Titlebook: Insoluble Proteins; Methods and Protocol Elena García-Fruitós Book 2015 Springer Science+Business Media New York 2015 Biotechnology.Express

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PRO

Functional Expression of Plant Membrane Proteins in ,o provide detailed protocols for (1) the expression of plant peripheral or intrinsic membrane proteins and then for (2) their solubilization, from . membranes, for further purification steps and biochemical characterization.

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Frequency

High Cell-Density Expression System: Yeast Cells in a Phalanx Efficiently Produce a Certain Range ofs to the stationary phase in non-inducing condition, (2) suspend the cells to a small aliquot of inducing medium to form a high cell-density suspension or “a phalanx,” and then (3) give a sufficient aeration to the phalanx. Factors and pitfalls that affect the system’s performance are also described.

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Insect Cells–Baculovirus System for the Production of Difficult to Express Proteinse the procedure, starting from the cloning of a gene of interest into an expression transfer baculovirus vector, followed by generation of the recombinant virus by homologous recombination, evaluation of protein expression, and scale-up. Handling of insect cell cultures and preparation of bacmid for co-transfection are also detailed.

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Presbycusis

Beyond the Cytoplasm of ,: Localizing Recombinant Proteins Where You Want Themcan often be overcome by targeting protein expression to extracytoplasmic compartments (e.g., membrane, periplasm) or to the culture medium. This chapter discusses various strategies for exporting proteins out of the cytoplasm as well as tools for monitoring and optimizing these different export mechanisms.

OUTRE

Cleavable Self-Aggregating Tags (cSAT) for Protein Expression and Purificationed intein self-cleavage reaction releases the target protein into solution. These cleavable self-aggregating tags (cSAT, intein-18A/ELK16) provide a quick and efficient route for the production of proteins with modest purity (around 90 % in the case of intein-ELK16). Two application examples are included in the chapter.

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General Introduction: Recombinant Protein Production and Purification of Insoluble Proteins,luble and functional recombinant proteins is among the main goals in the biotechnological field. In this context, it is important to point out that under stress conditions, protein folding machinery is saturated and this promotes protein misfolding and, consequently, protein aggregation. Thus, the s

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Overcoming the Solubility Problem in ,: Available Approaches for Recombinant Protein Productionneous product are still unsolved. Although several strategies were developed to overcome these obstacles, at present there is no magic bullet that can be applied for all cases. Indeed, several key expression parameters need to be evaluated for each protein. Among the different hosts for protein expr