Protein synthesis in vitro

MUELLER-URI, F., PARTHIER, B., NOVER, L., Jasmonate-induced alteration of gene expression in barley leaf segments analyzed by in vivo and in vitro protein synthesis, Planta, 1988,176,241-247. 

Adapting techniques based on in vitro protein synthesis to the isolation of enzymes requires establishing a link between a nucleic acid-protein complex and product formation. Methods based on binding, analogous to those developed for phage displayed libraries, may be used to enrich catalysts from noncatalysts. In addition, Tawfik and Griffiths (1998) exploited the aqueous core of reverse micelles as artificial compartments... 

Hong, S.C. Layman, D.K. (1984) Effects of leucine on in vitro protein synthesis and degradation in rat skeletal muscles. J. Nutr. 114, 1204-1212. 

Di GiAMBATnSTA, M., Hummel, H., Bock, A., Cocito, C., Action of synergi-mycins and macrolides on in vivo and in vitro protein synthesis in archaebacteria. Mol. General Genetics 1985, 199, 323-329. 

Lom-AKH-I suppresses fat body protein synthesis in locusts. Administration of glandular lobe extracts or synthetic Lom-AKH-I inhibits protein synthesis by 60% in immature and adult locusts (63). Lom-AKH-I does not mobilize lipids in larval locusts (36), and it is speculated that the major action of Lom-AKH-I in immatures is to regulate protein synthesis. The inhibitory effect of Lom-AKH-I on protein synthesis is observed for adult locusts in vivo at doses that are lower than those needed for lipid mobilization (64), and both arylphorin and vitellogenin synthesis are suppressed. TTie inhibitory action of Lom-AKH-I is also confirmed for in vitro protein synthesis by the muscle and gut, as well as the fat body. In preparations of dispersed fat body cells, synthetic Lom-AKH-I stimulated cAMP synthesis and lipid release but inhibited protein synthesis by 70% (38). 

Peptides, for example, are needed for the production of antibodies and for epitope mapping proteins can be produced in radioactively labelled forms by in vitro translation for studies of protein-protein interactions and other applications. In vitro protein synthesis is a technique that can be readily carried out in any biochemistry laboratory, whereas peptide synthesis, like peptide sequencing, is a specialist technique using dedicated equipment, which is nowadays routinely automated. 

Increasing evidence suggests that Met-tRNAf is also the initiator tRNA in eukaryotic systems (C35, G16). The failure of previous experiments to demonstrate the role of this Met-tRNAf for the in vitro protein synthesis is probably due to the lack of protein initiation factors. Mi, Ma, and Ms, which are present in a ribosomal salt-wash protein fraction (P24, S35, S36). The most recent experiments by Anderson and co-workers (C34, C35) show that the Met-tRNAf binds the initiation factors Ml and Ms to form an initiation complex with messenger RNA. The binding of this complex requires CTP and Mg + ions. A methionyl-valine dipeptide production is the next step in the biosynthesis of the chain the synthesis of this bond requires Mg + ions, an additional initiation... 

After establishing that L-tryptophan alone stimulated overall hepatic protein synthesis, it became important to determine which proteins were involved. Increased synthesis of albumin due to L-tryptophan was reported by Rothschild et al.65 and by Jorgensen and Majumdar.47/18 The latter investigators also reported that tryptophan increased the synthesis of transferrin, fibrinogen, and ferritin. Thus, extracellular as well as intracellular proteins that are synthesized by the liver were affected by L-tryptophan. Furthermore, it was demonstrated that after L-tryptophan administration, both free and membrane-bound polyribosomes of the liver showed a shift toward heavier aggregation, more marked for free polyribosomes than for membrane-bound polyribosomes.62 Also, in vitro protein synthesis revealed a greater increase with free polyribosomes than with membrane-bound polyribosomes of the livers of tryptophan-treated animals in comparison with similar fractions of livers of control animals. 

Cordycepin (3 -deoxyadenosine), an agent that suppresses polyadenylation with a marked reduction of mRNA in cytoplasmic polyribosomes, was investigated with L-tryptophan. Administration of L-tryptophan to fasted rats (1 h before kill) pretreated (2 h) with cordycepin induced a shift in hepatic polyribosomes toward heavier aggregation and an increase in in vitro protein synthesis.120 Also, treatment with L-tryptophan (2.5 h before killing) and cordycepin 0.5 h later enhanced hepatic polyribosomal aggregation and protein synthesis.188 Thus, L-tryptophan acted in a preventative and curative manner. 

Effect of L-Tryptophan before or after Treatment with Hepatotoxic Compounds or Drugs on Hepatic Protein Synthesis (Status of Polyribosomes and in Vitro Protein Synthesis)... 

Harley SM and Beevers H (1982). Ricin inhibition of in vitro protein synthesis by plant ribosomes. Proc Nat Acad Sci USA, 79, 5935-5938. 

The third approach used repeating ribonucleotide polymers containing known repeating sequences (Fig. 26.4C). When these were used as templates for in vitro protein synthesis, it was found that each ribonucleotide polymer could specify as many as three different repeating polypeptide products. Of the 64 possible codons, 61 were found to specify amino acids and 3 were later defined as termination codons. Figure 26.5 shows the genetic code for protein synthesis in E. coli, which is for the most part applicable to mRNA translation in mammalian cells. Note that methionine and tryptophan are only specified by single codons, whereas almost all the other amino acids have from two to four codons (except leucine and serine which are encoded by six codons). Methionine is the first amino acid in essentially all proteins however, methionine residues are also found within the polypeptide sequence. The amino-terminal methionine is called the initiator methionine. 

Thompson W, Scovill J, Pace J. Drugs that show protective effects from ricin toxicity in in-vitro protein synthesis assays. Natural Toxins. 1995 3 369-377. 

Residue type and sequential assignments obtained from specifically labeled samples, when combined with 3D heteronuclear data, can significantly increase the efficiency and accuracy of the assignment process, the first step in structure determination by NMR. A protocol for the design of specifically labeled samples with high information content has been developed by Shi et aV. In vitro protein synthesis methods were used to produce four specifically labeled samples of the 23.5 kDa protein phosphoserine phosphatase (PSP) from Methanoccous jannas-chii. Each sample contained two C/ N-labeled amino acids and one N-... 

The effect of 8 on in vitro protein synthesis in which poly(U) functions as a messenger RNA (where a UUU sequence is a codon for phenylalanine) was also examined. In the presence of 0.558 mM (r = 1) and 5.58 mM of 8 (r = 10), the polyphenylalanine synthesis underwent 15 % and 26 % inhibition, respectively. We propose a new mechanism for the inhibition of protein synthesis as depicted below. 

In vitro Protein Synthesis in the Chloroplasts isolated from the... 

Lied E, Lund B, von der Decken A (1982) Protein synthesis in vitro by epaxial muscle polyribosomes from cod, Gadusmorhua. Comp Biochem Physiol 72B 187-193 Lied E, Rosenlund G, Lund B, Von der Decken A (1983) Effects of starvation and refeeding on in vitro protein synthesis in white trunk muscle of Atlantic of (Gadus morhua). Comp Biochem Physiol 76B 777-781... 

Lied E, Lie O, Lambertsen G (1985) Nutritional evaluation in fish by measurement of in vitro protein synthesis in white trunk muscle tissue. In Cowey CB, Mackie AM, Bell JG (eds) Nutrition and feeding in fish. Academic Press, London... 

C25H35N5O7 517.581 Nucleoside antibiotic. Prod, by Strepto-myces plicatus. Potent inhibitor of in vitro protein synthesis. Inhibitor of gram-positive and gram-negative bacteria. Needies eOH aq.). 

In Vitro Protein Synthesis 107 Amino Acid Activation 107 Amino Acyl RNA Synthetase 108 tRNA General Properties Sequence Configuration... 

Liou YF, Hall IH, Okano M, Lee KH, Chaney SG (1982) Antitumor agents XLVIII Structure - activity relationships of quassinoids as in vitro protein synthesis inhibitors of P-388 lymphocytic leukemia tumor cell metabolism. J Pharm Sci 71 430-435... 

In vitro protein synthesis in the presence of added ribosomes and mRNA (or polysomes) and ATP/GTP can be supported by supernatant (cytoplasmic, or post-ribosomal) fractions obtained from dry peanut seeds (Table 5.6) and dry wheat embryos [76]. Consequently, components of the cytoplasm essential for protein synthesis (e.g. initiation and elongation factors, tRNA, amino acids, and aminoacyl tRNA-synthesizing enzymes—synthetases) must be present in the dry seed, presumably in sufficient quantities to permit resumption of protein synthesis in the seed upon imbibition. 

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