Nuclease protein system

II. Nuclease A131A Local Structure A. The Protein System... 

The mature red blood cell cannot synthesize protein. Reticulocytes are active in protein synthesis. Once reticulocytes enter the circulation, they lose their intracellular organelles (ribosomes, mitochondria, etc) within about 24 hours, becoming young red blood cells and concomitandy losing their ability to synthesize protein. Extracts of rabbit reticulocytes (obtained by injecting rabbits with a chemical—phenylhydrazine—that causes a severe hemolytic anemia, so that the red cells are almost completely replaced by reticulocytes) are widely used as an in vitro system for synthesizing proteins. Endogenous mRNAs present in these reticulocytes are destroyed by use of a nuclease, whose activity can be inhibited by addition of Ca +. The system is then pro-... 

ARCAs are incorporated into RNA exclusively in the correct orientation to an extent that is similar to the standard cap (see previously), which makes them potentially useful compounds in terms of increasing translational efficiency when incorporated into RNA. Similarly, they should be effective for inhibiting protein synthesis as free analogs. To test the influence of the ARCAs on protein synthesis in vitro, we use the microccocal nuclease treated rabbit reticulocyte lysate system (RRL system) optimized for cap-dependent translation (Cai et al., 1999). Highly cap-dependent translation is achieved at 100 mM potassium acetate and 1.4 mM magnesium chloride. 

The RTS system includes two different technology platforms for cell-free protein expression as well as a number of tools for finding optimal conditions (Scheme 1.1). All expression systems use the T7-polymerase for transcription and an E. coli lyzate with reduced nuclease and protease activity for translation. The conditions are optimized for a coupled transcription/translation reaction so that the DNA can be directly used as the template. 

An in-depth study of DNA repair systems (Aravind et al., 1999a) has concluded that few, if any, repair proteins occur with identical collinear domain arrangements in all three kingdoms of life. Approximately 10 enzyme families of adenosine triphosphatases (ATPases), photolyases, helicases, and nucleases were identified that are all likely to have been present in the cenancestor. These enzymatic domains are accompanied in DNA repair proteins by numerous regulatory domains. This indicates that the domain architectures of these proteins are labile, with incremental addition and/or subtraction of domains to conserved cores to be a common phenomenon except in the most closely related species. 

The leader sequence of the Serratia marcescens extracellular nuclease has been removed and the gene for the resulting nontransportable protein cloned behind the leftward promoter (PL) of lambda (Ahrenholtz, Lorenz Wackernagel, 1994). In the presence of a temperature-sensitive repressor (cl857), the cells can be induced to produce this altered enzyme by an increase in temperature. This produces the nuclease within the cell and, because it cannot be exported, its intracellular concentrations rise, rapidly degrading the cellular genome. Limitations to this system are that cell survival is reduced to only 2 x 10 5. 

Nuclease is a second case in which fragments could be obtained which yield productive complementation (47, 48, 80, 81) (Fig. 7). Observations on such complementations have contributed to the understanding of protein folding and, as with ribonuelease, have provided convenient systems for the synthetic approach to the study of the structure-function relationship in nuclease. 

Moreover, it was recently shown that the deliberate removal of phosphatases from the in vitro translation mixture by immunoprecipitation results in increased protein yields (Shen et al., 1998). As proposed earlier (Jermutus et al., 1998), the elimination of one of the many causes of fast ATP and GTP depletion extends the time of synthesis, and, as a consequence, the total amount of produced protein. It should be possible to similarly remove proteases and nucleases (see above). Together with new ATP regeneration systems to keep the biochemical energy level at a high steady state (Kim and Swartz, 1999), the optimization measures mentioned should increase the level of synthesis of most proteins, and this should directly improve in vitro selection. 

Abstract. Walter Kauzmann stated in a review of protein thermodynamics that volume and enthalpy changes are equally fundamental properties of the unfolding process, and no model can be considered acceptable unless it accounts for the entire thermodynamic behaviour (Nature 325 763-764, 1987). While the thermodynamic basis for pressure effects has been known for some time, the molecular mechanisms have remained rather mysterious. We, and others in the rather small field of pressure effects on protein structure and stability, have attempted since that time to clarify the molecular and physical basis for the changes in volume that accompany protein conformational transitions, and hence to explain pressure effects on proteins. The combination of many years of work on a model system, staphylococcal nuclease and its large numbers of site-specific mutants, and the rather new pressure perturbation calorimetry approach has provided for the first time a fundamental qualitative understanding of AV of unfolding, the quantitative basis of which remains the goal of current work. 

The Kirkwood-Buff integrals and the excess (or deficit) number of molecules of water and PEG around a protein molecule were calculated for 3-lactoglobulin (p-LG), bovine serum albumin (BSA), lysozyme, chymotrypsinogen and ribo-nuclease A (RNase A). Experimental data regarding and V2 for these systems are available in the literature [11,12,14]. The partial molar volumes V and E3 in the aqueous solutions of PEGs were calculated using the experimental data and the correlations suggested in Ref [51]. 

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