Folding, green fluorescent proteins

More recently Michnick and co-workers have introduced a dihydrofolate reductase complementation system, which seems to be particularly robust [61 - 65], They attribute the success of this system to the fact that the N-terminal (1 - 105) and C-terminal (106 - 186) DHFR fragments do not fold until they are dimerized. In addition to the obvious selection for essential metabolites dependent on the reduction of dihydrofolate to tetrahydrofolate, protein-protein interactions are detected based on the retention of a fluorescein-methotrexate conjugate. Several other enzymes have been employed for the design of complementation assays, including green fluorescent protein, which allows screens based on fluorescence or FRET [66 - 68]. As with the bacterial transcription assays, these complementation systems are new. It will be interesting to see if, as the selections are optimized, these systems prove competitive with the Y2H assay. 

Waldo GS, Standish BM, Berendzen J, Terwilliger TC. Rapid protein-folding assay using green fluorescent protein. Nat. Biotechnol. 1999 17 691-695. 

There have been quite a few papers published which deal with fluorescence methods in prion research. Generally, three different approaches have been utilized. First, PrP chimeras with fluorescent proteins (e.g., green fluorescent protein, GFP) have been produced to study various aspects of cellular questions or protein folding. For example, fluorescence imaging techniques have been applied to study prion propagation in yeast (reviewed in [45]). Other examples concern attempts to study protein folding and the nature of aggregates. Kawei-Noma et al. analyzed the... 

Vasiljevic S, Ren J, Yao Y, Dalton K, Adamson CS, Jones IM (2006) Green fluorescent protein as a reporter of prion protein folding. Virol J 3 59... 

Other reports show that intracellular expression of misfolded or unstable proteins can be dramatically improved by directed evolution. For example, directed evolution increased the expression of disulfide-containing antibody fragments in E. coli 50-fold, to reach a level of more than 0.5 g/L 212L The expression of a wide spectrum amidase of B. stearothermophilus in E. coli was improved 23-fold by two mutations 2101. And, in vivo fluorescence of the green fluorescent protein was improved 45-fold by increasing its solubility and native folding in E. co/t[1401. 

Most organelles have a characteristic architecture that is important for function, but may serve as an additional impediment to diffusion within the cell compartment. Organelle membranes within the cell can serve as local barriers (Table 4.9), which affect the rate of diffusion of molecules locally. This effect has been estimated for diffusion in the mitochondrial matrix and the endoplasmic reticulum (Figure 4.28a and b). The mitochondrial matrix was modeled as a closed cylinder with multiple barriers occluding the lumen to simulate the mitochondrial cristae the presence of multiple barriers produced a modest decrease in the rate of diffusion down the axis of the cyUnder (Figure 4.28a) this calculation is consistent with recent measurements of green fluorescent protein (GFP) diffusion in the mitochondrial matrix, which was approximately three-fold slower than diffusion in saline. The endoplasmic reticulum was modeled as an array of interconnected cylinders with a continu-... 

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