Enzyme Technology
Chromatography
Enzyme preparations that have been
clarified and concentrated are now in a suitable state for further purification
by chromatography. For enzyme purification there are three principal types of
chromatography utilising the ion-exchange, affinity and gel exclusion properties
of the enzyme, usually in that order. Ion-exchange and affinity chromatographic
methods can both rapidly handle large quantities of crude enzyme but
ion-exchange materials are generally cheaper and, therefore, preferred at an
earlier stage in the purification where the scale of operation is somewhat
greater. Gel exclusion chromatography (also sometimes called 'gel filtration' or
just 'gel chromatography' although it does not separate by a filtering
mechanism, larger molecules passing more rapidly through the matrix than smaller
molecules) is relatively slow and has the least capacity and resolution. It is
generally left until last as an important final purification step and also as a
method of changing the solution buffer before concentration, finishing and sale.
Where sufficient information has been gathered regarding the size and variation
of charge with pH of the required enzyme and its major contaminants, a rational
purification scheme can be devised. A relatively quick analytical method for
obtaining such data utilises a two-dimensional process whereby electrophoresis
occurs in one direction and a range of pH is produced in the other; movement in
the electric field determined by the size and sign of a protein's charge, which
both depend on the pH. As the sample is applied across the range of pH, this
method produces titration curves (i.e., charge versus pH) for all proteins
present.
A large effort has been applied to the development of chromatographic
matrices suitable for the separation of proteins. The main problem that has had
to be overcome is that of ensuring the matrix has sufficiently large surface
area available to molecules as large as proteins (i.e., they are macroporous)
while remaining rigid and incompressible under rapid elution conditions. In
addition, matrices must generally be hydrophilic and inert. Although the
standard bead diameters of most of these matrices are non-uniform and fairly
large (50 - 150 mm), many are now supplied as uniform-sized small beads (e.g., 4 - 6
mm diameter) which allows their use in very efficient
separation processes (high performance liquid chromatography,
HPLC), but at exponentially increasing cost with decreasing bead
size. Relatively high pressures are needed to operate such columns necessitating
specialised equipment and considerable additional expense. They are used only
for the small-scale production of expensive enzymes, where a high degree of
purity is required (e.g., restriction endonucleases and therapeutic enzymes).
Column manufacturers now supply equipment for monitoring and
controlling chromatography systems so that it is possible to have automated
apparatus which loads the sample, collects fractions and regenerates the column.
Such equipment must, of course, have fail-safe devices to protect both column
and product.
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This page was established in 2004 and last updated by Martin
Chaplin on
6 August, 2014
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