Wheat germ lysates

Before embarking on a particular expression strategy it is useful to have some information regarding the hands-on time required before the first results can be obtained. The fastest turnaround times are achieved with cell lysates, such as the reticulocyte and wheat germ lysates, which only need to be primed with in vitro synthesized RNA (or DNA in the case of a coupled transcription/translation system). These systems potentially provide answers within a few hours but are rarely used because of the small quantities that they yield. Plasmid-based systems used for expression in E. coli, yeast, and in combination with the vaccinia-T7 vector system are relatively fast as well. These strategies require subcloning of the gene(s) of interest into a plasmid vector, which is then transfected into the... 

The posttranslational import of yeast prepro-alpha factor into the ER was stimulated by the addition of a yeast postribosomal supernatant. This assay was used to purify two cytosolic members of the yeast Hsp70 family (Chirico et al., 1988). Similarly, while the import of a preprotein translated from wheat germ lysate could not be imported into yeast mitochondria, addition of yeast cytosolic Hsp70 (as well as another unidentified component) could restore this defect (Murakami etal., 1988). 

FIGURE 1. Insertion of LHCP into the thylakoids in the correct orientation following transport into the chloroplast by the transit peptide pS. RNA was transcribed from genes coding for pLHCP and the hybrid protein was translated in a wheat germ lysate, and 5ie products (lanes 1, panel A, pLHCP panel B, hybrid) were incubated with chloroplasts in an import reaction, llie total membrane-associated proteins are shown before (lanes 2) and after (lanes 3) trypsin treatment. 

Translation extracts Rabbit reticulocyte lysates (RRL) (Promega), wheat germ (WG) extract (Promega), bacterial S30 extract (Promega). Extracts from Krebs-2 cells were prepared as described (Svitkin and Sonenberg, 2004). 

Preparation of Krebs-2 translation extracts Krebs-2 extracts are an ideal system to screen for compounds that inhibit translation because they faithfully recapitulate the cap dependency and the cap-poly(A) synergism associated with eukaryotic mRNA translation (Svitkin and Sonenberg, 2004), unlike standard rabbit reticulocyte lysates (RRL) (Borman et al., 2000). Furthermore, the translation of many types of IRESes is supported in Krebs-2 extracts. The use of commercially available translation competent extracts prepared from RRL, wheat germ, and E. coli is extremely useful in assessing selectivity of inhibitors identified in primary screens. 

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). 

In a further experiment we assayed for the presence of a cap structure on the mRNAs for both Inhibitors I and II by competitive inhibition by 7-methyl-guanosine 5 -monophosphate (m G p) of the in vitro translation of these messengers. Concentrations of 40 pM m G p inhibited by 50% the in vitro translation of total tomato leaf poly(A)" " mRNA (Fig. 7A). This level is 40-fold lower than that required to similarly inhibit rabbit globin mRNA translated in a rabbit reticulocyte lysate (17) and 4-fold lower than that required to inhibit the same mRNA in a wheat germ system (18). It was of interest that the translation of Inhibitor I is inhibited to 50% by 20 pM m G p while 50% inhibition of Inhibitor II requires less than 10 pM (Fig. 7B). The basis of this difference is not understood but... 

Different in vitro translation systems are used depending on whether the mRNA is of prokaryotic or eukaryotic origin. For eukaryotic mRNA, translation kits from reticulocyte lysates or wheat germ extract are recommended, and for prokaryotic mRNA, the E. coli 30 S supernatant, all of which are commercially available. These translation kits can be supplemented with [3H]-leudne or... 

Transcription, translation, and import reactions. The procedures used have been previously described (1). A wheat gene (9) was inserted 3 to the SP6 promoter and the transcripts produced using SP6 polymerase were translated in either a wheat germ or reticulocyte lysate. After import reactions, the pea chloroplasts were either left untreated and hypotonically lysed, or treated with thermolysin to remove proteins on the exterior. Membrane and soluble fractions were separated by centrifugation and analyzed by SDS-PAGE. 

FIGURE 1. Products of in-vitro translation in reticulocyte lysate (a), in wheat germ extract (b) or in S-30 extract from E.,Coti (c) primed with total RNA (a,1), ptRNA (a, 2 b, 2 c, 1) or poly-A RNA (a,3 b,1) from the wild-type or primed with poly-ARNA (a,6 b,3) or ptRNA (c,2) from the mutant followed by immunoprecipitation with CMp15.2 1 (a), CMXVII 3G4 (b) or CMpChl -PI 1 (c). 

FIGURE 2. Products from pGEM-SUII (tomato)(a) or pGEM-SUIII (barley)(b) after coupled in-vitro transcription/translation and SDS-PAGE. Translation was performed either in reticulocyte lysate (a,1 and 2 b,3) or in wheat germ extract (b, 2 and 4-6). For details see text. 

The hybrid arrest of in vitro translation using some rabbit reticulocyte lysate preparations may not be reliable without added RNase H (41). Freshly prepared lysates are reported to contain 1-2% of the level of RNase H present in actively dividing cells, an amount sufficient to achieve near 100% cleavage of hybrid mRNAs (39). In contrast to some reticulocyte lysates, the wheat germ cell-free translation system apparently contains a sufficient amount of endogenous RNase H activity. 

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