© 1991 Oxford University Press
Nucleic Acids Research, Vol. 19, No. 15 4295
Casein is a potent enhancer for restriction enzyme activity
Klaus Dreyer and Heinrich Schulte-Holthausen
Institute of Molecular Biology, Tumor Research, University (GHS) of Essen, D-4300 Essen 1, FRG
Submitted June 7, 1991
Restriction enzymes class II are ATP independent and require
magnesium ions for their activity. Bovine serum albumin (BSA)
and spermidine are added in some reaction buffers. In our
experience addition of spermidine sometimes causes severe
problems with high molecular weight DNA and BSA has only
a slight effect on enzyme activity. Here we show that casein,
the major protein component of milk is a potent stimulator of
restriction enzyme activity.
The bovine casein (Merk/Darmstadt) used for the experiments
was a purified preparation specified electrophoretically unique
by the supplier. The casein is dissolved in 10 mM Tris/HCl,
pH 8.0 at a concentration of 1 mg/ml. Stock solution is added
to the reaction mixture to give a final concentration of 50 to
100 /tg/ml casein. Autoclaving of casein solutions is not
recommended.
Casein was found active in all restriction buffer systems tested
so far, the ones according to Maniatis et al. (1), the potassium
glutamate buffer (2) and those obtained from the enzyme
suppliers.
Figures 1 and 2 show the enhancing effect of casein for the
restriction enzymes EcoRI and Hindm. Various enzyme
concentrations were tested in the presence or absence of casein.
Different kinds of restriction enzymes and enzymes of different
suppliers were tested. We detected an enhancement for all
restriction enzymes tested so far. The effect is seen with DNA
of eucaryotic-, plasmid- or viral origin commercially obtained
or prepared by ourselves.
Unspecific activity leading to unspecific cleavage or damage
of DNA was not observed.
Concentration experiments show that with casein concentrations
down to 1.5 ng/ml effects can be seen.
We have not elucidated the mode of action but we suppose
that the effects are of allosteric nature acting directly on the
enzyme and/or the DNA.
1 2
1011 12
Figure 1. EcoRI restriction digest with and without casein. 1 /ig Lambda-DNA
was digested in 50 /il reaction volume at 37°C in 50 mM Tris/Cl pH 7.5, 10
mM MgC^, 100 mM NaCl, 1 mM dithioerythritol for one hour. Reactions were
stopped by adding EDTA to a final concentration of 25 mM. An aliquot of each
reaction mixture was separated on a 0.5% agarose gel. Lanes 1 and 7: 2 units
EcoRI, lanes 2 and 8: 1 unit EcoRI, lanes 3 and 9: 0.5 units EcoRI, lanes 4
and 10: 0.25 units EcoRI, lanes 5 and 11: 0.125 units EcoRI, lanes 6 and 12:
0.0625 units EcoRI; lanes 1 - 6 contained no, lanes 7-12 100 jig/ml casein.
1 2 3 4 5
6 7 8 9
10
REFERENCES
1. Maniatis.T., Fritsch.E.F. and SambrookJ. (1982) Molecular Cloning, A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, NY.
2. McCelland.M., HanishJ., Nelson.M. and Patel,Y. (1988) Nud. Adas Res.
16, 364.
The content of this paper is subject to a patent application by Dreyer.K. and SchuheHolthauscn.H. (1991). Deutsches Patentamt, Offenlegungschrift DE 4000247A.
Figure 2. HindUl restriction digests with and without casein. 1 /ig Lambda-DNA
was digested in 50 pi reaction volume at 37°C in 10 mM Tris/Cl pH 8.0, 5 mM
MgCl2, 100 mM NaCl, 1 mM 2-mercaptoethanol for 1 hour. Reactions were
stopped by adding EDTA to a final concentration of 25 mM. An aliqout of each
reaction mixture was separated on a 0.5% agarose gel. Lanes 1 and 6: 2 units
HindUl, lanes 2 and 7: 1 unit HindUl, lanes 3 and 8: 0.5 units HindUl, lanes
4 and 9: 0.25 units Hindm, lanes 5 and 10: 0.125 units Hindu!; lanes 1-5
contained no, lanes 6 - 1 0 100 /ig/ml casein.