Proteins cellular expression

The calculation of protein proximity and hence association on the basis of sensitized emission or FSPIM requires correction for direct acceptor excitation and donor bleed through using several mathematical models and instrument correction factors [22, 59-61], which is difficult to control [22] (see also Chapters 7 and 8). A high detected acceptor to donor signal ratio in these techniques may also reflect other phenomena than FRET. For instance, this ratio is dependent on cellular expression levels and subcellular localizations, which are difficult to control. Additionally, for the widely used... 

The staphylokinase gene has been cloned in E. coli, as well as various other recombinant systems. The protein is expressed intracellularly in E. coli at high levels, representing 10-15 per cent of total cellular protein. It can be purified directly from the clarified cellular homogenate by a combination of ion-exchange and hydrophobic interaction chromatography. 

Glutamate transporters in brain are coded by five different but closely related genes, SLC1A1-4 and SLC1A6. There are several trivial names for each of the corresponding proteins. The transporters can all symport one Glu, with three Na+ and one H+, and antiport one K+ within each cycle, but they differ in their cellular expression. 

Proteins can undergo different rounds of palmitoylation and depalmitoylation, either constitutively or as a response to signals." " Here the Ras proteins are the most commonly discussed examples. As described above, all Ras proteins are expressed with the CAAX-box and are subject to post-translational modifications. First, they get farnesylated and after proteolysis and methylation of the C-terminus, H-/N-Ras as well as K-Ras 4A get further palmitoylated at additional cysteines present in their C-terminus. Palmitoylation occurs in the Golgi apparatus and via vesicular transport the farnesylated and palmitoylated proteins are directed to the plasma membrane (PM). The palmitoyl thioester is hydrolyzed at multiple cellular sites and the protein is transported back to the Golgi via a nonvesicular pathway (Scheme 3)." ... 

The host bacteria used for production of recombinant proteins are usually E. coli, or Bacillus subtilis they may express proteins at 1 % to over 50% of the cellular protein, depending on such variables as the source, promoter structure, and vector type. Generally the proteins are expressed intracellularly, but leader sequences for excretion may be included. In the latter case, the protein is generally excreted into the periplasmic space, which limits the amount that can be produced. Excretion from grampositive species such as B. subtilis sends the product into the culture medium, with little feedback limitation on total expression level. 

Kim G. M., Xu J., Xu J. M., Song S. K., Yan P., Ku G., Xu X. M., and Hsu C. Y. (2001). Tumor necrosis factor receptor deletion reduces nuclear factor-kappa B activation, cellular inhibitor of apoptosis protein 2 expression, and functional recovery after traumatic spinal cord injury. J. Neurosci. 21 6617-6625. 

Simak J, Holada K, D Agnillo F, Janota J, Vostal JG. Cellular prion protein is expressed on endothelial cells and is released during apoptosis on membrane microparticles found in human plasma. Transfusion 2002 42 334-342. 

Our laboratory has provided significant contributions in the area of myristoyla-tion. We discovered and purified the myristoyl-CoA binding protein (MCBP) from bovine cardiac muscle (Raju and Sharma 1997). In cardiac tissues there is a high level of cAMP-dependent protein kinase expression whose catalytic subunit is myristoylated (Carr et al. 1982). The catalytic subunit of cAMP-dependent protein kinase and the beta subunit of calcineurin are myristoylated proteins localized in the cytoplasm (Selvakumar et al. 2006, 2002 Rajala et al. 2000 Johnson et al. 1994 Carr et al. 1982 Aitken et al. 1982). Recently it has been shown that dephosphorylation of the catalytic subunit of myristoylated and nonmyristoylated cAMP-dependent protein kinase at Thr-197 by cellular protein phosphatase and protein... 

Recently progress has been made in studying various aspects of one particular stress, i.e., the process by which plants protect themselves from heat. Under normal conditions, it has been found that plants produce enzymes required for normal cellular metabolism (Figure 23A). Figure 23B illustrates the situation when the plant senses an increase in temperature. New genes, called the "heat shock genes", are turned on, with the subsequent production of heat shock proteins and expression of normal metabolism genes is reduced. 

Fig. 4.3. Farnesylated CaaX proteins are more dependent on carboxylmethylation by Icmt for correct membrane localization than are geranylgeranylated CaaX proteins. (A) The indicated GFP-tagged Ras isoform was expressed in MEFs deficient in Icmt (-/-) or wild-type MEFs from httermates (+/+). All three Ras isoforms showed marked mislocalization with accumulation of GFP-Ras in the fluid phase cytosol. (B) GFP-tagged Rho proteins were expressed in the same MEFs as in (A). Activated forms of Racl, RhoA, and Cdc42 were utilized for clarity since these forms do not bind to the cytosolic chaperone RhoGDI but instead associate with the cellular membranes to which their C-termini are targeted. In contrast to farnesylated Ras proteins, geranylgeranylated Rho proteins are localized with similar patterns in both +/+ and -/- MEFs.

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