Reticulocyte lysate protein synthesis

ARCAs are incorporated into RNA exclusively in the correct orientation to an extent that is similar to the standard cap (see previously), which makes them potentially useful compounds in terms of increasing translational efficiency when incorporated into RNA. Similarly, they should be effective for inhibiting protein synthesis as free analogs. To test the influence of the ARCAs on protein synthesis in vitro, we use the microccocal nuclease treated rabbit reticulocyte lysate system (RRL system) optimized for cap-dependent translation (Cai et al., 1999). Highly cap-dependent translation is achieved at 100 mM potassium acetate and 1.4 mM magnesium chloride. 

Protein synthesis inhibition. Chromatographic fraction of the dried seed, in cell culture, was active on reticulocyte lysate of rabbits, inhibitory concentrationjf, 15.25 ng/ mL vo84. 

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). 

Fig. 10. Cell-free synthesis of t-PA glycoforms. The niRNA coding for t-PA was translated in a rabbit reticulocyte lysate in the presence of dog pancreas microsomes. Microsonies were isolated posttranslationally and the translocated, glycosylated products were separated by SDS-PAGE. Translation was carried out under conditions that either prevented (lane 2) or allowed (lane 3) proper folding of the t-PA molecule, yielding enzymatically active protein that was sensitive to natural inhibitors and stimulators.

Some members of the human RNase A superfamily of proteins are known to have host defense activities (reviewed in ref. 9). These include, for example, two of the eosinophil cytotoxic granular proteins, eosinophil cationic protein (ECP), and eosinophil-derived neurotoxin (EDN) (10). Angiogenin, a protein 65 % homologous to pancreatic RNase (11,12) that was originally isolated on the basis of its angiogenic activity (13), is a potent inhibitor of protein synthesis in the rabbit reticulocyte lysate (14) and when injected into Xenopus oocytes (15). We have therefore sought to fuse RNases to MAbs to evaluate their usefulness as immunotoxins (16,17). 

The presented transcription-translation method is based on T7 RNA polymerase and the micrococcal nuclease-treated reticulocyte lysate as described by Jackson and colleagues,22 and is suitable for a semi-preparative synthesis of recombinant protein. If few reactions on an analytical 10 pi scale are to be carried out the TnT kit from Promega can be recommended. 

Clarke PA, Sharp NA, Clemens MJ. Translational control by the Epstein-Barr virus small RNA EBER-1. Reversal of the double-stranded RNA-induced inhibition of protein synthesis in reticulocyte lysates. EurJBiochem 1990 193(3)i635-641. 

For in-vitro synthesis of the compartment-specific phosphorylase forms polyadenylated RNA (mRNA) was isolated from cotyledons of either developing or germinating seeds and was used to program a reticulocyte lysate. When varying amounts of a mRNA preparation were applied to the translation mixture a decrease of the rate of total protein synthesis was observed at higher mRNA concentrations (Fig. 1). Although the mRNA concentration applied did not affect the size distribution of the translation products, for each mRNA preparation the optimal concentration was determined empirically. 

Not surprisingly, the toxin s liver toxicity is reflected by the ability of micromolar concentrations of cylindrospermopsin to kill in vitro liver cells such as rat hepatocytes and the human hepatoblastoma cell line HEP-G2. Cylindrospermopsin is known to potently inhibit cellular protein synthesis that can be measured in vitro using rabbit reticulocyte lysate. 

While the reticulocyte synthesizes equal amounts of both a and /J chains, the production of both of these proteins comes to a halt if the cells are incubated in vitro in a medium without iron (for heme synthesis) or without heme itself (Waxman and Rabinovitz, 1965 for further references see Rabinovitz et al., 1969). The decrease in globin synthesis during heme deficiency is associated with a disaggregation of polysomes to 80 S monomers, which is rapidly reversed on readdition of heme. This suggests that the supply of heme controls the rate at which ribosomes can attach to mRNA and initiate protein synthesis and has no effect, for example, on the stability of mRNA. Reticulocyte lysates also require heme to synthesize globin for longer than 5-10 minutes, and lack of heme leads to polysome disaggregation. 

In this section we shall present evidence that indeed eRF is a component of the system which regulates protein synthesis in rabbit reticulocyte lysate. First of all we will focus attention on the formation of the ternary complex because protein factors influencing the activity of eIF-2 in ternary complex formation have been described. Two factors, called ESP (5) and (3o-eIF-2A (6, 7) enhance the activity of eIF-2 in ternary complex formation two other factors, Co-eIF-2B (8, 9) and Co-eIF-2C (9) seem to modulate the effect of Mg on this reaction. Co-eIF-2B promotes the dissociation of the ternary complex at high Mg + concentration, >1 mM (8, 9). Co-eIF-2C, on the other hand, prevents this dissociation and appears to counteract Co-eIF-2B (8). In addition it was shown that eIF-2, phosphorylated by HRI, was irresponsive to the above described effects (5, 10). 

One way of searching for the presence of inhibitors of polypeptide initiation in infected cells was to add cytoplasmic fractions from virus infected cells to a cell-free system from rabbit reticulocytes. This system initiates the synthesis of new polypeptide chains at a very high rate. Cytoplasm from poliovirus infected HeLa cells, but not from uninfected cells, inhibited protein synthesis in the reticulocyte lysate (59) The inhibitor was isolated and identified as double-stranded (ds) RNA (60). To study the effect of ds RNA on host and viral protein synthesis, a cell-free system from HeLa cells was developed which initiated translation on endogenous cellular or viral mRNA. When added to this system, the ds RNA was found to inhibit the translation of both cellular and viral mRNAs (61). Furthermore, measurement of the amount of ds RNA present in cells early in infection (61, 62) revealed that an insufficient quantity was present to act as a direct agent of protein synthesis inhibition. 

EHRENFELI), E. and HUNT, T. Double-stranded poliovirus RNA inhibits initiation of protein synthesis by reticulocyte lysates. Proc. Natl. Acad. Sci. U.S.A. (l97l), 68f 1075-1078. 

Ehrenfeld and Hunt (12) showed that the inhibition of protein synthesis in reticulocyte lysates by extracts of polio-infected cells was due to the presence of double-stranded RNA (dsRNA) of viral origin. It was later established that low levels of dsRNA activate a protein kinase which blocks initiation of protein synthesis by phosphorylating a subunit of the initiation factor eIF-2 (155 a detailed discussion, see chapters 10 and 11 of this book). The inactivation of eIF-2 seemed to be responsible for the inhibition of the binding Met-tRNA (initiator tRNA) to the 40 S native ribosomal subunit (40 observed after the addition of synthetic... 

Inhibitors of well known metallo-, sulfhydi yl and serine proteases block viral proteolysis in infected cells. The use of iodoacetamide (lAM) to stabilize poliovirus and foot-and-mouth virus precursors has been described (36, 52). The preciirsors were subsequently cleaved in vitro to yield normal-sized products (36)1 and polio RHA replicase activity was stabilized in cell-free extracts of lAM-treated cells (55) H-ethyl maleimide inactivates an EMC virus-specific protease in reticulocyte lysates (20). These results could signify that a sulfhydryl-type protease, e.g., cathepsin B, is required for some of the cleavage reactions. However, when such compounds are added to cells, numerous proteins react, and there is substantial inhibition of protein synthesis. Energy metabolism in such cells is depressed, and ATP-reqiiiring proteolytic reactions blocked. 

The association of ( S) methionyl-tRRA with the 4O-S subunit was strongly inhibited by poly(l) poly(c) in extracts of interferon-treated cells. In extracts of control cells poly(l) poly(c) had no significant effect upon the formation of methionyl-tRHA 40-S subunit complexes. Addition of crude rabbit reticulocyte initiation factors greatly reduced the poly(l) poly(c)-induced inhibition of (55s) methionyl-tRRA 4O-S subunit complex formation. The effect was most marked when the initiation factors were added during the incubation with dsRRA, strongly suggesting that in interferon-treated cell extracts dsRHA acts at the level of a protein synthesis initiation factor which binds methionyl-tRNA to native 4O-S ribosomal subunits, a situation analogous to that of the rabbit reticulocyte lysate (20-22 and chapter 10 of this volume). 

In order to identify initiation factors involved in mRNA competition, a number of studies have employed reconstituted cell-free systems (Golini et al., 1976 Ray et al., 1983). A major problem with such systems is that certain components may be present in excess, while others may be limiting or partially inactivated, precluding efficient initiation. However, meaningful studies of translation initiation frequency in vitro can only be done in systems where ribosomes can cycle rapidly and repeatedly over mRNA. To date, the only cell-free systems that translate mRNA at high efficiency are the reticulocyte lysate (see Jackson, 1982) and the micrococcal nuclease-treated reticulocyte lysate (Pelham and Jackson, 1976). The latter system offers several advantages over reconstituted cell-free systems. It responds to translational control signals (see below), it is capable of extensive and efficient initiation in conditions more likely to be representative of protein synthesis in intact cells, and except for mRNA, it contains all other components for translation in a proportion much closer to that of the intact cell. 

On the other hand, Asselbergs et al. (1980) observed translational competition between globin mRNA, lens crystallin mRNAs, and turnip yellow mosaic virus RNA in the micrococcal nuclease-treated reticulocyte lysate, but not after microinjection into Xenopus oocytes (Asselbergs et al., 1979). The reason for this discrepancy is not clear, as Laskey et al. (1977) have shown that in oocytes, a component of the translation machinery, and not mRNA, limits the rate of protein synthesis. 

Protein synthesis in reticulocyte lysates is regulated by the availability of heme, as it is in whole cells (Zucker and Schulman, 1968 Adamson et al, 1968). In the absence of added heme, initiation of translation continues abated for about 5-10 min, but then stops abruptly. During this period, the level of 40 S/Met-tRNAf complexes declines (Legon et al., 1973). The lesion in initiation can be restored by the addition of an initiation factor (Kaempfer and Kaufman, 1972) identified later as eIF-2 (Kaempfer, 1974(i Clemens, 1976). The absence of heme leads apparently to the inactivation of eIF-2. 

These observations can account for the greater sensitivity of translation to dsRNA in extracts from interferon-treated cells. Apparently, interferon treatment induces a rise in the level of dsRNA-dependent eIF-2-kinase, but since the enzyme is inactive in the absence of sufficient dsRNA, protein synthesis in interferon treated, uninfected cells continues normally. During infection, however, virus-generated dsRNA potentiates the kinase and the resulting extensive phosphorylation of eIF-2 leads to a general inhibition of initiation of translation. As noted for reticulocyte lysates, in extracts of interferon-treated cells, too, there is no good correlation between the extent of phosphorylation of eIF-2 and inhibition of translation (Jacobsen et al., 1983). 

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