by partial or incorrect folding, probably due to the reducing environment of theE. coli
cytoplasm. However, it was soon realised that this phenomenon could be used to
advantage in protein purification. The inclusion bodies can be separated from a large
proportion of the bacterial cytoplasmic protein by centrifugation, giving an effective
purification step. The recovered inclusion bodies must then be solubilised and
denatured and subsequently allowed to refold slowly to their active, native configur-
ation. This is normally achieved by heating in 6 M guanidinium hydrochloride
(to denature the protein) in the presence of a reducing agent (to disrupt any disulphide
bridges). The denatured protein is then either diluted in buffer or dialysed against buffer,
at which time the protein slowly refolds. Although the refolding method is not always
100% successful, this approach can often produce protein that is 50% or more pure.
Having carried out an initial fractionation step such as that described above, one
would then move towards using higher resolution chromatographic methods.
Chromatographic techniques for purifying proteins are summarised in Table 8.4,
and some of the more commonly used methods are outlined below. The precise
practical details of each technique are discussed in Chapter 11.
Charge
Proteins differ from one another in the proportions of the charged amino acids
(aspartic and glutamic acids, lysine, arginine and histidine) that they contain. Hence
proteins will differ in net charge at a particular pH. This difference is exploited in ion-
exchange chromatography (Section 11.6), where the protein of interest is bound onto
a solid support material bearing charged groups of the opposite sign (ion-exchange
resin). Proteins with the same charge as the resin pass through the column to waste,
after which bound proteins, containing the protein of interest, are selectively released
from the column by gradually increasing the strength of salt ions in the buffer passing
through the column or by gradually changing the pH of the eluting buffer. These ions
compete with the protein for binding to the resin, the more weakly charged protein
being eluted at the lower salt strength and the more strongly charged protein being
eluted at higher salt strengths.Size
Differences between proteins can be exploited in molecular exclusion (also known as
gel filtration) chromatography. The gel filtration medium consists of a range of beads
with slighly differing amounts of cross-linking and therefore slightly different pore
sizes. The separation process depends on the different abilities of the various proteins
to enter some, all or none of the beads, which in turn relates to the size of this protein
(Section 11.7). The method has limited resolving power, but can be used to obtain a
separation between large and small protein molecules and therefore be useful when
the protein of interest is either particularly large or particularly small. This method
can also be used to determine the relative molecular mass of a protein (Section 11.7.2)
and for concentrating or desalting a protein solution (Section 11.7.2).Affinity
Certain proteins bind strongly to specific small molecules. One can take advantage
of this by developing an affinity chromatography system where the small molecule324 Protein structure, purification, characterisation and function analysis