Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0694563 (eds)
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The thioredoxin gene (trxA) from Escherichia coli K12 has been cloned on a 3-kilobase pair PvuII fragment in a derivative of pBR325 (pBHK8). Thioredoxin protein production was amplified 150-200-fold in a strain containing pBHK8 (SK3981), with the greatest increase/cell observed after cultures reached stationary phase. A simple purification procedure, involving DEAE and AcA-54 column chromatography, yielded homogeneous protein with approximately 70% yield. The high amplification of thioredoxin in these cells (i.e. 10(6) copies/cell representing 40% of total cell protein) approaches the maximum yields seen in genetically constructed cloning vehicles (Bernard, H.U., and Helinski, D.R. (1980) in Genetic Engineering (Setlow, J. K., and Hollaender, A., eds) Vol. 2, pp. 133-167, Plenum Press, New York). This tremendous overproduction of thioredoxin protein is attributed to the high plasmid copy number observed in SK3981 (1700/cell). These results suggest a role for thioredoxin in plasmid DNA replication.
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PMID:Amplification and purification of plasmid-encoded thioredoxin from Escherichia coli K12. 638 86

Lipopolysaccharide (LPS) from Escherichia coli K12 W3100 is known to contain several glycoforms, and the basic structure has been investigated previously by methylation analyses (Holst, O. (1999) in Endotoxin in Health and Disease (Brade, H., Opal, S. M., Vogel, S. N., and Morrison, D., eds) pp. 115-154; Marcel Dekker, Inc., New York). In order to reveal dependences of gene activity and LPS structure, we have now determined the composition of de-O-acylated LPS by electrospray ionization-Fourier transform ion cyclotron-mass spectrometry (ESI-FT-MS) and identified 11 different LPS molecules. We have isolated the major glycoforms after de-O- and de-N-acylation and obtained four oligosaccharides that differed in their carbohydrate structure and phosphate substitution. The main oligosaccharide accounted for approximately 70% of the total and had a molecular mass of 2516 Da according to ESI-FT-MS. The dodecasaccharide structure (glycoform I) as determined by NMR was consistent with MS and compositional analysis. One minor oligosaccharide (5%) of the same carbohydrate structure did not contain the 4'-phosphate of the lipid A. Two oligosaccharides contained the same phosphate substitution but differed in their carbohydrate structure, one (5%) which contained an additional beta-D-GlcN in 1-->7 linkage on a terminal heptose residue (glycoform II) which was N-acetylated in LPS. A minor amount of a molecule lacking the terminal L-alpha-D-Hep in the outer core but otherwise identical to the major oligosaccharide (glycoform III) could only be identified by ESI-FT-MS of the de-O-acylated LPS. The other oligosaccharide (20%) contained an alpha-Kdo-(2-->4)-[alpha-l-Rha-(1-->5)]-alpha-Kdo-(2-->4)-alpha-Kdo branched tetrasaccharide connected to the lipid A (glycoform IV). This novel inner core structure was accompanied by a truncation of the outer core in which the terminal disaccharide L-alpha-D-Hep-(1-->6)-alpha-D-Glc was missing. The latter structure was identified for the first time in LPS and revealed that changes in the inner core structure may be accompanied by structural changes in the outer core.
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PMID:Structural analysis of oligosaccharides from lipopolysaccharide (LPS) of Escherichia coli K12 strain W3100 reveals a link between inner and outer core LPS biosynthesis. 1281 7