Permeabilization, chemical, protein

Protein Release from Chemically Permeabilized Escherichia coli... 

An important factor complicating the recovery of recombinant proteins from Escherichia coli is their intracellular location. An alternative to the commonly used method of releasing these proteins by mechanical disruption is to chemically permeabilize the cells. The objective of this research was to characterize the protein release kinetics and mechanism of a permeabiliza-tion process using guanidine-HCl and Triton-XIOO. The protein release kinetics were determined as a function of the guanidine, Triton, and cell concentrations. Some of the advantages over mechanical disruption include avoidance of extensive fragmentation of the cells and retention of the nucleic acids inside the cell structure. 

From these results, three major differences between chemical permeabilization and mechanical disruption can be identified. First, the release occurs by fundamentally different mechanisms. With mechanical disruption the cells are essentially torn apart, whereas with chemical treatment the cell structure is still present but has been altered to allow release of intracellular components. Second, there is a nearly complete preferential release of protein over DNA. Third, there is a partial selective release of protein over RNA. This selectivity may result from a molecular sieving mechanism. The average protein molecular weight is 40,000 whereas the cellular DNA has a molecular weight of 2.5 x 10s (12). The molecular weight distribution of RNA 18% is 25,000, 27% is 500,000, and 55% is 1,000,000 is such that most of the RNA is also significantly larger than proteins (12). 

I. HETTWER AND WANG Protein Release from Chemically Permeabilized E. coli 5... 

Cheung, C.W., Cohen, N.G., Raijman, L. (1989). Channeling of urea cycle intermediates in situ in permeabilized hepatocytes. J. Biol. Chem. 264,4038-4044. Cohen, P.P. (1954). Nitrogen metabolism of amino acids. In Chemical Pathways in Metabolism (Greenberg, D.M., Ed.), Vol. 2, pp. 1-46. Academic Press, New York. Fisher, R.B. (1954). Protein Metabolism. Methuen, London. 

The localization of proteins and carbohydrates within cells and tissues with specific antibodies has long been proven to be a valuable technique. Immuno-localization procedures allow one to detect not only well-characterized cellular structures but also provide information about newly characterized proteins and carbohydrates. This chapter will review some of the advantages and drawbacks of common chemical fixation and permeabilization methods used for immuno-localization at the level of light microscopy. 

Another bio-inspired approach is to design polymersomes as enclosed reaction compartments for the development of nanoreactors, nanodevices, or artificial organelles, in which active compounds are not only protected from the environment, but also allowed to act in situ. For such function, membrane permeability is of crucial importance, since it allows the exchange of substrates/products with the environment of the pol)maersomes. Various methods have been reported to generate polymersomes with permeable membranes (i) polymers forming intrinsically porous membranes, (ii) polymer membranes that are permeable to ions as e.g. specific oxygen species, (iii) pore formation in pH responsive polymer membranes by chemical treatment, (iv) polymer membrane permeabilization by UV-irradiation, and (v) biopores or membrane proteins inserted into polymer membranes. ... 

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