PubMed · 8713888
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P Littlebury. 1996. Pouring linear and buffer-gradient sequencing gels.. https://doi.org/10.1385/0-89603-402-x%3A389
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Phosphatidate phosphatase is an important enzyme in glycerolipid biosynthesis, but difficult to purify. A purified preparation of N-ethylmaleimide-sensitive phosphatidate phosphatase from the yeast Saccharomyces cerevisiae was obtained by a five-step protocol, using chromatography on DE-53/DEAE FF, Affi-Gel Blue, hydroxyapatite, Mono-Q, and Superdex 200. A protease-deficient yeast strain gave preparations similar to those of the wild-type strain. In exclusion chromatography, the enzyme activity of all preparations eluted at approximately the same position as albumin. However, the behavior on SDS/PAGE differed considerably among preparations, suggesting a multimeric subunit structure or degradation during purification. A 35-kDa and a 40-kDa protein band which coincided with activity were found in all preparations. Glycerol in the buffers could be excluded without rapid loss of enzyme activity, and Tris could be substituted for ammonium bicarbonate, while at least 0.6% sodium cholate in the buffers was essential.
Co-expression of di-alpha-globin and beta-globin in Escherichia coli in the presence of exogenous heme yielded high levels of soluble, functional recombinant human hemoglobin (rHb1.1) and, under certain conditions, large amounts of insoluble globin protein. Insoluble rHb1.1 accumulated in large, amorphous inclusion bodies visible by electron microscopy. The half-life of soluble rHb1.1 in E. coli, measured by pulse-chase experiments, was at least 11 h for each globin subunit. The in vivo half-life for insoluble globin was about fivefold shorter than that for soluble rHb1.1. We expressed significant amounts of each subunit, di-alpha-globin and beta-globin, independently with exogenous heme. The half-life of the soluble subunits alone was approximately 1 and 4 h, respectively, shorter than when they were expressed together as rHb1.1. Individually, the insoluble di-alpha-globin subunit had a half-life of just under 1 h when exogenous heme was added, but under 20 min when exogenous heme was not provided. The greater persistence of insoluble subunits in the presence of heme indicated that heme may stabilize the insoluble globin protein. The soluble rHb1.1 persistence in the E. coli cytoplasm during long periods of stationary phase growth indicated that once assembled, rHb1.1 is extremely resistant to proteolysis.
For the purposes of the antisense strategy oligodeoxyribonucleotides can be protected against serum and cell nuclease digestion by tagging at their 3'-end with a sequence naturally forming a very stable hairpin, d(GCGAAGC). This nuclease-resistant hairpin is also known for its high thermostability. We demonstrate in this study that attachment of d(GCGAAGC) at the 3'-end of an oligodeoxyribonucleotide does not hinder hybridization of the 5'-part of this oligonucleotide to a complementary DNA strand. Moreover, the hairpin is in equilibrium between a folded and an open structure, with an energy minimum in favor of pairing if it is possible, even with mismatches.