Rodionov et al. 10.1073/pnas.0609279104. |
Fig. 8. (A) Predicted inositol-1-phosphate cytidylyltransferases (IMPCT) domain similarities in the SWISSPROT database. (B) Multiple alignment of IMPCT and other characterized proteins from the sugar nucleotidyltransferases family.
Fig. 9. (A) Predicted DIP synthase domain similarities in the SWISSPROT database. (B) Multiple alignment of the predicted DIP synthases and other characterized proteins from the CDP-alcohol phosphatidyltransferase class I family. (C) Hydropathy profile of the putative DIP synthase DipB from T. maritima.
Fig. 10. (A-C) 31P NMR (202.2 MHz) spectra of the phosphodiester region of E. coli BL21-DIP lysed cells incubated for 3 h with 3 mM L-I-1-P and 6 mM CTP at 60°C (A), 75°C (B), and 85°C (C). (D) Arrhenius plot of DIP production at the three temperatures.
SI Materials and Methods
Bacterial Strains, Plasmids, and Reagents.
E. coli strains BL21, BL21/DE3 (Stratagene, La Jolla, CA), and DH5a, TOP10 (Invitrogen, Carlsbad, CA) were used for gene cloning and protein overexpression. For expression of all genes in E. coli, a pET-derived vector containing the T7 promoter, His6 tag, and TEV-protease cleavage site (36) was used. Enzymes for PCR and DNA manipulations were from New England Biolabs (Beverly, MA). Plasmid purification kits were from Promega (Madison, WI). PCR purification kits and nickel-nitrilotriacetic acid resin were from Qiagen (Valencia, CA). Oligonucleotides for PCR and sequencing were synthesized by Sigma-Genosys (The Woodlands, TX). L-I-1-P was generated enzymatically from recombinant A. fulgidus IPS as described previously (15). All other chemicals, including the assay components, glucose-6-P, CTP, NADH, NADP+, ATP, UTP, GTP, and 2-deoxy-CTP, were purchased from Sigma-Aldrich (St. Louis, MO).Cloning Overexpression and Purification of T. maritima IMPCT.
The predicted full-size gene dipA (TM1418a) was PCR-amplified by using T. maritima MSB8 genomic DNA (kindly provided by Scott Lesley, Joint Center for Structural Genomics, Genomics Institute of the Novartis Research Foundation, San Diego, CA) and oligonucleotide primers ggggtcaTGAgagaggccattgtgctggctt and ggggtcgacTCAgcatgccactccctcctttatctt, introducing restriction sites BspHI at the 5' end and SalI at the 3' end (underlined). The produced PCR product of 741 bp was cloned into the pET-derived vector digested by NcoI and SalI, and its entire DNA sequence was verified. The recombinant protein was overexpressed as an N-terminal His6-tag fusion using E. coli BL21 (DE3) expression strain and partially purified from 50 ml of culture by using a standard Ni-NTA agarose minicolumn protocol. Briefly, cells were grown in LB medium containing 100 mg/liter ampicillin and induced by 0.8 mM IPTG for 3 h. Harvested cells were resuspended in 20 mM Hepes buffer (pH 7) containing 100 mM NaCl, 0.03% Brij 35, and 2 mM 2-mercaptoethanol supplemented with 2 mM PMSF and a protease inhibitor mixture (Sigma-Aldrich). Lysozyme was added to 1 mg/ml, and the cells were lysed by freezing-thawing followed by sonication. After centrifugation at 18,000 ´ g, the Tris·HCl buffer (pH 8) was added to the supernatant (50 mM, final concentration), and it was loaded onto a Ni-NTA acid agarose minicolumn (0.2 ml). After washing with the starting buffer containing 1 M NaCl and 0.3% Brij-35, bound proteins were eluted with 0.3 ml of the starting buffer containing 250 mM imidazole. Protein size, expression level, distribution between soluble and insoluble forms, and extent of purification were monitored by SDS/PAGE.