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PROTEINS: Structure, Function, and Genetics 27:160–161 (1997)
Crystallization and Preliminary X-Ray Analysis of
Recombinant Staphylokinase
Anja Rabijns,1 Katrien Baeyens,1 Hendrik L. De Bondt,1 and Camiel De Ranter1*
1Laboratorium voor Analytische Chemie en Medicinale Fysicochemie, Faculteit Farmaceutische Wetenschappen,
Katholieke Universiteit Leuven, Leuven, Belgium
Diffraction quality crystals of
recombinant staphylokinase (STAR) have been
grown by the hanging drop vapor diffusion
technique from a solution containing MgCl2,
Tris buffer (pH 8.5), and polyethylene glycol
4000. The crystals belong to the monoclinic
space group C2 with unit cell dimensions a 5
60.6 Å, b 5 43.7 Å, c 5 54.3 Å, and b 5 115.6°. A
complete native data set to 1.8 Å resolution has
been collected using synchrotron radiation.
Proteins 27:160–161 r 1997 Wiley-Liss, Inc.
Key words: protein crystallization; x-ray crystallography; thrombolytic agents;
staphylokinase; STAR
Staphylokinase (STA), a protein produced by certain strains of Staphylococcus aureus, has been
shown to have profibrinolytic properties.1 Recently,
its mechanism of action2,3 and its potential for thrombolytic therapy4–7 have been the subject of intensive
Like streptokinase (SK), which is a routinely used
thrombolytic agent, STA is not an enzyme, but it
activates plasminogen indirectly by forming a 1:1
complex with plasmin, which in turn activates other
plasminogen molecules. In contrast to SK, however,
STA is able to induce clot lysis in a plasma milieu
without causing systemic plasminogen activation
and marked fibrinogen breakdown, thereby decreasing the risk of severe bleeding.8,9 Furthermore, in
experimental animal models, STA is relatively more
efficient than SK for the dissolution of plateletenriched and/or retracted clots, and STA appears to
be less immunogenic than SK.10,11 All these observations prove that STA may be promising as an alternative to streptokinase for the treatment of acute
myocardial infarction.
Mature STA consists of 136 amino acids (calculated Mr 5 15.5 kDa), but also lower molecular
weight derivatives of mature STA, lacking the 6 or
the 10 NH2-terminal amino acids, were obtained.12,13
Recent studies have shown that the amino acid at
position 26 in STA appears to be important for the
activation process of plasminogen by STA. Indeed,
substitution of the Met residue at position 26 with
Arg or Val results in total loss of the functional
activity of STA, while substitution with Leu or Cys
has little or no influence.14,15
In order to get a better understanding of the
mechanism of the nonenzymatic plasminogen activation, we report here the crystallization and some
preliminary x-ray data obtained from crystals of
recombinant staphylokinase (STAR).
Production and Purification of STAR
STAR was produced and purified as described
previously.5 The final material was obtained at a
concentration of 2 to 3 mg/ml. NH2-terminal amino
acid sequence analysis revealed a main sequence of
Ser-Ser-Ser-Phe, and a minor sequence (approximately 8%) of Gly-Lys-Tyr-Lys.5
Finally, the material was dialyzed and concentrated against 50 mM Hepes (pH 7.5) to a final
concentration of 35 mg/ml.
Crystallization and Data Collection
Crystallization was carried out by the hanging
drop vapor diffusion technique at 4°C using Linbro
multiwell tissue plates. Each well was filled with 700
µl of reservoir solution that contained 0.2 M MgCl2,
0.1 M Tris buffer (pH 8.5) and 35% polyethylene
glycol 4000. Drops consisting of 1.5 µl protein solution (35 mg/ml protein in 50 mM Hepes buffer, pH
7.5) and 1.5 µl of reservoir solution, were placed on
coverslips and set to equilibrate against the reservoir solution in the wells. Chuncky x-ray quality
crystals of STAR generally appeared after 2 or 3
months. These spontaneous crystals could be utilized for microseeding. To produce a seeding solution,
a crystal was placed in 10 µl of the original reservoir
solution and crushed with a glass rod. Next, tenfold
serial dilutions of the crushed crystal were made in
the reservoir solution, and hanging drops consisting
of 1.5 µl protein solution and 1.5 µl seeding solution
(104-fold dilution) were assembled.
Diffraction data were collected at DESY/EMBL
*Correspondence to: Camiel De Ranter, Laboratorium voor
Analytische Chemie en Medicinale Fysicochemie, Faculteit
Farmaceutische Wetenschappen, Katholieke Universiteit Leuven, Van Evenstraat 4, B-3000 Leuven, Belgium.
Received 25 April 1996; accepted 17 May 1996.
1. Santos-Oliveira, R., Dias-Baruffi, M., Thomaz, S.M.O., Beltramini, L.M., Roque-Barreira, M.C. A Neutrophil Migration Inducing Lectin from Artocarpus integrifolia. J. Immunology 153:1798–1807, 1994.
2. Rot, A. Neutrophil Attractant/Ativation protein-1 (Interleukine-8) Induces in vitro Neutrophil Migration by Haptotatic
Mechanism. Eur. J. Immunology 23:303, 1993.
3. Sharon, N., Lis, H. Carbohydrates in Cell Recognition.
Scientific American, January, 82–89, 1993.
4. Travis, J. Biotech Gets a Grip on Cell Adhesion. Science
260:906–908, 1993.
5. Mathews, B.W. Solvent content of protein crystals. J. Mol.
Biol. 33:491–497, 1968.
6. Higashi, T. The processing of diffraction data taken on a
screenless Wissenberg camera for macromolecular crystallography. J. App. Cryst. 22:9–18, 1989.
7. Sakabe, N. X-ray diffraction data collection system for
modern protein crystallography in a Weissenberg camera
and an imaging plate using synchrotron radiation. Nucl
Instrum. Methods, A303, 448–463, 1991.
8. CCP4—Collaborative Computer Project, Number 4. The
CCP4 Suite: Programs for Protein Crystallography. Acta
Cryst. D50:760–763, 1994.
9. Crowther, T. The Fast Rotation Function. In: Rossmann,
MG, ed. Molecular Replacement Method. N. York, Gordon
and Breach, 173–178, 1972.
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