Sequence Of Chromatographic Steps Research Articles

Functional expression and purification of cytokinin dehydrogenase from Arabidopsis thaliana (AtCKX2) in Saccharomyces cerevisiae

Cytokinin dehydrogenase (CKX) is responsible for regulating the endogenous cytokinin content by oxidative removal of the side chain and seven distinct genes, AtCKX1 to AtCKX7, code for the enzyme in Arabidopsis thaliana. The recombinant enzyme AtCKX2 was produced in Saccharomyces cerevisiae after expressing the corresponding gene from a plasmid (pDR197) or following chromosomal integration, under either the constitutive promoter PMA1 or the inducible promoter GAL1. The recombinant protein was purified from yeast culture media using a sequence of chromatographic steps. The purified enzyme had a molecular mass of 61 kDa and a typical flavoprotein spectrum. The specific activity of the enzyme was 87.8 µkat g−1, with isopentenyladenine as a substrate and 2,3-dimethoxy-5-methyl-p-benzoquinone as an electron acceptor. The pH optimum lay between 7.0 and 8.0, depending on the electron acceptor used. AtCKX2 reacts both with isoprenoid and aromatic cytokinins, the activity with isoprenoid cytokinins being two to three orders of magnitude higher. AtCKX2 prefers p-quinones and the synthetic dye 2,6-dichlorophenol indophenol as electron acceptors, although low reactivity with oxygen can also be observed. This study presents the first purification and characterization of the enzyme from Arabidopsis thaliana.

High-throughput process development for recombinant protein purification

Methods development in chromatographic purification processes is a complex operation and has traditionally relied on trial and error approaches. The availability of a large number of commercial media, choice of different modes of chromatography, and diverse operating conditions contribute to the challenging task of accelerating methods development. In this paper, we describe a novel microtiter-plate based screening method to identify the appropriate sequence of chromatographic steps that result in high purities of bioproducts from their respective culture broths. Protein mixtures containing the bioproduct were loaded on aliquots of different chromatographic media in microtiter plates. Serial step elution of the proteins, in concert with bioproduct-specific assays, resulted in the identification of "active fractions" containing the bioproduct. The identification of a successful chromatographic step was based on the purity of the active fractions, which were then pooled and used as starting material for screening the next chromatographic dimension. This procedure was repeated across subsequent dimensions until single band purities of the protein were obtained. The sequence of chromatographic steps and the corresponding operating conditions identified from the screen were validated under scaled-up conditions. Various modes of chromatography including hydrophobic interaction, ion exchange (cation and anion exchange) and hydrophobic charge-induction chromatography (HCIC), and different operating conditions (pH, salt concentration and type, etc.) were employed in the screen. This approach was employed to determine the sequence of chromatographic steps for the purification of recombinant alpha-amylase from its cell-free culture broth. Recommendations from the screen resulted in single-band purity of the protein under scaled-up conditions. Similar results were observed for an scFv-beta-lactamase fusion protein. The use of a miniaturized screen enables the parallel screening of a wide variety of actual bioprocess media and conditions and represents a novel paradigm approach for the high-throughput process development of recombinant proteins.

The Sleep Inducing Factor Oleamide Is Produced by Mouse Neuroblastoma Cells

Cis-9,10-octadecenoamide (oleamide) was isolated from the cerebrospinal fluid of sleep-deprived mammals and shown to induce sleep in rats. The enzyme catalyzing the hydrolysis of the amide bond of oleamide as well as of anandamide, the putative endogenous ligand of cannabinoid receptors, was purified from rat liver, cloned, shown to be expressed also in brain and named fatty acid amide hydrolase (FAAH). The enzymatic synthesis of oleamide from oleic acid and ammonia by rat brain microsomes has been also described. However, no evidence has been reported so far on the neuronal origin of oleamide, necessary in order to postulate for this compound a role as a neuromodulator. Here we show for the first time that oleamide is produced by a neuronal cell type and that its biosynthesis in intact neurons is not likely to occur through the direct condensation of oleic acid and ammonia. A lipid metabolite was extracted and purified from mouse neuroblastoma N18TG2cells through a sequence of chromatographic steps and characterized as oleamide by means of gas chromatography/electron impact mass spectrometry (GC/EIMS). The amount of oleamide, as estimated by GC analyses carried out in comparison with known amounts of synthetic oleamide, was 55.0±09.5 pmols/107cells, compared to less than 0.7 pmol/107cells for anandamide in the same cells. When N18TG2cells were prelabeled with [14C]oleic acid and the lipids extracted and purified, a radioactive component with the same chromatographic behavior as oleamide was found whose levels: (1) were not significantly influenced by stimulation with ionomycin; (2) were slightly increased by incubation with FAAH inhibitor phenyl-methyl-sulphonyl-fluoride (PMSF); (3) appeared to correlate with [14C]oleic acid incorporation into phospholipids but not with free [14C]oleic acid levels. N18TG2cell membranes were shown to contain an enzymatic activity catalyzing the synthesis of oleamide from oleic acid and ammonia. This activity was inhibited by FAAH selective inhibitors arachidonoyltrifluoromethylketone and methylarachidonoylfluorophosphonate, as well as by an excess of anandamide, and by PMSF at the same concentration which increased oleamide formation in intact cells. These data suggest that a FAAH-like enzyme working “in reverse” may be responsible for the formation of oleamide in cell-free preparations but not in whole cells.

Purification and characterization of a high molecular weight histone deacetylase complex (HD2) of maize embryos.

The dynamic state of core histone acetylation is maintained by histone acetyltransferases and deacetylases. In germinating maize embryos, four nuclear histone deacetylases can be distinguished. From a chromatin fraction prepared at 72 h after start of embryo germination, we have purified the nuclear histone deacetylase HD2 to homogeneity. Using a sequence of chromatographic steps, we achieved the purification of an enzymatically active high molecular weight protein complex with an apparent molecular mass of 400 kDa, as determined by gel filtration chromatography. The purified enzyme was characterized in terms of enzymatic and kinetic properties, and sensitivity to several histone deacetylase inhibitors. In SDS-polyacrylamide gels, HD2 split into three polypeptides of 45, 42, and 39 kDa, suggesting that the native enzyme is a multimer-protein complex. Electrophoresis under nondenaturing conditions in combination with second dimension SDS-gel electrophoresis indicated that all three protein components of the HD2 complex were enzymatically active. Polyclonal antibodies against each of the three polypeptides were raised in rabbits. Each antiserum reacted with all three polypeptides on Western blots, suggesting that p45, p42, and p39 are highly homologous. This homology was confirmed by amino acid sequencing of peptides generated from each of the three HD2 components.