Protein denaturation limits

Protein extraction procedures employing chemicals such as detergents are effective in many instances, but they suffer from a number of drawbacks, not least of which is that they often induce protein denaturation and precipitation. This obviously limits their usefulness. Furthermore, even if the chemicals employed do not adversely affect the protein, their presence may adversely affect a subsequent purification step (e.g. the presence of detergent can prevent proteins from binding to a hydrophobic interaction column). In addition, the presence of such materials in the final preparation, even in trace quantities, may be unacceptable for medical reasons. 

The simplest approach to minimizing protein-wall interaction is to use a buffer pH at which interactions do not occur. At acidic pH the silanols on the surface of the capillary are protonated, and the net charge of the proteins is positive. At high pH, the wall is negatively charged, and so are the sample components. Both conditions result in electrostatic repulsion. Problems associated with operation at pH extremes include the potential instability of proteins (denaturation, degradation, and precipitation) and the limited pH range in which to achieve resolution. Additionally, operation at extreme pH does not eliminate all nonspecific interactions. 

One of the most important causes of poor resolution in protein and large peptide analysis with conventional phases is slowness of mass transport and of sorption/desorption. When selecting a column for a protein separation, it is important to look for obtaining rapid separations in order to limit protein denaturation. 

The third approach has provided several successful results, though its application is usually limited to low molecular-weight peptides or non-peptide drugs. The liposome formulations, however, suffer from difficulties of storage or reconstruction and unpredictable distribution. Immobilization into insoluble polymeric particles is difficult in the case of structured proteins, as it normally involves the use of organic solvents. Moreover, protein denaturation and degradation may occur... 

It is not always possible to obtain data over too broad a temperature range because the rates may be too slow at low temperatures, while protein denaturation can become a limiting factor at temperatures in excess of 40 or 55 C. 

The most important step of the inclusion body protein recovery scheme is the refolding of the denatured protein to form a biologically active product. Refolding can be relatively simple, for small monomeric proteins, or quite complicated when the protein consists of more than one polypeptide chains or contains several disulfide bonds. Difficult refolding processes can result in overall low recovery yields of active protein. This limits the use of inclusion body processes for the production of some recombinant products. 

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