Ptyb21 protein expression plasmid

Wild-type S. flexneri corresponds to the serotype 2a 2457T strain originally isolated in 1954 [27 (link)]. The S. flexneri Spa47 null strain was engineered by Abdelmounaaïm Allaoui as described by Jouihri et al [28 (link)]. Rabbit polyclonal antibodies against IpaC and IpaD and the pWPsf4 expression plasmid were generous gifts from William Picking and Wendy Picking (University of Kansas). E. coli strains and 2x ligation mix were from Novagen (Madison, WI). Restriction enzymes, the pTYB21 protein expression plasmid, polymerase chain reaction (PCR) buffer, Phusion High-Fidelity polymerase, and chitin resin were purchased from New England Biolabs (Ipswich, MA). Oligonucleotide primers and the synthesized spa47 gene were from Integrated DNA Technologies (Coralville, IA). The N-terminal GFP pWPsf4 vector was designed in house and synthesized by General Biosystems (Morrisville, NC). The Superdex 16/600 size exclusion and 5-ml Q FF columns were purchased from General Electric (Pittsburgh, PA). ATP was from Sigma-Aldrich, and α-³²P-ATP was from PerkinElmer Life Sciences. Dithiothreitol (DTT) and ampicillin were from Gold Biotechnology (St. Louis, MO). Defibrinated sheep red blood cells were from Colorado Serum Company (Denver, CO). All other solutions and chemicals were of reagent grade.