Restriction Enzymes 111
enzymes. In general, two or three chromatographic steps yield restric-
tion enzyme preparations that are devoid of nonspecific nucleases
(25,26). In combination, they might yield a homogeneous enzyme
preparation. Quite often, however, it is necessary to add an affinity
chromatography step on blue sepharose, which contains an immobi-
lized Cibacron dye, as well as DNA-cellulose or variants thereof, as
final purification steps in the isolation of restriction enzymes. Recently,
an eicosomer (oligonucleotide) affinity matrix containing the recog-
nition sequence of EcoRI was produced that allows the enzyme to be
purified to near homogeneity in a single step starting from a crude cell
extract (27). It remains to be shown whether this procedure is of gen-
eral utility. The possibility of cloning the genes for restriction enzyme
into expression vectors has led to a considerable simplification of the
isolation procedures for these enzymes. Typically, 100-1000 mg of
homogeneous protein can be isolated from 1 kg of wet cell paste using
two chromatographic steps only (e.g., 28,29). Care has to be taken that
overproduction does not lead to aggregation and inactivation (30) (see
note added in proof at end of chapter).
2.2. Molecular Properties
To date about 30 Type II restriction enzymes have been sequenced
(Table 2). Some of them show a high degree of homology, such as
EcoRI and RsrI (47),TaqI and TthHB81 (Zebala, Mayer, and Barany,
personal communication), and BsuBI and PstI (Xu, Kapfer, Walter, and
Trautner, personal communication). Others have stretches of homol-
ogy, such as BcnI and MvaI (Butkus, Padegimiene, Menkevicius,
Butkiene, Timinskas, and Janulaitis, personal communication), as well
as BsuFI and MspI (31). It is noteworthy that these pairs of restriction
enzymes are isoschizomers. This indicates that their common function
at least in part is reflected in a common structure. A similar signifi-
cance can be attributed to our finding that several restriction enzymes
(DpnlI, EcoRV, HinclI), which cleave the DNA within the sequence
G-/-C or G--/--C where the dashes stand for As or Ts, have a common
protein sequence motif--Ser Gly X X X Asn X lie-. This sequence in
EcoRV is involved in DNA recognition as shown by site-directed
mutagenesis studies. In addition, EcoRV shows a stretch of sequence
homology to SmaI. In EcoRV, this stretch corresponds to a region impli-
cated in backbone contacts outside the recognition sequence. Otherwise,