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Download BioNMR in Drug Research by O. Zerbe PDF

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By O. Zerbe

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In particular, in the RTS 100 HY system the exonuclease activity is reduced, so that the direct use of PCR-generated DNA templates is possible. To facilitate the generation of the PCR templates there is a special product available (linear template kit), which introduces all regulatory elements [T7-promotor, gene10 enhancer sequence and the Shine-Dalgarno (RBS) sequence]. Consequently, RTS 100 HY can be used for the rapid evaluation of the best template, without spending time with cloning, and for optimization of the reaction conditions (e.

A naturally occurring split intein, DnaE from Synechocystis sp. PCC6803, was first successfully used for cyclization. However, similarly to the IPL/EPL approach, a mixture of linear and circular forms is obtained, presumably because of hydrolysis of an intermediate [73, 75]. On the other hand, artificially split inteins such as PI-PfuI, DnaB, and the RecA intein have been successfully applied for in vivo cyclization, and only circular forms were observed [80–82], suggesting that the circular permutation approach is more suitable for cyclization.

Pastoris has also been used as a host for expression of GPCRs [109]. The mouse 5-HT5A receptor and the human b2-adrenergic receptor were fused to the prepropeptide sequence of the S. cerevisiae a-factor, which enhanced the expression levels by a factor of three. Multiple chromosomal integrations further improved the expression twofold. In the case of the b2-adre- 23 24 1 Modern Methods for the Expression of Proteins in Isotopically Enriched Form nergic receptor, addition of the antagonist alprenol to the culture medium increased the number of specific binding sites.

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