Drugs and the pharmaceutical sciences

590 Elwell
refrigerated overnight. The resultant precipitate was pelleted by centrifugation and gently resuspended
in a small volume of b-galactosidase assay buffer. This assay buffer contains a final
concentration of 0.4 mM dithiethreitol (DTT). The reducing agent, DTT, is necessary to
disrupt disulfide bonds, thereby disaggregating b-galactosidase into its constituent,
enzymatically active monomers. The tetrameric form of this large enzyme (465,412 Da)
would be excluded from conventional polyacrylamide gels. The suspension was exhaustively
dialyzed against multiple exchanges of assay buffer and the resultant “mud”colored
suspension was transferred to a clean tube or small bottle and refrigerated until further use.
ENZYME PURIFICATION USING GEL-FILTRATION
CHROMATOGRAPHY
General Considerations
Chromatography is the technique of choice for high-resolution purification. These
methods, normally involving glass columns containing chromatographic media, are
universally used for the concentration and purification of protein preparations. In
choosing the appropriate chromatographic technique a number of considerations must be
taken into account. For protein products these factors include molecular mass, isoelectric
point, hydrophobicity and inherent biological affinities. Each of these properties can be
usefully exploited by specific chromatographic methods that may be scaled-up for use in
an industrial unit-process context.
Choice of Matrix
As discussed earlier, one of the chief limitations of gel-filtration chromatography is that
the separation may be slow and that the resolution of the emerging peaks is limited.
Resolution is limited because proteins do not bind to the matrix. As a consequence,
the column matrix for gel-filtration must be chosen with great care to insure the best
resolved separation of the target macromolecule from contaminants. The matrix should
be chosen so that the molecular weight of the target protein falls near the middle of the
matrix fractionation range. The fractionation range defines the approximate protein or
peptide molecular weights that can be separated with that particular chromatography
medium. Tables 1 and 2 provide information pertinent to the selection of column media.
Sephacryl-Hr Gel-Filtration Matrices
For this experiment Sephacryl HR (high resolution) medium has been chosen. As shown
in Table 1, this particular gel filtration matrix is a cross-linked copolymer of allyl dextran
and N, N-methylenebisacrylamide. According to Pharmacia, the narrow particle size
distribution of this medium, together with its steep selectivity curve, make Sephacryl HR
resolution matrices particularly useful for routine separations, especially when dealing
with relatively large amounts of crude sample.
Sephacryl 300-HR was chosen for two specific reasons. First, the molecular weight
of the b-galactosidase monomer (116,000 Da) fell within the fractionation range of this
matrix (10–1500 kDa). The second reason was that Craven et al. (1965) had reported the
efficient purification of E. coli b-galactosidase using a Sephadex G-200 column; the
fractionation range for Sephadex G-200 is close to that of Sephacryl 300-HR (Table 2).
The large size of our target protein was advantageous with respect to its ultimate
purification. Because of the relatively low resolution power of gel-filtration