Protein Chromatography by Dermot Walls & Sinéad T. Loughran

Author:Dermot Walls & Sinéad T. Loughran
Language: eng
Format: epub
Publisher: Springer New York, New York, NY

Key words

Ammonium sulfate precipitationBioinformaticsInclusion body solubilizationProtein refoldingTrichloroacetic acid precipitationThree phase precipitation

1 Introduction

Protein precipitation can be caused by the differential solubility between a protein-rich soluble phase and a solid chemical precipitant. Soluble proteins can be insolubilized by interaction with a suitable precipitant that decreases the protein’s attraction to the solvent and increases the protein’s attraction to other protein molecules, resulting in protein accumulation and eventually precipitation. The addition of low-molecular-weight substances, such as glycerol , polyethylene glycol , and sucrose, and high molecular weight substances such as serum albumin, can have significant effects on protein structure and stability. Preferential hydration of a protein molecule caused by the presence of these additives can increase the protein’s stability . Certain salts can also exert a stabilizing effect by “salting out” hydrophobic residues of a protein, causing the molecule to adapt a more compact, stable structure [1] frequently resulting in precipitation. The use of such protein precipitating molecules is an empirical process, the effects of any given substance on a protein must be determined experimentally. The use of additives can not only be used as a simple approach to increase the stability of a given protein, but also to actively effect protein precipitation. Protein precipitation can be used as a crude protein clean-up method from cell lysates, readily employed after bacterial over-expression of recombinant proteins.

Differential solubilization of proteins is often employed for proteomic analyses [2, 3], but it too can offer an alternative purification technique for nonsoluble recombinant proteins expressed in heterologous hosts. Recombinant proteins expressed as inclusion bodies can be readily separated from the host cell protein matrix, however careful solubilization and refolding are critical for obtaining suitable recombinant proteins for further downstream processes.

Protein modeling and in silico analysis can assist in the experimental design process, for example, in reducing the empirical experimentation required. Bioinformatic analyses can be undertaken to gain an understanding of the physicochemical properties of the target protein (e.g., amino acid composition, secondary structure prediction) and its propensity to aggregate at the outset of a protein expression project. The solubility of a protein upon expression is heavily dependent on its primary amino acid sequence and initial solubility prediction methods were based on the content of charged and turn-forming residues. In recent years, more advanced prediction software has been developed utilizing training sets of soluble and insoluble proteins. These tools are used to predict solubility before performing wet lab experiments thus saving effort, time and cost (recently reviewed elsewhere; [4]).

By way of a worked example, a typical recombinant protein precipitation and resolubilization procedure, preceded by judicious bioinformatic analyses, is outlined.

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