Protein Chemistry. Structure, Properties, Research Methods - Shendryk A.N. 2022

Methods for Experimental Study of Protein Structure
Protein Purification Methods

For any protein research, whether determining its Structure or biological function, the protein must first be obtained in a purified form free of contaminants. Achieving this in practice is challenging because nature does not synthesize Proteins outside of living Cells. Consequently, Cell Disruption invariably yields a highly complex mixture. As a rule, the protein Isolation and Purification Procedure involves multi-stage Processing of the starting biological material. At each successive step, a portion of extraneous substances is removed until the protein is obtained in pure form. Naturally, each stage entails not only purification but also the inevitable loss of a greater or smaller amount of the protein. Typically, one manages to recover 5-10% of the initial protein in pure form.

There is no universal method for Protein Purification. Each subsequent step is chosen by trial and error, and success depends on the expertise, precise knowledge, and intuition of the experimenter. An acceptable purification pathway is considered to be one that yields (retains) the maximum amount of protein in the highest state of purity. If this result can be achieved in 4-6 stages, one can be quite satisfied.

As an example, let us examine the results obtained by Anraku in isolating a galactose-binding protein from E. coli cells (see Table 4.2). The isolated protein binds simple sugars and facilitates their Transport Across the bacterial cell membrane.

Protamine Treatment (stage 2) does not result in significant removal of contaminant proteins (2,352 out of 2,600 mg remain). Protamine is used to remove DNA and RNA from the homogenate via precipitation. The third step, ammonium sulfate fractionation, is a widely used technique in Protein Isolation. Ammonium sulfate decreases Protein solubility (the salting-out effect).

The final three stages involve chromatographic Separation on ion exchangers and Gel filtration on DEAE-Sephadex.

Class="center">Table 4.2 Sequence of the isolation process for the galactose-binding protein from E. coli cells

* Yield (%) is The ratio of the total activity at a given stage to the total activity of the initial extract

Total protein content was determined by the Lowry Method. Total activity was measured as follows: the protein fraction was placed in a semipermeable dialysis tubing and dialyzed for several hours against a solution containing a known amount of ¹⁴C-galactose. The radioactivity of the sample induced by the binding of labeled galactose to the protein was then measured.