140

NOTES & TIPS

Anomalous Estimation of Protease Molecular

Weights Using Gelatin-Containing SDSPAGE
Katrina M. Hummel, Alan R. Penheiter,1
Allen C. Gathman, and Walt W. Lilly2
Department of Biology, Southeast Missouri State
University, Cape Girardeau, Missouri 63701
Received August 29, 1995

Since first proposed by Heussen and Dowdle (1),

substrate-containing polyacrylamide gel electrophoresis has proven to be a valuable method for dissecting the proteolytic systems of many organisms
(2 6). Several variations of this basic technique have
been adapted to specific systems and situations; however, one technique, casting gelatin into an SDS3
polyacrylamide gel, has become particularly popular.
Normally, samples are loaded unheated in SDS-loading buffer lacking b-mercaptoethanol. Following
electrophoresis, the gel is equilibrated in Triton X100 to remove the SDS and allow refolding of the
protein. After an appropriate incubation, the gels are
stained with Coomassie blue and destained. Clear
areas show where proteases have digested the substrate. The method has numerous advantages, including high resolution and adaptability (with care)
to a quantitative assay (7, 8). In addition, gelatin
SDS PAGE has been used often to estimate the molecular weights of unknown proteases in complex extracts (2, 3, 6, 9). It was in performing exactly this
latter activity that we discovered the anomalous behavior of a protease from the basidiomycete fungus
Schizophyllum commune. This protease, ScPrB, is a
metalloprotease of ca. 57 kDa based on standard
SDS gel electrophoresis of the purified protein (10).
When compared with standard markers in gelatin
SDS PAGE, its molecular weight appears to be 70
kDa. Because of this discrepancy, we decided to test
several other proteases by comparing their relative
molecular weights (based on a standard curve) obtained from gelatin SDS PAGE to that determined
by standard SDS PAGE. The results suggest that
molecular weights determined in gelatin SDS
PAGE may not be reliable.
The effect of gelatin on the mobilities and relative
molecular weights of five proteases was tested. ScPrB
1
Present address: Department of Biochemistry, University of Nebraska, Lincoln, NE.
2
To whom reprint requests should be addressed. Fax: (314) 6512223. E-mail: C749SCB@SEMOVM.SEMO.EDU.
3
Abbreviations used: SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; BAEE, N-a-benzoyl-L-arginine ethyl
ester.

ANALYTICAL BIOCHEMISTRY

from crude extracts of S. commune was obtained and

diluted as described in Gordon and Lilly (7). Chymotrypsin (EC 3.4.21.1; Type 1-S), trypsin (EC 3.4.21.4;
Type IX), papain (EC 3.4.22.2; Type II), and elastase
(EC 3.4.21.36; Type I) were obtained from Sigma
Chemical Company. Samples were mixed with loading buffer lacking b-mercaptoethanol (4 parts sample:1 part loading buffer) and loaded directly onto
gels without heating. The following amounts of the
commercial proteases were loaded: chymotrypsin, 40
mg (2.1 BAEE units); trypsin, 2.5 mg (2.8 BAEE
units); papain, 200 mg (0.58 units); elastase, 6.25 mg
(0.625 BAEE units). One BAEE unit is defined as the
amount of enzyme required to hydrolyze 1 mmol Na-benzoyl-L-arginine ethyl ester per minute at 257C;
for papain, one unit is defined as the amount of enzyme required to hydrolyze 1 mg of elastase in 20
min at 257C. In addition to proteases, a 10-kDa protein ladder (Gibco-BRL) was run in each gel. Mobilities were determined relative to the dye front, and
relative molecular weights were established on the
basis of a standard curve derived from plotting the
relative mobilities vs log Mr of the standards.
Standard SDSPAGE was performed in miniformat
gels using the Laemmli (11) buffer system. Different
percentages of T gel concentrations were made using a
40% T, 37.5:1 acryamide:bisacrylamide stock solution.
Protease activities were visualized by blotting the separated proteins to a second polyacrylamide gel. The blot
gel was made as follows: 7.5% T acrylamide, 0.5%
(w/v) gelatin, 0.1 M Tris buffer, pH 7.0. The original
separating gel was equilibrated for 30 min in 2.5% (v/v)
Triton X-100 in 0.1 M citrate buffer, pH 6.0. Following
equilibration, the gel was placed on three sheets of 3mm paper soaked in 0.1 M Tris, pH 7.0. The blot gel
was placed directly on top of the separating gel followed
by a single nylon membrane three sheets of 3-mm paper and a 2-in. stack of paper towels. Capillary transfer
of protease activity was allowed to proceed for 3 h, at
which time both gels were stained with Coomassie blue
and destained.
Gelatin SDSPAGE was performed as described by
Gordon and Lilly (7). Gelatin was added to 0.5%
(w/v) final concentration by substituting an appropriate
amount of 4% (w/v) gelatin for a portion of the water in
the gel recipe. Following electrophoresis the gels were
equilibrated in 0.1 M citrate, pH 6.0, /2.5% (v/v) Triton
X-100 (for ScPrB, chymotrypsin and elastase) or in 0.1
M tris, pH 8.0, /2.5% (v/v) Triton X-100 (for trypsin).
A second 30-min wash in equilibration buffer without
Triton X-100 was followed by incubation in buffer only
for 1 to 3 h. The gels were stained with Coomassie blue
and destained.
The addition of gelatin to SDS polyacrylamide
gels appears to reduce the migration of the standard
proteins by 15 to 20% (Fig. 1). In 7.5% T acrylamide,

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NOTES & TIPS

the addition of gelatin reduced mobility of the standards by 13 { 3.4%; in 10% T, the reduction was 16
{ 3.8%; and in 12.5% T, the reduction was 15 { 2.8%.
As this effect is consistent regardless of acrylamide
concentration, it is likely due to the added sieving
effect of gelatin polymerized into the matrix of the
gel. We have also routinely observed this same level
of retardation with nonprotease proteins from S.
commune (data not shown). In contrast, proteases migrate much more slowly in gelatin SDS PAGE than
would be predicted by the retardation of standards.
For the proteases we tested, the reduction of mobility
due to addition of gelatin in a 10% T acrylamide gel
was 48% for chymotrypsin, 44% for elastase, 43% for
trypsin, 41% for papain, and 28% for ScPrB. The degree of retardation is not constant from one protease
to another. This means that it is impossible to predict
directly what the position of a protease band will be
in a gelatin SDS gel based on its molecular weight.
In addition, it suggests that molecular weights of proteases cannot be estimated accurately in gelatin
SDS gels.
The degree of overestimation of relative molecular
weights is shown in Table 1. The magnitude of the
overestimation appears to be reduced with increasing
acrylamide concentration in the gels. This would be
expected because of the increased sieving effect of the
higher acrylamide concentration. It is also important
to note that the standard SDS PAGE may not give
an accurate measurement of Mr when samples are
not reduced or heated. Chymotrypsin consistently
migrated to Mr 36,000 when loaded unheated and
run on SDS PAGE, even though its correct Mr is
approximately 24,000. Unfortunately, few, if any,
proteases can be reactivated following electrophoresis if they have been first completely denatured.

TABLE 1

Effect of Gelatin on Estimated Molecular

Weights of Proteases
Mr estimate

Sample
Elastase
Mr 22500a
Chymotrypsin
Mr 24,000
ScPrB
Mr n/a

Gel
%T

SDS
PAGE

Gelatin
SDSPAGE

Overestimate
(%)

7.5
10
12.5
7.5
10
12.5
7.5
10
12.5

ndb
21,300
24,500
35,500
36,300
33,800
56,200
57,500
57,500

nd
39,400
36,300
79,400
60,300
53,703
79,400
74,100
65,300

nd
84
48
123
66
58
41
29
14

a
Mr as determined from published amino acid sequences. The
amino acid sequence of ScPrB has not been determined.
b
nd, not determined.

The mechanism of specific retardation of proteases

in gelatin SDS gels is unknown; however, we suspect an affinity interaction between the enzymes active site and gelatin. This is supported in part by our
observation that ScPrB has an Rm of 0.07 in a native
gelatin-containing polyacrylamide gel (10% T) and
that, loaded in high concentration, it actively digests
gelatin during the electrophoretic run (12, 13). Regardless of the mechanism of interaction, our data
suggest that great caution must be used in assigning
molecular weights to proteases on the basis of relative migration in gelatin SDS polyacrylamide gels.
Acknowledgments. The authors thank Robert Bilbrey for useful
discussions. This work was supported by a grant from National Science Foundation (DMB 93-03879).

REFERENCES
1. Heussen, C., and Dowdle, E. B. (1980) Anal. Biochem. 102, 196
202.
2. Garcia-Carreno, F. L., Dimes, L. E., and Haard, N. F. (1993)
Anal. Biochem. 214, 6569.
3. Harrington, D. J., and Russel, R. B. (1994) FEMS Microbiol.
Lett. 121, 237242.
4. Michaud, D., Faye, L., and Yelle, S. (1993) Electrophoresis 14,
9498.
5. North, M. J., Cotter, D. A., and Franek, K. J. (1990) J. Gen.
Microbiol. 136, 835840.
6. Rodier, M-H., El Moudni, B., Ghazali, M., Lacroix, C., and Jacquemin, J-L. (1994) Exp. Mycol. 18, 267270.
7. Gordon, L. J., and Lilly, W. W. (1995) Curr. Microbiol. 30, 337
343.
FIG. 1. Migration of standard protein markers (10-kDa ladder,
Gibco-BRL) in standard SDSPAGE (10% T) and gelatinSDS
PAGE (0.5% gelatin, 10% T).

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8. Kleiner, D. E., and Stetler-Stevenson, W. G. (1994) Anal. Biochem. 218, 325329.

9. North, D. J., Scott, K. I., and Lockwood, B. C. (1988) Biochem.
J. 254, 261268.

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NOTES & TIPS

10. Penheiter, A. R. (1995) M.S. thesis, Southeast Missouri State

University, Cape Girardeau, MO.
11. Laemmli, U. K. (1970) Nature (London) 277, 680685.
12. Lilly, W. W., Higgins, S. M., and Wallweber, G. J. (1990) Mycologia 82, 505508.
13. Lilly, W. W., Bilbrey, R. E., Williams, B. L., Loos, L. S., Venable,
D. F., and Higgins, S. M. (1994) Mycologia 86, 564570.

Generation of Site-Directed Mutagenesis by

Extralong, High-Fidelity Polymerase Chain
Reaction
Nianli Sang,*,, Gianluigi Condorelli,
Antonio De Luca,*,, Timothy K. MacLachlan,*,,
and Antonio Giordano,*,,,1
*Jefferson Cancer Institute, and Departments of
Microbiology/Immunology and Pathology, Thomas
Jefferson University, Philadelphia, Pennsylvania 19107
Received September 15, 1995

Site-directed mutagenesis is one of the most powerful

tools in molecular biology and genetics. It is particularly useful to study the relations between the structure and function of the cloned genes and/or gene products. The classical site-directed in vitro mutagenesis
involves many complicated procedures (1, 2), although
various modifications have been made to improve its
efficiency, versatility, and simplicity (36) since its
principles were first described (1). To overcome those
disadvantages, several researchers developed PCR-mediated protocols (7, 8) to directly incorporate mismatched primer or 5*-add-on sequences into PCR amplified gene fragments. However, the application is
often limited by the absence of suitable restriction sites
and by the primer lengths that can be achieved by oligonucleotide synthesizers. Even the recently developed,
PCR-based techniques still require multiple steps and
sometimes need more than one pair of primers to generate deletions (8, 9). In addition, high frequency of undesirable mutation is also an obstacle to its widespread
application, especially for large DNA fragments. When
a panel of 12S E1A (an oncoprotein encoded by the
early region 1A of the adenoviral genome) mutants was
required for our research, we noticed that recently, an
extralong, high-fidelity PCR technique had been developed and reported (10, 11). Based on that technique,
we adapted the inverse PCR method for site-directed
1
To whom correspondence should be addressed at Jefferson Cancer Institute, BLSB, Rm. 531, Thomas Jefferson University, Philadelphia, PA 19107. Fax: 215-923-9626.

ANALYTICAL BIOCHEMISTRY

mutagenesis (Fig. 1A) and tested its applicability by

using this strategy to generate a panel of 12S E1A
mutants (Fig. 1B).
A cDNA fragment encoding 12S E1A was inserted
into the pGBT9 vector which was obtained from Clontech as part of the Matchmaker kit for the two-hybrid
system. This led to an in-frame fusion of the GAL4
DNA binding domain, which is encoded by sequences
in the vector, to 12S E1A and this construct was further
used as the template for subsequent mutagenic PCR.
As the size of the pGBT9 vector is 5.5 kb and the insert
is 0.72 kb, the size of this template construct is 6.22
kb. Primers used were outlined in Table 1. For each
mutant, 50 ml PCR reaction was set up containing 11
XL reaction buffer, 1 unit rTth DNA polymerase (Perkin Elmer), 1.1 mM Mg(OAc)2 , 15 pmol of each primer,
and 200 mM of each dNTP. To ensure the fidelity and
specificity, the hot start technique was employed. After
initial holding of the reaction mixtures at 947C for 2
min, we performed a two-temperature PCR reaction
(947C, 30 s, 657C, 6 min and 30 s, autoextension 10 s
per cycle) for 2530 cycles. The proper amount of the
PCR product was incubated with Klenow (Promega)
and dNTPs at 377C for 30 min to ascertain that all
the ends were blunt. Then the products were purified
through a 0.6% agarose gel with Genclean Kit II (Sun
Bioscience Inc). Phosphorylation of the ends of the PCR
fragment was done at 377C for 1 h with T4 polynucleotide kinase in 11 provided buffer (Promega) in the presence of 1 mM ATP. The kinase reaction was terminated
by incubation at 657C for 10 min. One hundred nanograms of end- phosphorylated PCR products was circularized by 5 units T4 DNA ligase (Promega) in 20 ml
reaction at 167C overnight. Four microliters of the ligation was used to transform DH5a competent cells
(Gibco). The plasmids prepared from initial transformants were first analyzed by restriction digestion
and the results showed that more than four of five of
the colonies contained the intended inserts. The fidelity
and specificity were confirmed by subsequent sequencing analysis of the E1A insert and part of the vector,
the GAL4 DNA binding domain of each mutant. Four
different transformants for each mutant were sequenced and among them no unintended mutation was
found. This is consistent with previous reports which
suggest that the rTth catalyzed extralong PCR has high
specificity and fidelity (10, 11). It is important to point
out that each joining site was precisely ligated, maintaining the correct frame for fusion protein translation
(Fig. 2). It is very difficult to achieve this point by using
homologous termini. However, if no frame is required,
using homologous termini may obviously simplify the
steps in vitro and increase the efficiency. To examine if
the mutant constructs could express GAL4E1A fusion
proteins in yeast as expected, we prepared the total
cell lysate from each yeast transformant. After resolu-

233, 142144 (1996)

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