Proteins temperature sensitive

Temperature-sensitive mutations usually arise from a single mutation s effect on the stability of the protein. Temperature-sensitive mutations make the protein just unstable enough to unfold when the normal temperature is raised a few degrees. At normal temperatures (usually 37°C), the protein folds and is stable and active. However, at a slightly higher temperature (usually 40 to 50°C) the protein denatures (melts) and becomes inactive. The reason proteins unfold over such a narrow temperature range is that the folding process is very cooperative—each interaction depends on other interactions that depend on other interactions. 

A modification of the conventional soy protein isolate process has been investigated on a small pilot-plant scale. It is based on the absorption of water from the aqueous protein after extraction at pH 8.5 using temperature-sensitive polyisopyropylacrylamide gels, followed by spray drying to give a 96% protein isolate (111). 

Freeze drying, or lyophilization, is normally reserved for temperature-sensitive materials such as vaccines, enzymes, microorganisms, and therapeutic proteins, as it can account for a significant portion of... 

Using a random mutagenesis approach, respiratory-deficient (34) and temperature-sensitive (46, 47) mutants of the Rieske protein of the yeast bc complex have been selected. A large fraction of the point mutants had changes of residues in the bottom of the cluster binding subdomain (the loop /S7-/38) and in the Pro loop comprising residues 174-180 of the ISF (Fig. 9 see Section III,B,3) this indicates the importance of the Pro loop for the stability of the protein. Amino... 

Temperature-sensitive mutations are those which allow a virus to replicate at one temperature and not at another, due to a mutational alteration in a virus protein that renders the protein unstable at moderately high temperatures. For instance, temperature-sensitive mutants are known in which the phage will not be replicated in the host at 43 °C but will at 25 °C, although the host functions at both temperatures. Such mutations are called conditionally lethal, since the virus is unable to reproduce at the higher temperature, but replicates at the lower temperature. 

For a number of temperature-sensitive mutations it is possible to find (or make) a seond mutation in the protein that will suppress the effects... 

The biochemical reactions of living cells are controlled by enzymes, which are defined as temperature-sensitive, organic catalysts. Chemically, the enzymes are proteins that are specific in that they will act as catalysts to accelerate specific reactions. Some of these enzymes are associated with the protoplasm of the cell and perform their function within the cell. They are called intracellular enzymes. Other enzymes are excreted by the cell into the surrounding medium and are known as extracellular enzymes. 

Other cell lines used in permeability studies include the T84 human colonic adenocarcinoma colonic crypt cell model. This line has a reduced carrier expression, secrets mucus, and has very high resistance [31, 32], The IEC cell line is a rat fetal intestinal epithelium cell with higher permeabilities than Caco-2 cells [33], LLC PKi is a pig kidney epithelial cell line with low expression of efflux systems, but expression systems for transport proteins [32], 2/4/A1 cells are a conditionally immortalized rat fetal intestinal epithelium line with crypt cell-like morphology and temperature-sensitive differentiation [34], They form differentiated monolayers with tight junctions, increased brush border enzymes when grown on extracellular matrices with laminin. Transport of drugs with LP in 2/4/A1 monolayers was comparable to that in the human jejunum and up to 300 times faster than that in Caco-2 monolayers. In contrast, the permeability of HP drugs was comparable in both cell lines [34],... 

The need to transport temperature-sensitive raw materials and products, such as cell line, medium, large molecule drugs, and vaccines, means that some form of control during transportation is needed. For example, a working cell bank for the production of proteins may be transported in liquid nitrogen (-196 °C) and that of protein and vaccines in dry ice (-78 °C) in order to protect the integrity of the materials. Data loggers are used to record the temperature... 

Sefton BM, Hunter T, Beemon K. 1980. Temperature-sensitive transformation by Rous sarcoma virus and temperature-sensitive protein kinase activity. J Virol 33 220-229. 

The Sos recruitment yeast two-hybrid system was developed by Aronheim and colleagues (1997). It is now known as the CytoTrap yeast two-hybrid system and marketed by Stratagene (La Jolla, California, USA). The CytoTrap system differs from both the GAL4 and the LexA systems in that it is not dependent on transcription factor activation in the nucleus for the detection of protein-protein association. Instead, protein interactions are detected in the cytoplasm and involve the reconstitution of the Sos/Ras signaling pathway in conjunction with the temperature-sensitive yeast strain, cdc25H. 

From these experiments it is not possible to find out whether the El protein has its separate signal peptide, mainly because of the low yields of the El protein in the synchronized translation experiments. However, evidence for such a peptide has come from studies with a temperature-sensitive mutant of SFV in which the cleavage between the capsid and the p62 protein is blocked (Hashimoto etal., 1981). In cells infected with this mutant at the restrictive temperature, the El protein is assembled in the correct orientation into the membrane of the ER. The uncleaved protein containing the capsid and the p62 sequences (Mr = 87,000) is left in the cytoplasm. These findings suggest that the El protein has its own signal peptide which might be located in the 6K protein. 

A successful tool in the early studies of metabolic pathways was blocking the pathway at some specific point. This could be done by the use of either mutants or inhibitors. Schekman et al have isolated a number of yeast mutants with blocks in their secretion pathway (Schekman, 1982). It is not yet known which proteins these mutations affect, but this is clearly a most promising approach for identifying those components involved in transport. In animal cells there are no cellular mutants with blocks in the intracellular transport of protein from the ER to the cell surface. There are, however, genetic diseases which affect the routing of lysosomal enzymes to the lysosomes (Neufeld et al, 1975 Sly and Fischer, 1982). For viruses it has been possible to isolate temperature-sensitive mutants in which a mutation in the viral glycoprotein arrests... 

Molecular studies explained this apparent paradox when the temperature-sensitive G a Ala366Ser mutation of the G a protein was identified. At 32°C, the G a 366Ser mutation results in the constitutive cAMP accumulation that causes the testosterone secretion that is the hallmark of the testotoxicosis phenotype. At 37°C, however, the G a 366Ser mutation results in loss of adenylyl cyclase signaling, causing PHP-Ia. As a result, a single mutation that performs differently in different tissues causes precocious puberty and abnormalities of PTH and TSH (91). 

No cysteine residues are found for alpha(sl) and P-caseins do. If any S-S bonds occur within the micelle, they are not the driving force for stabilization. Caseins are among the most hydrophobic proteins, and there is some evidence to suggest that they play a role in the stability of the micelle. It must be remembered that hydrophobic interactions are very temperature sensitive. 

AOT/isooctane/ buffer Horse liver alcohol dehydrogenase Microemulsion system that is temperature sensitive to phase separate was used for recovery of proteins and enzymes [283]... 

Hydrolysis of primary caseins by plasmin. In 1969, Groves and coworkers showed that the y-casein fraction, as isolated by Hipp et al., is very heterogeneous, containing at least four distinct proteins y-casein, temperature-sensitive casein (TS, which is soluble in the cold but precipitates above 20°C), R-casein and S-casein. These four proteins were shown to be C-terminal fragments of /3-casein. In 1976, the nomenclature of the y-casein group was revised, as shown in Figure 4.7 and Table 4.3. 

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. 

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