The specific enzyme (AK) is feedback inhibited by its end product either by lysine or threonine and activated by metabolite from a competing branch (Asp). The presence of lysine in the structure of CaAK (despite lysine is not part of crystallization buffer) speculate that CaAK is more sensitive to be inhibited by lysine than the threonine. The crystal structure of CaAK provides a unique view of compact cooperative tetrameric oligomers check details which yield insights into the molecular determinants for catalytic and regulatory roles of the
widespread and biotechnologically important aspartate kinase enzymes. Purified native and selenomethionine-substituted (SeMet) CaAK protein was obtained from the New York SGX Research Center for Structural Genomics (PSI TargetTrack: NYSGXRC-6204b). The protein with selenomethionine labeling was expressed in E. coli high yield (HY) media and purified by standard NYSGXRC protocol , ,  and . The HY media is prepared using following procedure. To a 2 L baffled flask containing 950 ml autoclaved Milli-Q water add one packet of M9 salts, 10 mL of mineral supplement, 1 mL of vitamin supplement, Epacadostat chemical structure 1 mL of antibiotic (kanamycin −30 mg/mL solution) and 10 mL 50% glycerol. Mix flask and allow salts to go in to solution before using. Briefly, a full-length cDNA fragment of aspartate kinase (GenBank
AE001437) was amplified by polymerase chain reaction (PCR) from C. acetobutylicum (strain ATCC 824) genomic DNA using forward (AAAATCGTAGTAACAAAGTTTGG) and reverse (CATTAAATGCATTGTATATGGATTTAACAGC) primers. The PCR product was cloned into a vector pET modified for topoisomerase directed cloning (Invitrogen) and designed to express the protein of interest followed by a C-terminal hexa-histidine
tag then was transformed into TOP10 cells. The resulting clone was grown by adding 500 mL of Luria–Bertani (LB) medium containing 500 μL of 30 mg/mL kanamycin, 25 mL of 10% glucose, and a small amount of transformed cell glycerol stock scraping to a 2 L baffled flask at 30 °C with overnight shaking (250 rpm). 10 mL of the resulting culture was added to each of six flasks containing similar culture medium for large scale expression. The cultures were subjected to shaking under similar conditions until Idoxuridine the OD595 reached to the range of ∼0.8. The protein expression was induced by adding 200 μL of 1 M isopropyl-D-thiogalactopyronoside (IPTG). After overnight vigorous shaking (250 rpm, 21 °C), the cells were pelleted by centrifugation (in 1 L spin bottles; at 6500 rpm for 10 min). The pellets were collected into 50 mL conical tubes and re-suspended in a lysis buffer (35 mL/10 g), 50 μL of protease inhibitor cocktail tablet (Sigma and 5 μL of benzonase (Novagen). The cells were lysed by repeated sonication (with intervals of cooling) followed by centrifugation (38,900 g for 30 min).