Protein synthesis fractionated cell-free systems

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. 

A wheat germ, cell-free, translation extract was fractionated into three concentrated parts using ammonium sulfate the 0 - 40 % saturated fraction, the 40 - 60 % saturated fraction, and the ribosome fraction. These fractions were tested for their ability to enhance the translational activity of the wheat germ, cell-free extract for dihydrofolate reductase. The fortified cell-free system supplemented with the 0 - 40 % ammonium sulfate fraction enhanced the efficiency of protein synthesis by 50 %. 

The catalytic activities of the fortified wheat germ cell-free systems supplemented with each fraction were investigated (Fig. 2). As shown in Fig. 2, only 0 - 40 % ammonium sulfate fraction showed an enhancement in DHFR protein synthesis. This enhancement of protein experimental results and the fact that the various eukaryotic initiation factors are contained in synthesis was also confirmed by SDS-PAGE and autoradiography (Fig. 3). From the above 0-40 % ammonium sulfate fraction [5, 6], it can be concluded that the amount of initiation factors in a conventionally prepared wheat germ cell-fi extract is deficient for the translation of DHFR with internal ribosome entry site. Therefore, it needs to supplement a wheat germ cell-free extract with the fraction containing the limited initiation factors for the efficient protein translation, and this fortified cell-free system can be easily made by simple... 

Cell-free systems capable of synthesising polypeptides have been prepared from protoscoleces of E. granulosus (7), larval T. crassiceps (588) and H. diminuta (633). In general, these studies have demonstrated that protein synthesis in cestodes, although showing some specificity, is similar to that in mammals in that it requires polysomes, amino acid adenylates, aminoacyl-tRNAs, pH 5 fraction, ATP, GTP, magnesium and either sodium or potassium ions. 

In 1962 Speyer, Basilio and I proved the validity of a hypothesis by Spotts and Stanier according to which the difference between streptomycin-sensitive, resistant and dependent E. coli. resides in the structure of their ribosomes. We established that streptomycin inhibits poly(U) directed polyphenylalanine synthesis in a fractionated cell-free protein synthesizing system only if the ribosomes in the system are taken from streptomycin-sensitive cells. These results indicated that in sensitive cells streptomycin inteiieres with ribosome action. As subsequent studies revealed, streptomycin is only one among many antibiotics whose site of action is the ribosome. 

Progress has been made in demonstrating synthesis of specific proteins by cell-free systems, e.g., on the synthesis of cytochrome c by isolated rat liver mitochondria (S3, cf. 383) and on the synthesis of serum albumin by the isolated microsome fraction of rat liver 34, cf. 335,336). Campbell et al. 34) concluded that while specific serum albumin is, indeed, synthesized on the ribonucleoprotein particle fraction of the microsomes, it is not readily released in soluble form. In other words, the isolated microsomes have lost their ability to promote substrate turnover. The same is true for the hemoglobin-synthesizing RNP particles from rabbit reticulocytes 35, cf. 138). Ogata and associates 36) have essentially confirmed the results of Campbell et al. 34) on the synthesis of serum albumin by liver microsomes they have also studied the relative effect of both stimulatory and inhibitory factors on the incorporation of different amino acids into total ribonucleoprotein and into serum albumin, and showed that the requirements for the two processes were generally the same it may be noted, however, that pretreatment of the pH 5 enzymes with ribonuclease, which caused a 95% inhibition of the ineorporation into ribonucleoprotein, inhibited the corresponding incorporation into serum albumin by only 55%. 

In most systems in which either whole cells, protoplasts, or cell fractions are used, peptide and protein synthesis can be differentiated various amino acid analogues can selectively suppress the former whereas enzymes and antibiotics that specifically inhibit either DNA or RNA function or RNA or protein synthesis can selectively suppress the latter. In one cell-free system, however, peptide and protein synthesis cannot be differentiated in this system, formation of the peptide antibiotics require the same ingredients (and are sensitive to the same inhibitors) as those needed for (or antagonistic to) production of cell proteins. One of the essential ingredients consists of a messenger RNA molecule of 36 2 nucleotides. The reasons for the differences between systems in which differentiation can or cannot be achieved are presently unknown. 

Fig. 3. Autoradiograph of SDS-PAGE of in vitro translated dihydrofolate reductase (DHFR) in the wheat germ cell-free protein synthesis systems with (n) 4 pi of ribosome fiaction, (III) 4 pi of 0 -40 % ammonium sulfate fraction, or (IV) 4 pi of 40 - 60% ammonium sulfate fraction, respectively. Lane I is control dihydrofolate reductase produced in the normal wheat germ cell-free protein synthesis system.

Another approach to inexpensive labelling of proteins is by means of cell-free protein synthesis.48 50 These in vitro expression systems use a crude Escherichia coli fraction as a source of ribosomes and other factors necessary for the synthesis of proteins, T7 RNA polymerase for transcription, and dialysis to maintain levels of amino acids and nucleotides. In principle, the technology is capable of labelling proteins at specific sites without isotope scrambling and also of producing labelled proteins from small volumes of labelled medium. 

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). 

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