Protein Solubilization is a process that transforms a non-soluble form of protein into a homogenous solution. 

     

    The initial state of the protein is some form of an aggregate. This can be a cloudy solution, containing precipitated protein or a sample of dry lyophilized protein 'cake'. The aggregated state often appears turbid in solution and tends to clog fine mesh filters. The particulate matter formed by aggregated protein either does not pass through a gel permeation matrix or runs in the 'void' - the exclusion volume in SEC (Size Exclusion Chromatography). Dynamic Light Scattering (DLS) and Analytical Ultracentrifugation can be used to characterize the average particle size and shape.

    The soluble protein solution is homogenous and protein molecules diffuse as individual entities, monomer, dimers etc. Such particles do not sediment spontaneously, they can not be filtered easily and typically form symmetrical elution peaks when passed through a Size Exclusion Chromatography column. Dynamic Light Scattering and Analytical Ultracentrifugation usually show mono-disperse distribution of sizes, corresponding to single or systematic oligomeric species (dimer, trimer etc.).

    The process of transferring the aggregated protein into a homogenous protein solution - solubilization - involves breaking up interaction that hold the aggregate together. Depending on the aggregate the solubilization process may involve breaking of chemical bonds (i.e. reduction of disulfide bonds in intramolecularly bonded protein aggregate), breaking of ionic interactions, hydrogen bonds, van der Waals interactions and interactions that are based on the Hydrophobic Effect.  Certain reagents promote the splitting of such bonds, i.e. beta-mercapto ethanol breaks disulfide bonds and urea can solubilize precipitated protein.

     

    There are different  reasons to solubilize protein samples that are rooted in the subsequent use of the protein sample. And, depending on the utility of the resulting sample, specific solubilization methods are preferred and others to be avoided.

     

    Table: Utility of protein solubilization for different purposes

     

 

Purpose

Solubilization Considerations

Produce sample for SDS-PAGE

Remove large particulate matter and dissolve protein sample into a homogenous solution; folding and function do not matter; may be run as reduced (broken disulfide bonds) on unreduced forms (disulfide bonds intact).

Prepare for Chromatographic Protein Purification

Dissolve reversibly precipitated protein (i.e. Ammonimu sulfate induced aggregation or 'inclusion bodies') and produce a homogenous solution; if folding does not matter this can be done with urea; if properly folded protein is required, additives may be used to break up the precipitate.

Preparation of injectable biologics drug

A lyophilized protein 'cake' is dissolved in buffer with a minimal fraction of aggregated protein in the final solution.  Extensive formulation and optimized lyophilization techniques are necessary to produce a protein 'cake' that readily dissolves into solution.

Functional characterization, (enzymatic) assay

Proteins work by interacting with other molecules, requiring proper fold and tertiary and quarternary structure. Functional assays require proper solubilization to determine specific activity and produce reproducible data.

 

     

    Membrane Protein Solubilization

     

    This is a special case where the membrane protein is transferred from its native environment, i.e. cell membranes, to an artificial environment, such as a protein detergent complex (micelle). Initially the membrane protein is dissolved in two media: aqueous and membraneous. During its solubilization the membrane protein environment changes from a lipid environment (membrane) to that of a non-native environment where lipid molecules are replaced by detergent molecules. Membrane Protein Solubilization is a prerequisite for membrane protein purification employing chromatographic methods.

     

     

    Practical Aspects


    There are many ways to transform protein aggregate into the proper solubilized state. Depending on the state of the aggregated protein material and the sought after utility of the solubilized protein material  the solubilization conditions will likely require screening different solubilization reagents (additives) and solubilization techniques. Appropriate analytical tools are required to assess the progress of such an optimization trial.

     

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