BioNMR in Drug Research by O. Zerbe

By O. Zerbe

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Low et al. demonstrated protein ligation by introducing a cleavable thiolcontaining auxiliary group, 1-phenyl-2-mercaptoethyl, at the alpha-amino group of a chemically synthesized peptide, which is removed upon protein ligation [64]. Unfortunately, this modification at the N-terminus could be difficult to introduce into proteins which are prepared from bacterial expression systems, and hence its use could be limited to situations where the C-terminal peptide can be chemically synthesized. Nevertheless, the removal auxiliary approach presents an opportunity for segmental isotope labeling regardless of the primary sequence.

Cerevisiae, no budding occurs, and the yeast only reproduces by means of fission and by spores [98]. Two types of expression vectors have been developed for S. pombe. The chromosomal integration type of vector maintains the foreign gene stably in the chromosome [99], and the episomal vector replicates autonomously in yeast cells [100]. Some mammalian promoters like the human chorionic gonadotropin and CMV promoters are functional in S. pombe [101]. The fission yeasts possess many similar features to mammalian cells.

Cerevisiae and other yeast species. A wide range of mammalian proteins have been expressed in S. pombe. In a successful example, the human lipocortin I comprised 50% of the total soluble proteins in yeast cells and showed high activity, indicating that the post-translational modifications were mammalian-like [104]. Membrane proteins including cytochrome P450 were expressed at ten times the levels of those in other yeast systems [105]. Also, GPCRs have been expressed in S. pombe, where the human dopamine D2 receptor was correctly inserted into the yeast cell membrane and demonstrated expression levels three times those of S.

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