Preparation and partial characterization of cell-free protein-synthesizing extracts from Tetrahymena pyriformis.

1. We have examined methods necessary for preparing post-mitochondrial supernatants from Tetrahymena pyriformis strain HSM that are capable of efficient cell-free protein synthesis. 2. The requirements for optimum synthesis in these extracts are described. 3. Data relating to the kinetics of protein synthesis and the initiation capacity of these supernatants are presented.     

Synthesis of Phosphopeptides in the Fmoc Mode

The synthesis of phosphopeptides has played a major role in the characterization of protein phosphorylation/dephosphorylation. The current range of synthesis protocols available provides a variety of possible routes by which to approach specific synthetic challenges, and this review article discusses these methods for the preparation of phosphopeptides and provides synthesis notes for each method. Phosphopeptide synthesis is achieved by either introduction of the phosphate group via post-synthetic (‘global’) phosphorylation of a resin-bound peptide or the incorporation of a pre-phosphorylated derivative into the growing peptide chain. Protocols and synthesis notes are provided for the synthesis of phosphoramidites, phosphotyrosyl, -seryl and -threonyl peptides and their mimetics, including thiophosphopeptides. The aim of this review was to provide a synthesis reference guide for Fmoc-based synthesis of both singly and multiply phosphorylated peptides, with particular emphasis given to the most successful and generally applicable methods.     

Gene Order of the Poliovirus Capsid Proteins

Two methods were used to determine the genetic map of the poliovirus capsid proteins. The first method uses pactamycin, a drug which selectively inhibits the initiation of protein synthesis and causes a change in the relative amounts of capsid proteins synthesized. This differential effect on each of the capsid proteins is interpreted as indicating the relative distance of each protein from the initiation site of protein synthesis. The second method involves an analysis of coat precursor molecules released from polyribosomes after a series of short pulses of different length terminated by addition of emetine, a drug which stops all protein synthesis almost immediately after its addition. As the pulse length is increased, each of the capsid proteins within the precursor gains radioactivity with different kinetics. From these kinetics, it is possible to determine the gene order of the capsid proteins within the precursor as well as a rate of protein synthesis. Both methods indicate a gene order for the region of the ribonucleic acid coding for the capsid proteins as (5' --> 3') VP 4 - VP 2 - VP 3 - VP 1.     

Chemical synthesis and expression of a gene coding for hirudin, the thrombin-specific inhibitor from the leech Hirudo medicinalis

A DNA containing the coding sequence for the proteinase inhibitor protein hirudin from the leech Hirudo medicinalis has been obtained by enzymic ligation of chemically synthesized deoxyoligonucleotides. The 226 bp synthetic gene carries signals for the translation initiation and termination. Fragment synthesis was performed by the Khorana ligation method as well as by the fill-in method. Efficiencies of these two methods are compared. The synthetic gene was expressed in E. coli as a fusion protein with beta-galactosidase under the control of the lac-promoter as well as a non-hybrid protein under the control of the lambda PL-promoter. The non-hybrid expression product was shown to have similar biological properties as the authentic protein isolated from the leech.     

Synthesis of of beta-amyloid precursor peptide and presenilin segments.

It seems likely that the beta-amyloid precursor protein (APP) and the presenilins (PS-1/2) play important roles in the development of Alzheimer's disease (AD). Attempts to mimic the biochemical actions of these proteins are often made by the application of fragments of these proteins. However, the synthesis of these segments by conventional methods of peptide synthesis is problematic. We have synthesized several C-terminal fragments of APP and PS-1/2 by solid-phase synthesis through combination of automatic and manual methods of synthesis. This permits solution of the 'difficult sequences' in the solid-phase synthesis of these peptides. Some details of the syntheses of nine segments are presented in this paper.     

Recent advancements in mass spectrometry–based tools to investigate newly synthesized proteins

Tight regulation of protein translation drives the proteome to undergo changes under influence of extracellular or intracellular signals. Despite mass spectrometry–based proteomics being an excellent method to study differences in protein abundance in complex proteomes, analyzing minute or rapid changes in protein synthesis and abundance remains challenging. Therefore, several dedicated techniques to directly detect and quantify newly synthesized proteins have been developed, notably puromycin-based, bio-orthogonal noncanonical amino acid tagging–based, and stable isotope labeling by amino acids in cell culture–based methods, combined with mass spectrometry. These techniques have enabled the investigation of perturbations, stress, or stimuli on protein synthesis. Improvements of these methods are still necessary to overcome various remaining limitations. Recent improvements include enhanced enrichment approaches and combinations with various stable isotope labeling techniques, which allow for more accurate analysis and comparison between conditions on shorter timeframes and in more challenging systems. Here, we aim to review the current state in this field.     

A proteomic method for the analysis of changes in protein concentrations in response to systemic perturbations using metabolic incorporation of stable isotopes and mass spectrometry

While several techniques exist for assessing quantitative differences among proteomes representing different cell states, methods for assessing how these differences are mediated are largely missing. We present a method that allows one to differentiate between cellular processes, such as protein synthesis, degradation and PTMs which affect protein concentrations. An induced systemic perturbation of a cell culture was coupled to a replacement of the growth medium to one highly enriched in the stable isotope 15N. The relative abundance of the 15N- and 14N-enriched forms of proteins, isolated from cell cultures harvested at time points following the onset of the perturbation, were determined by MS. Alterations in protein synthesis and degradation were quantified by comparing proteins isolated from perturbed and unperturbed cultures, respectively. The method was evaluated by subjecting HeLa cells to heat stress. As expected, a number of known heat shock proteins (Hsp) increased in concentration during heat stress. For Hsp27, increased de novo synthesis accounted for the concentration increase, while for Hsp70, decreased degradation accounted for the increase. A protein that was detected only after prolonged heat stress, vimentin, was not primarily synthesized de novo, but appeared rather as a result of PTM.     

A new cloning strategy for generating multiple repeats of a repetitive polypeptide based on non-template PCR

A new cloning method for generating multiple repeats of amino acids is described which can be used as biomaterials, protein polymers and biomedical applications. Although several traditional methods for cloning multiple repeats are still exploited, these are laborious and complicated because they must go through several consecutive cloning steps. To solve these problems, synthetic gene libraries encoding repetitive patterns were constructed by using non-template PCR. As a result, a ‘length library’ with fourteen different ELP repeating genes was constructed and expressed in a cell-free protein synthesis system. These results showed our novel cloning method is efficient, and has the potential capacity for synthesizing repetitive genes by PCR to be cloned in any commercial expression vectors.     

An Improved Method for Estimating Macromolecular Synthesis

Estimating rates of DNA, RNA, and protein synthesis has beengreatly facilitated by the use of radioactive precursors. Currentlyavailable methods to assess macromolecular synthesis in higherplants are time-consuming and imprecise. In the procedure outlinedhere, the conventional method is simplified by processing thetissue intact. This method allows a rapid analysis and considerablyreduces the inherent variability associated with the conventionalmethod.     

Synthesis of palmitoylated Ras-peptides and -proteins

In this review, an overview is given and details are provided for the synthesis of lipidated Ras (rat-adeno-sarcoma)-peptides and -proteins. The progress made in the synthesis of the lipidated peptides from the Ras superfamily is discussed with special emphasis on the recently developed solid-phase synthesis methods, since these methods have turned out to be the preferred synthesis method for the majority of the required peptides. Solid-phase lipopeptide synthesis has given access to native and modified peptides on a scale that allows peptide-consuming studies like for ligation to proteins and concomitant X-ray crystal structure determination. The access to these peptides has also enabled biological questions concerning these peptides and proteins to be resolved. The review describes different solid-phase methods, which are individually suited for different types of lipopeptides, differing for example in lipidation pattern or amino acid side-chain functionality, and their ligation to proteins. Finally, an example is provided how these peptides can serve to resolve biological aspects of the Ras family GTPases.     

Role of RNA and protein synthesis in memory formation

A brief review is given of experiments which are concerned with the hypothesis that brain RNA and protein synthesis are directly involved in the establishment of long-term memory. It is concluded that these experiments neither support or refute this hypothesis. A convincing demonstration is lacking of interanimal memory transfer by injection of macromolecular extracts. The majority of experiments which attempt to correlate increased macromolecular synthesis with learning use radioactive precursor methods and these studies do not exclude possible changes in precursor specific activity as the cause of the increased labeling. Although some studies find directly observable changes in brain macromolecules in response to training, their relationship to memory formation is unclear. It is possible that these changes represent only an enhanced production of constitutive macromolecules in response to an increase in cerebral metabolism during training, rather than molecular changes that are directly involved with modifying synaptic connectivity. Inhibitors of cerebral protein synthesis block memory formation, but these drugs are not pharmacologically specific and this complicates the interpretation of these studies.     

Design and selection of ligands for affinity chromatography

Affinity chromatography is potentially the most selective method for protein purification. The technique has the purification power to eliminate steps, increase yields and thereby improve process economics. However, it suffers from problems regarding ligand stability and cost. Some of the most recent advances in this area have explored the power of rational and combinatorial approaches for designing highly selective and stable synthetic affinity ligands. Rational molecular design techniques, which are based on the ability to combine knowledge of protein structures with defined chemical synthesis and advanced computational tools, have made rational ligand design feasible and faster. Combinatorial approaches based on peptide and nucleic acid libraries have permitted the rapid synthesis of new synthetic affinity ligands of potential use in affinity chromatography. The versatility of these approaches suggests that, in the near future, they will become the dominant methods for designing and selection of novel affinity ligands with scale-up potential.     

Application of a nonradioactive method of measuring protein synthesis in industrially relevant chinese hamster ovary cells

Due to the high medical and commercial value of recombinant proteins for clinical and diagnostic purposes, the protein synthesis machinery of mammalian host cells is the subject of extensive research by the biopharmaceutical industry. RNA translation and protein synthesis are steps that may determine the extent of growth and productivity of host cells. To address the problems of utilization of current radioisotope methods with proprietary media, we have focused on the application of an alternative method of measuring protein synthesis in recombinant Chinese hamster ovary (CHO) cells. This method employs puromycin as a nonradioactive label which incorporates into nascent polypeptide chains and is detectable by western blotting. This method, which is referred to as SUnSET, successfully demonstrated the expected changes in protein synthesis in conditions that inhibit and restore translation activity and was reproducibly quantifiable. The study of the effects of feed and sodium butyrate addition on protein synthesis by SUnSET revealed an increase following 1 h feed supplementation while a high concentration of sodium butyrate was able to decrease translation during the same treatment period. Finally, SUnSET was used to compare protein synthesis activity during batch culture of the CHO cell line in relation to growth. The results indicate that as the cells approached the end of batch culture, the global rate of protein synthesis declined in parallel with the decreasing growth rate. In conclusion, this method can be used as a “snapshot” to directly monitor the effects of different culture conditions and treatments on translation in recombinant host cells. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1043–1049, 2013     

Conformation-directed recombination of enzyme-activated peptide fragments: a simple and efficient means to protein engineering. Its use in the creation of cytochrome c analogues for structure-function studies

Protein fragments have been activated by the addition of amino acid esters using proteolytic enzymes under conditions where the equilibria are shifted in the direction of synthesis. Because of the natural propensity of large protein fragments to form complexes approximating native conformation, these activated fragments have been induced to recombine by formation of the missing peptide bond. Although the incorporated ester is only weakly activating, the complex, mimicking an enzyme, provides proximity and orientation at the reacting termini, so that coupling yields are high. In other words, the protein catalyzes its own resynthesis. What distinguishes our technique from the rare natural examples of this phenomenon is that it operates at a variety of cleavage sites and with varying chain lengths. There seems to be no particular limitations on the amino acid esters that can be added, and serine proteases with a wide range of specificities can be used. It thus appears that we have a truly general method for the condensation of large fragments in protein synthesis, be they natural or the products of synthetic or genetic methods. This approach has the advantages over conventional methods of great specificity, high efficiency, and mild conditions of use. With our model protein, cytochrome c, we have used this approach to make analogues that illuminate structure-function relations. Both the highly conserved lysine 39 and the functionally invariant threonine 40 have been replaced by a range of substitutions. The results show how crucial these residues are to the structural and functional integrity of the bottom omega-loop of the protein.     

Effects of several inhibitors of macromolecule synthesis upon maturation of marine invertebrate oocytes

Protein, RNA and DNA syntheses, during oocyte maturation in Asterias glacialis and Chaetopterus, have been studied with cytochemical and biochemical methods. The effects of several inhibitors of the biosynthesis of these macromolecules have been investigated. The results show that protein synthesis is required for maturation: fusidic acid and puromycin, which strongly inhibit protein synthesis, prevent maturation. However, a paradoxical effect of cycloheximide was observed in Chaetopterus oocytes: this drug, like KCl, induces activation of the eggs. DNA and RNA are synthesized during maturation, but studies with inhibitors show that these syntheses are not necessary for the completion of maturation. Protein synthesis was followed during maturation and activation of Chaetopterus oocytes. It was observed that protein synthesis, which stops at the end of maturation, is not readily restored by activation. The significance of these results is discussed.     

Effect of testosterone on the amount of serous-like granules in convoluted tubular cells of mouse submandibular glands.

The effect of testosterone on the amount of granules present in convoluted tubular cells of the submadibular glands of mice was studied by the following two methods; 1) an immunochemical method using antiserum specific to the granular components, and 2) a histographic method. The results obtained by these two methods were in agreement. The amounts of the granules in normal female and castrated male mice were one-tenth to one-twentieth of that in normal male mice. When male mice were castrated, the amount of granules decreased, reaching a minimum 20 days after the aperation the injection of the male hormone, testosterone, into castrated male mice caused an increase in the amount of granules; this increase reached a maximum 15 days after the injection. The increase of granules caused by testosterone injection was almost completely prevented by inhibitors of protein synthesis, actinomycin D and puromycin. This suggests that protein synthesis was indispensable to the increase in the amount of granules. In male mice, the injection of female hormones scarcely affected the amount of granules. Kinetic analysis of the decrease and increase of granules on castration and testosterone injection suggested that the male hormone stimulated granule synthesis, but it hardly influenced the loss of granules.     

Schistosoma mansoni: analysis of cercarial transformation methods

Four methods of transforming cercariae to schistosomulae in vitro in ELAC buffer (pH 7.2, 37 C, 0-6 hr incubation) were compared in relation to biochemical and ultrastructural characteristics. The transformation methods used were chemical (3 mM linoleate), mechanical (centrifuge/vortex), mechanical/chemical, and heat (incubation at 37 C). Ultrastructural characteristics examined were based on the presence or absence of glycocalyx, heptalaminate membrane, cyton granules, and nuclear condition. Two EM fixation methods were used. Biochemical parameters assayed were loss of water tolerance (uptake of trypan blue dye), eicosanoid biosynthesis (PGE, LTB4, and 5-HETE), protein synthesis (leucine uptake), RNA synthesis (uracil and orotic acid uptake), and DNA synthesis (thymidine uptake). EM characteristics were remarkably similar for all transformation methods except heat incubation, with transformed cercariae evidencing the characteristics of schistosomulae (cyton granule migration, absence of glycocalyx and heptalaminate membrane); however, euchromatic nuclei could not be demonstrated using in vivo or in vitro transformation methods. Despite the ultrastructural similarities between transformation methods, biochemical data demonstrated that the resultant organisms were quite different. The chemical transformation method gave the highest rate of loss of water tolerance and eicosanoid production. RNA and protein synthesis were not correlated to ultrastructural changes and were highest in those organisms undergoing mechanical transformation methods, significantly higher than in those cercariae transformed by the chemical method. DNA synthesis was not demonstrated using any transformation method, although thymidine uptake did occur. Our data indicate substantial biochemical differences exist between morphologically similar organisms. Thus, experiments using any type of artificially transformed schistosomule must be interpreted with caution until additional biochemical and physiological studies on cercarial transformation are undertaken.