S30 system

Initially, display of a peptide libraiy on prokaiyotic polysomes in the E. cdi S30 system was described (6, 7). To produce a population of stalled polysomes, agents such as rifampicin or chloramphenicol, which block prokaiyotic translation, were used. (The term stalled implies that the process of translation has been interrupted such that the ribosome, mRNA, and nascent protein remain associated.) The main example was of screening a large peptide libraiy with 10 members and selection of epitopes by a specific antibody. Peptide libraries have also been expressed and selected in the wheat germ system (8). 

A cell-free extract (S30) from E. coli K12 has been developed as an efficient coupled transcription/translation system which performs protein synthesis in vitro from the genes cloned in plasmids under the T7, T3, or SP6 promoter. Capping of the mRNA for eukaryotic proteins is apparently unnecessary with the S30 system. The coupled transcription/translation system, which was originally developed as the S3 0 translation system (122), can be used in a batchwise or continuous-flow mode (123,124). Use of the ribosome fraction collected from the S30 extracts is reported to improve the yield and efficacy further and more advantageously with nonlinearized plasmids than with linearized plasmids (125). 

Fig. 8. Salt dependence of enzyme synthesis in an S30 system. (Experiment together with H. J. Rahmsdorf.) 50 ml of an exponentially growing culture of strain XA 7007 (suA) in rich medium were harvested at O.D.goo = 0.4. After washing once with TMA buffer, the cells were suspended in 0.4 ml TMA buffer and lysed by ultrasonication. The debris were removed. The S30 incubation mixture contained, in addition to 20 (d of the S30 extract, the following components in a final volume of 0.05 ml tris HCl pH 8.0 51 mM K-acetate 50 mM amino acids 0.2 mM each ATP 2mM UTP, CTP, GTP 0.5 mM each PEP 20 mM dithiothreitol 2.5 mM tRNA 500 (xg/ml T3 DNA 50 xg/ml MgClg 11 mM (the S30 extract adds to 15 mM MgClg in toto) varying concentrations of NH4CI. After 40 minutes at 37°, aliquots were tested for lysozyme (o-------o)...

ZuBAY and coworkers (Zubay and Chambers, 1969) have established a modification of the S30 system of Matthaei and Nirenberg (I96I), a preincubated S30 system. This system found wide application for the study of synthesis of E, coli enzymes. The S30 is mixed with a small volume of a solution containing all the components needed for translation (amino acids, ATP, ATP-regenerating system) and preincubated at 37° for 80 minutes. The S30 extract is then dialyzed against a desired buffer at 0°. Table 2 summarized the preparation of the S30 system and lists the constituents of the incubation mixture. 

Various components have been added to either an S30 system or the DEAE-system, e.g. RNA polymerase, CAP factor, repressors, regulatory proteins. In each case one must observe whether the conditions optimal for enzyme synthesis are also optimal for the action of the added component. For... 

In a crude extract or S30 system, no additional tRNA is required. The S30 or crude extract contains saturating amounts of tRNA. The DEAE-system is partially dependent on the addition of tRNA the protein fraction is almost free of tRNA, but tRNA is attached to the ribosomes. In fact, preincubated ribosomes still contain sufficient tRNA to allow 30-50% of the leucine incorporation which can be achieved upon addition of tRNA. However, some tRNA species are apparently limiting since meaningful protein synthesis is reduced to 5% (Gold and Schweiger, 1969a). Response to tRNA addition is improved by further purification of the ribosomes. 

In addition to magnesium, the concentration of calcium salts appears to be critical in the preincubated S30 system (Table 2 Zubay, personal communication). 

How does the rate of in vitro enzyme synthesis compare to in vivo synthesis Under optimal conditions, 0.2 [Jig / -galactosidase per ml is synthesized within 60 minutes of incubation (Chambers and Manley, 1973). Compared to maximal in vivo synthesis, this corresponds to an efficiency of 0.06%. In 08Odarg directed synthesis of N-a-acetyl-L-ornithinase, the in vitro rate was approximately 0.09% of the in vivo rate (Urm et al., 1973). T7 infected cells produce about 7X10 units of lysozyme per minute and per mg of ribosomes assuming that 3 X 10 cells contain 1 mg (1 500 O.D.geo) of ribosomes. The in vitro rate is 4 X 10 units per minute and per mg of ribosomes or 0.57% of the in vivo rate. All these data are of course approximations. Rates of enzyme synthesis in both the preincubated S30 system and the DEAE system are of the same order of magnitude. 

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