017607 DART
Keywords: Adherence and colonization, Bacterial, Infection, Plaque and Structure