The first PPR protein to be described was the Saccharomyces cerevisiae mitochondrial protein Pet309, found to participate in translation of cox1 ( Manthey & McEwen, 1995 Manthey et al., 1998 Tavares-Carreon et al., 2008). Pentatricopeptide repeat (PPR) proteins are the most numerous of these. RNA metabolism plays a particularly important role in organelle gene expression ( Stern et al., 2010) and a wide array of different RNA binding proteins are found in organelles. As plant mitochondria encode no machinery to manage their own RNA expression and post-transcriptional RNA modification processes, these essential steps are totally reliant on nuclear-encoded gene products ( Binder & Brennicke, 2003). Although these represent only a small proportion of all mitochondrial proteins, their importance to mitochondrial function means that incorrect regulation of these mitochondrial genes would severely affect the whole system. 40 proteins are encoded within the mitochondrial genome, most of which encode essential subunits of oxidative phophorylation enzymatic complexes and ribosomal proteins ( Kubo et al., 2000 Notsu et al., 2002 Handa, 2003 Ogihara et al., 2005 Sugiyama et al., 2005 Tian et al., 2006 Allen et al., 2007 Kubo & Newton, 2008 Fujii et al., 2010). For example, the plant mitochondrial proteome can be estimated to consist of c. Many of these functions are carried out by enzyme complexes with both organelle-encoded and nucleus-encoded subunits, and therefore coordination of the expression of organelle and nuclear genes is a critical matter. Organellar function is essential for eukaryotic life, and depends upon activities that are maintained by proteins either internally synthesized within the organelle (organelle-encoded) or imported from cytosol (nucleus-encoded). In this review, we will summarize our current knowledge about the evolution of PPR genes, and will discuss the relevance of the dramatic expansion in the family to the functional diversification of plant organelles, focusing primarily on RNA editing. PPR proteins are sequence-specific RNA-binding proteins involved in many aspects of RNA processing in organelles. This provides a rare opportunity to study selection pressures driving a 50-fold expansion of a single gene family. 10–20 in other eukaryotic organisms, including green algae, the family has obviously greatly expanded during land plant evolution. As the number of PPR genes is generally only c. From the wealth of sequence information now available from plant genomes, the PPR protein family is now known to be one of the largest families in angiosperm species, as most genomes encode 400–600 members. We prepared 600 g of purified enzyme with a low endotoxin content of sufficient quality for therapeutical use, with a 41% overall yield in the purification process.The pentatricopeptide repeat (PPR) is a degenerate 35-amino-acid structural motif identified from analysis of the sequenced genome of the model plant Arabidopsis thaliana. After recrystallization, the enzyme was purified by anion-exchange column chromatography to remove endotoxins and by gel filtration for polishing. The crystals were directly obtained from crude enzyme with 87% yield by a crystallization in the presence of 9.0% polyethylene glycol 6000, 3.6% ammonium sulfate, and 0.18 M sodium chloride using a 100-l crystallizer. The transformants produced the enzyme, which intracellularly accumulated at 2.1 mg/ml as an active form and accounted for 43% of the total proteins in the soluble fraction by simple batch fermentation using a 500-l fermentor. Plasmid pMGLTrc03, which has a trc promoter and a spacing of 12 nucleotides between the Shine-Dalgarno sequence and the ATG translation initiation codon, was selected as the most suitable plasmid. The plasmid was optimized with a promoter and the region of the ribosome-binding site. An efficient production process for the recombinant enzyme was constructed by using the overexpression plasmid in Escherichia coli, large-scale cultivation, and practical crystallization on an industrial scale. L-Methionine gamma-lyase is a pyridoxal 5'-phosphate-dependent enzyme which has tumor selective anticancer activity.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |