sulphate. Protamine sulphate is a mixture of small, highly basic (i.e. positively charged)
proteins, whose natural role is to bind to DNA in the sperm head. (Protamines are
usually extracted from fish organs, which are obtained as a waste product at canning
factories.) These positively charged proteins bind to negatively charged phosphate
groups on nucleic acids, thus masking the charged groups on the nucleic acids and
rendering them insoluble. The addition of a solution of protamine sulphate to the
extract therefore precipitates most of the DNA and RNA, which can subsequently be
removed by centrifugation. An alternative is to use polyethyleneimine, a synthetic long
chain cationic (i.e. positively charged) polymer (molecular mass 24 kDa). This also binds
to the phosphate groups in nucleic acids, and is very effective, precipitating DNA and
RNA almost instantly. For bacterial extracts, carbohydrate capsular gum can also be a
problem as this can interfere with protein precipitation methods. This is best removed by
totally precipitating the protein with ammonium sulphate (see below) leaving the gum in
solution. The protein can then be recovered by centrifugation and redissolved in buffer.
However, if lysozyme (plus detergent) is used to lyse the cells (see Section 8.3.3) capsular
gum will not be a problem as it is digested by the lysozyme.
The clarified extract is now ready for protein fractionation steps to be carried out.
The concentration of the protein in this initial extract is normally quite low, and in fact
the major contaminant at this stage is water! The initial purification step is frequently
based on solubility methods. These methods have a high capacity, can therefore be
easily applied to large volumes of initial extracts and also have the advantage of
concentrating the protein sample. Essentially, proteins that differ considerably in their
physical characteristics from the protein of interest are removed at this stage, leaving
a more concentrated solution of proteins that have more closely similar physical
characteristics. The next stages, therefore, involve higher resolution techniques that
can separate proteins with similar characteristics. Invariably these high resolution
techniques are chromatographic. Which technique to use, and in which order, is more
often than not a matter of trial and error. The final research paper that describes in
four pages a three-step, four-day protein purification procedure invariably belies the
months of hard work that went into developing the final ‘simple’ purification protocol!
All purification techniques are based on exploiting those properties by which
proteins differ from one another. These different properties, and the techniques that
exploit these differences, are as follows.Stability
Denaturation fractionation exploits differences in the heat sensitivity of proteins. The
three-dimensional (tertiary) structure of proteins is maintained by a number of forces,
mainly hydrophobic interactions, hydrogen bonds and sometimes disulphide bridges.
When we say that a protein is denatured we mean that these bonds have by some
means been disrupted and that the protein chain has unfolded to give the insoluble,
‘denatured’ protein. One of the easiest ways to denature proteins in solution is to heat
them. However, different proteins will denature at different temperatures, depending
on their different thermal stabilities; this, in turn, is a measure of the number of bonds
holding the tertiary structure together. If the protein of interest is particularly heat
stable, then heating the extract to a temperature at which the protein is stable yet other321 8.3 Protein purification