Translation targeting construct

Viral IRES elements can be useful tools to identify the sub-step in translation targeted by a given regulator (Jackson, 2005 Ostareck et al., 2001). The IRES elements listed in Table 6.1 are of particular interest, because careful biochemical and structural analyses have defined different initiation factor requirements for translation in each case (Borman and Kean, 1997 Fraser and Doudna, 2006 Hellen and Sarnow, 2001 Poyry et al, 2004). We and others have employed viral IRES-containing reporter constructs to... 

To obtain maximal protein productivity, it is necessary to construct an expression clone in which a protein coding region (open reading frame, mature region, domain, etc.) obtained from a cDNA of interest is inserted into the MCS of the pTD 1 vector. Typically, expression of the target protein at about 35-50 pg per mL of the translation reaction mixture can be obtained by using mRNA transcribed from the expression clone and the Transdirect insect cell kit. Furthermore, the expression clone can be effectively combined with other eukaryotic cell-free protein synthesis systems, such as rabbit reticulocyte lysate and wheat germ systems (tee Note 3). 

The process of translating the coded values to real values is illustrated in detail for the construction of the calibration set for product PI. The target values for each component in product PI are shown in Table 8.17, with UV-inactive components shown as shaded rows. 

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