Translation in a cell-free system

The formation of disulfide bonds in proteins synthesized in vitro can be followed by measuring enzymatic activity or by an increased mobility compared to the reduced protein during SDS-PAGF. This increased mobility arises from the fact that, as disulfide-bonded proteins are intra-molecularly cross-linked, they form a more compact structure and occupy a smaller hydrodynamic volume compared to the reduced protein (Gold-enberg and Creighton, 1984). An illustration of this increase in mobility is shown in Fig. 2. Here the mRNA for preprolactin was translated in a cell-free system optimized for the formation of disulfide bonds, and then analyzed by SDS-PAGF. The translocated protein forms disulHde bonds under these conditions whereas the protein synthesized under the same conditions but in the absence of microsomal membranes does not form disulfide bonds. Thus the nascent protein must be translocated into microsomal vesicles for disulfide bond formation to occur. 

Most of the information on the regulation of eIF-2 activity comes from studies of reticulocyte lysates. It appears, however, that very similar, if not identical mechanisms operate in a variety of mammalian cell types. Initiation of translation in a cell-free system from HeLa... 

Control of Character Formation by Nucleic Acids 7. Transcription and Translation in a Cell-free System... 

Roberts, B. E. and Paterson, B. M. (1973) Efficient translation of tobacco mosaic vims RNA and rabbit globin 9S RNA in a cell-free system from commercial wheat germ. Proc. Natl. Acad. Sci. USA 70,2330-2334. 

The third approach used mammalian sodium pumps expressing the a 3 and 3 1 subunits synthesized by in vitro translation in a cell-free expression system and then ineorporated into a planar phospholipid bilayer, where exposure to palytoxin elicited single-ehannel eurrents that had a conductance of about 10 pS (Hirsh and Wu 1997). 

The immediate segregation of proteins in the ER suggested that translocation across the membrane may be coupled to translation. The experiments of Redman and Sabatini (1966) supported this concept on a molecular level. After addition of puromycin, an inhibitor of protein elongation, to growing cells, N-terminal fragments of secretory proteins were found in the lumen of the ER, and not in the cytoplasm. In later work, Sabatini and Blobel (1970) showed that inclusion of microsomes of RER in a cell-free system translating mRNA coding for secretory pro-... 

We have reasoned that a novel approach to prolong the production phase of secondary metabolites should be based on an attempt to prevent or retard the process of lja vivo Inactivation of the enzymes (synthetases) catalyzing their formation In fermentations. Such an attempt would require an understanding of the chemical nature of the Inactivation. This knowledge could be subsequently translated Into process development and control In actual fermentations, which, for the most part, are carried out In batch reactors. If the object of the fermentation Is the recovery of the enzymes for further use In a cell-free system or the acquisition of active whole cells for repeated use In a fixed bed-type bloreactor, the prevention or retardation of the Inactivation process would ensure both an adequate margin of time for primary harvesting and a longer half-life of the activity for the blo-catalysts In the cells. 

Hackett and co-workers (57> 5 ) have also observed that in a cell-free system from mengovirus infected Ehrlich ascites tumor cells, host mENA was translated less well than in extracts from uninfected cells. F-urthermore, at supraoptimal concentrations of Mg and K, the system from virus infected cells supported the translation of mengovirus ENA but not host mENA, thereby demonstrating a selective translation of viral over host mENA. A large part of the selectivity found in the translational system derived from infected cells was due to the initiation factor fraction. 

Glover, J. F., and Wilson, T. M. A., 1982, Efficient translation of the coat protein cistron of tobacco mosaic virus in a cell-free system from Escherichia coli. Ear. J. Biochem. 122 485. 

NS proteins. Complete replication of full-length plus- and minus-strand nucleocapsids has been achieved in a cell-free coupled transcription-translation reaction in the presence of a required cellular factor(s) (Hill et al., 1981 Patton et ai, 1983). In fact, Wertz (1983) has been able to replicate the RNA of defective-interfering VSV nucleocapsids in a cell-free system coupled with VSV mRNA translation but free of infectious full-length nucleocapsids. 

The first evidence for control of translation in adenovirus-infected cells came when mRNA isolated from infected cells harvested during the late phase was translated in heterologous, cell-free systems, usually derived from reticulocyte lysates such mRNA preparations direct the synthesis of quite substantial quantities of cellular proteins in addition to large amounts of viral structural proteins (see, for example, Anderson et ai, 1974 Lewis et ai, 1975 Paterson et ai, 1977). The mRNA was prepared from cells collected at a time... 

Bossart, W., Nuss, D. L., and Paoletti, E., 1978, Effect of UV-irradiation on the expression of vaccinia virus gene products synthesized in a cell-free system coupling transcription and translation, J. Virol. 26 673. 

The primary translation product of proteins destined to be anchored by GPI contains both an A-terminal secretory signal sequence and a C-terminal signal sequence that specifies GPI addition. The latter is cleaved and replaced by the GPI precursor in the ER (Figure 3A). This transfer reaction is mediated by a transamidase and involves a nucleophilic attack by the EtN amino group of the GPI precursor on a carbonyl of a specific amino acid residue near the C-terminus. This residue, residing at the co site , will become the new C-terminus of the GPI anchored protein (Figure 3B). In a cell free system that reconstitutes anchor attachment, an alternative nucleophile such as hydrazine or hydroxylamine can substitute for a GPI in this reaction [89]. In a T. brucei cell free system, GPI addition to VSG does not require ATP or other cofactors [90], In a mammalian system, the transfer of the GPI precursor to a protein seems to be enhanced by an ATP-dependent chaperone [91,92],... 

Fig. 8. Heteronuclear single-quantum coherenc (HSQC) spectrum of the hypothetical protein of the flowering locus T protein produced in the cell-free system. The FT protein was synthesized in the same way as in Fig. 6 except that Ala, Leu, Gly, and Gin in both translation and substrate mixture were replaced with their -labeled forms (Isotec, Inc ). After incubation for 48 h, the reaction mixture (1 mL) was dialyzed against 10 mMphosphate buffer (pH 6.5) overnight, and then centrifuged at 30,000g for 10 min. The supernatant containing 30 xMof the protein was directly subjected to nuclear magnetic resonance spectroscopy. The spectrum was recorded on a Broker DMX-500 spectrometer at 25°C, and 2048 scans were averaged for the final H- WHSQC spectrum.

Several studies have used a cell-free system prepared from rabbit reticulocytes to translate cestode messenger RNA (mRNA) in vitro. Thus, a small portion of the proteins synthesised in this heterologous system, using mRNA from T. crassiceps, were shown to be antigenic (5). The molecular weights of the translated polypeptides were generally low (13 000-22 000),... 

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