Cloning, expression optimization, purification, crystallization, initial characterization and X-ray diffraction analysis

Deinococcus radiodurans is a bacterium with extreme resistance to desiccation and radiation. The resistance mechanism is unknown, but an efficient reactive oxygen species (ROS) scavenging system and DNA-repair and DNA-protection mechanisms are believed to play important roles. Here, the cloning and smalland medium-scale expression tests of a novel dye-decolourizing peroxidase from D. radiodurans (DrDyP) using three different Escherichia coli strains and three different temperatures in order to identify the optimum conditions for the expression of recombinant DrDyP are presented. The best expression conditions were used for large-scale expression and yielded ~10 mg recombinant DrDyP per litre of culture after purification. Initial characterization experiments demonstrated unusual features with regard to the haem spin state, which motivated the crystallization experiment. The obtained crystals were used for data collection and diffracted to 2.2 A - resolution. The crystals belonged to the trigonal space group P31 or P32, with unit-cell parameters a = b = 64.13, c = 111.32 A - , and are predicted to contain one DrDyP molecule per asymmetric unit. Structure determination by molecular replacement using previously determined structures of dye-decolourizing peroxidases with ~30% sequence identity at ~2 A- resolution as templates are ongoing.publishersversionpublishe

Phenotypic variation in patients heterozygous for familial defective apolipoprotein B (FDB) in three European countries

A glutamine-for-arginine substitution at amino acid position 3500 of apolipoprotein B (apo B) causes synthesis of LDL with reduced binding affinity to the LDL receptor (LDLR). The associated clinical syndrome has been named familial defective apolipoprotein B- 100 (FDB). In 205 FDB patients from Germany (n = 73). The Netherlands (n = 87), and Denmark (n = 45), we tried to assess determinants of variation in lipid concentrations. Besides age, sex, and geographic origin, variation in the LDLR gene was the most powerful determinant of variation in total cholesterol and LDL cholesterol levels. Polymorphic variation in the LDLR gene (SfaNI, exon 2; Nco I, exon 18) was associated with total cholesterol (TC) and LDL cholesterol (LDL-C) variation in women (SfaNI: P = .04 and .03 for TC and LDL-C, respectively; Nco I; P = .003 and .006, respectively), whereas the Ava II (exon 13) and the Pvu II (intron 15) polymorphisms were not. Combined information from all three LDLR exon polymorphisms showed that subjects with at least one S + A + N + allele had 13% to 20% higher TC than non-S + A + N + subjects (P = .02 [TC, men]; P = .01 [LDL-C, men]; P = .005 [TC, women]; and P = .004 [LDL-C, women]) and, together with age and geographic origin, accounted for 20% (women) and 19% (men) of the variation in LDL-C. The expected association of the apo E genotypes (e3e2, e3e3, and e3e4) with cholesterol concentrations was seen in S + A + N + but not in non-S + A + N + subjects and in P-P- but not in P + P + or P + P- subjects. With regard to clinical expression, FDB patients had lower TC and LDL-C levels and a lower prevalence of cardiovascular disease than 101 Danish patients with familial hypercholesterolemi