Download Factor Xa (Bovine)

INSTRUCTIONS
Factor Xa (Bovine)
32520
3747 N. Meridian Road
P.O. Box 117
Rockford, IL 61105
32521
0184
Introduction
Factor X is involved in blood coagulation by the intrinsic and extrinsic pathways. Factor Xa, an endoprotease, is formed by
the activation of Factor X. The active site of Factor Xa is thought to be very similar to the active site of trypsin.1 Its action
involves the conversion of prothrombin to thrombin, a protein essential to blood-clotting. Factor Xa will cleave any peptide
bond preceded by isoleucine-glutamic acid-glycine-arginine (Ile-Glu-Gly-Arg) or isoleucine-aspartic acid-glycine-arginine
(Ile-Asp-Gly-Arg) unless proline occupies the P´1 site2 (the site after the cleavage position).
The extremely specific proteolytic activity of Factor Xa (Bovine) makes this enzyme useful in the genetic engineering of
proteins. Many eukaryotic genes are difficult to overexpress in bacterial systems. One way of overcoming this problem is to
fuse the eukaryotic sequences to prokaryotic genes. The genes are then expressed as a hybrid or fusion protein in the
bacterial system. However, in order for the protein of interest to be useful for research or clinical applications, the bacterial
portion must be removed. Retrieving the eukaryotic protein from the fusion product can be difficult. The process can be
facilitated, however, by inserting specific peptide sequences between the hybridized proteins to provide recognition sites for
proteolytic enzymes. The tetrapeptide recognition sequence for Factor Xa (Bovine) is rare in protein sequences2 and,
therefore, offers excellent specificity with little risk of damaging the protein of interest by random or internal cleavage.
When constructing the fusion protein expression vector, oligonucleotides coding for the recognition sequence of Factor Xa
must be inserted between the fusion genes. The ligated plasmid is then transformed into a competent host strain where the
genes can be expressed. Appropriate selection techniques are used to determine which colonies carry the recombinant
plasmid. These include antibiotic resistance, chromogenic (enzyme-substrate) reactions and nucleic acid hybridization.
Once a correct colony has been identified it can be grown under conditions optimal for expression of the fusion product. The
hybrid protein is then purified and digested with Pierce's Factor Xa, releasing the eukaryotic portion for further study.
Points to consider:
•
Two complementary oligonucleotides coding for the Factor Xa recognition sequence must be synthesized (see Table 1).
• The protein of interest should not contain a Factor Xa recognition sequence.
•
In choosing the expression vector, consider variables which optimize transcription and translation such as promoters,
ribosome-binding sites (Shine-Dalgarno sequences4) and genetic markers to facilitate selection of vector carrying
colonies or capable of producing plaques.
•
In choosing the prokaryotic gene for the fusion, some thought should be given to how well the protein is normally
expressed in E. coli, toxicity of the protein to the host, and the presence of inducible genes which can aid in the selection
of positive colonies or plaques.
•
The Factor Xa recognition sequence and the eukaryotic gene must be inserted into the vector in the proper orientation
and in the correct translational reading frame.
Telephone 800-8-PIERCE or 815-968-0747
Fax 815-968-7316 or 800-842-5007
1
Product Description
NUMBER
DESCRIPTION
32520
Factor Xa (Bovine), is supplied as a solution in 5 mM MES (2-(N-Morpholino)
ethane sulfonic acid), 0.5 M NaCl, pH 6.0, 1 mM benzamidine•HCl.
Contains: 250 µg of the protein.
32521
Factor Xa (Bovine), is supplied as a solution in 5 mM MES (2-(N-Morpholino)
ethane sulfonic acid), 0.5 M NaCl, pH 6.0, 1 mM benzamidine•HCl.
Contains: 50 µg of the protein. Pierce's Factor Xa (Bovine) will remain stable for
at least 12 months, if stored frozen at -80°C. The enzyme loses activity with
repeated cycles of freezing and thawing, and upon storage in solution.
Note: Please see the specification sheet for additional and lot-specific information.
Activity of Factor Xa (Bovine)
The activity of Factor Xa (Bovine) is measured using the synthetic tetrapeptide S-2222 (Kabi Diagnostics, Franklin, OH).1
This tetrapeptide contains the specified amino acid sequence with p-nitroanilide attached to the arginine.
Factor Xa (Bovine) hydrolyzes S-2222 to release free p-nitroaniline, which is measured at 405 nm. The assay buffer contains
0.8 mM S-2222 and 0.05 µg/ml Factor Xa in Tris-Buffered Saline (TBS, 50 mM Tris, 200 mM NaCl, 5 mM CaCl2, pH 8.3).
The assay is performed at 37°C and the change in absorbance at 405 nm is recorded for 4-5 minutes. Pierce's Factor Xa
(Bovine) is free of extraneous activating proteins. Factor Xa, when compared to Factor Xa from two competitors, has higher
purity and specific activity (160 U/ml compared to 1 U/ml and 7 U/ml). This product can therefore be used at higher
dilutions.
Protocol for Using Factor Xa (Bovine) as a Restriction Endopeptidase
The following protocol has been adapted from Nagai and Thøgersen.2 In this protocol, the expression vector is used to fuse
the gene encoding the 31 amino-terminal residues of the λcII protein to the gene for human ß-globin. The oligonucleotide
sequence coding for the Ile-Glu-Gly-Arg recognition sequence of Factor Xa was inserted between the fusion genes. This
protocol may be optimized for your particular application.
Protocols for the construction of an appropriate expression vector can be found in Molecular Cloning, A laboratory manual
(Maniatis, T. et al. 1982), and Current Protocols in Molecular Biology (Eds. Ausubel, M. et al. 1987).
Cleavage of Fusion Proteins using Factor Xa
Note: The digestion time and the enzyme to substrate ratio may be optimized for your application.
1.
Dissolve or dialyze the protein to be hydrolyzed in TBS buffer (50 mM Tris, 100 mM NaCl, 6 mM CaCl2 pH 8.0).
2.
Add the diluted Factor Xa to the protein solution at an enzyme to substrate mass ratio of 1:100.
3.
Digest at 25°C for at least 2 hours. The digestion time can be increased if necessary. The progress of the hydrolysis can
be determined by removing aliquots from the digestion mixture and performing polyacrylamide gel electrophoresis.
Note: During extended incubation, cleavage at sites other than the peptide recognition sequence may be observed due to
protease contamination of the fusion protein.
4.
The hydrolyzed proteins can be analyzed on a polyacrylamide-SDS gel.
Telephone 800-8-PIERCE or 815-968-0747
Fax 815-968-7316 or 800-842-5007
2
References
1.
2.
3.
4.
5.
Aurell, L., Friberger, P., Karlsson, G. and Claeson, G. (1977). A new sensitive and highly specific chromogenic peptide substrate for Factor Xa.
Thrombosis Res. 11, 595-609.
Nagai, K. and Thøgersen, H. C. (1984). Generation of ß-globin by sequence specific proteolysis of a hybrid protein produced in Escherichia coli.
Nature 309, 810-812.
Lehninger, A.L. (1982). Principles of Biochemistry, Worth Publishers, New York (S. Anderson, J. Fox eds.), p. 897.
Maniatis, T., Fritsch E.F. and Sambrook, J. (1982). Molecular Cloning, A laboratory manual. Cold Spring Harbor Laboratory.
Ausubel, F.M., Brent, R., Kingston R.E., Moore D.D., Smith J.A., Seidman J.G. and Struhl, K. (Editors). (1987). Current Protocols in Molecular
Biology, Greene Publishing Associates and Wily-Interscience, New York..
© Copyright Pierce Chemical Company, 1995. Printed in U.S.A.
Table 1.
DNA triplets for the Ile-Glu-Gly-Arg Sequence (5' --› 3')3.
Ile
Glu
Gly
Arg
AAT
TTC
ACC
ACG
GAT
CTC
GCC
GCG
CCC
TCG
TAT
CCG
TCT
CCT
Telephone 800-8-PIERCE or 815-968-0747
Fax 815-968-7316 or 800-842-5007
3