Isopropyl beta d thiogalactopyranoside iptg

Preparation of recombinant protein has been described previously (9) (link). Briefly, genomic DNA was isolated from the M. avium subsp. paratuberculosis JTC303 strain, and the DNA encoding MAP1305 was amplified by PCR. After purification of the PCR product, it was ligated into the pET-22b(+) vector digested with the NdeI and XhoI enzymes. Expression of MAP1305 in transformed BL21(DE3) cells was induced with isopropyl-beta- D-thiogalactopyranoside (IPTG) (Promega, Madison, WI). The soluble MAP1305 was extracted after cell disruption by sonication. MAP1305 containing a C-terminal histidine tag was purified using Ni-nitrilotriacetic acid (NTA) resin (Qiagen, Chatsworth, CA). The MAP1305 was dialyzed five times in 10 mM phosphate-buffered saline (PBS) (pH 7.2) using a Slide-ALyzer dialysis cassette with a 3-kDa cutoff (Pierce, Rockford, IL). After dialysis, contaminating endotoxin was removed using Detoxi-Gel Affinity Pak columns (Pierce, Rockford, IL). MAP1305 was incubated with endotoxin removal resin overnight to remove LPS and concentrated with a Centricon device (2,000-Da cutoff; Millipore). Also, endotoxin was assayed under endotoxin-free experimental conditions using a Limulus amebocyte lysate pyrogen kit (Biowhittaker, Walkersville, MD). The experiments were conducted according to the manufacturer's protocol. The quantity of endotoxin in the MAP1305 was ≤0.01 ng/mg.