FIELD OF INVENTION
The present invention relates to the use of Staphylokinase (SAK) as a proteolytic tool. Also, the
present invention relates to use of autocatalytic (self cleavage) activity of SAK by cloning and
expression of SAK gene fused with a protein gene of interest and the present invention also
relates to the development of a method of cloning SAK gene with a gene of protein of interest.
The present invention further relates to preparation of a kit of a vector containing SAK gene
wherein a gene of protein of interest can be fused with SAK and expressed for purification of
protein of interest.
BACKGROUND OF THE INVENTION
SAK is a 136 amino acid long bacteriophage encoded protein of 15.5-kDa size and is devoid of
disulphide linkages. SAK is presently undergoing clinical trials for blood clot-lysis in the
treatment of thrombovascular disorders due to its ability to convert plasminogen, (an inactive
proenzyme of the fibrinolytic system) into plasmin, which is a protease. SAK has gained
importance as a potential therapeutic thrombolytic protein and is an extracellular protein
produced by Staphylococcus aureus strains It is also produced by S.lyicus, S.simulans,
S.seweri and S.xylosus
Various plasminogen activators reported in the literature are streptokinase (SK), SAK, tissue
type plasminogen activator (t-PA), prourokinase (u-PA). These plasminogen activators are
reported to be efficient thrombolytic agents, which are most widely used clinically.
SK is the most widely prescribed thrombolytic agent for acute myocardial infarction (AMI) in
many parts of the world due to it's low cost. However, SK is generally accepted since it
demonstrates lower efficacy in terms of arterial patency and an absolute difference of 1% in
mortality compared to more expensive agents such as t-PA.
Schlott et al have disclosed that SAK is not an enzyme, but rather a cofactor; it forms a 1:1
stoichiometric complex with plasmin-(ogen) that converts other plasminogen molecules to
plasmin, a potent enzyme that degrades proteins of the extracellular matrix. The high affinity of
the SAK-plasminogen complex for fibrin makes it a promising thrombolytic agent.

US pat 5496549 discloses u-PA has both protease and thrombolytic activity. u-PA expression
delayed tumor progression and had antiangiogenic and antiproliferative effects that may be
mediated by u-PA's protease activity.
Jackson and Tang have reported that SK has been shown to be homologous to serine proteases
although it does not have any protease activity of its own. Also, Nicole et al have studied that t-
PA has a serine protease domain.
Sakharav et a I had reported that SAK structurally resembles plasminogen activators, has
plasminogen-binding site and serine protease domain.
Although SAK has serine a protease domain, it has been not reported to have protease activity.
OBJECTIVES OF THE PRESENT INVENTION
A primary objective of the present invention is to use SAK as a proteolytic tool.
Another objective of the present invention is to use autocatalytic activity of SAK by cloning and
expression of SAK gene with a fused protein gene of interest.
Yet another objective of the present invention is to develop a method of cloning SAK gene with
a gene of protein of interest.
Yet another objective of the present invention is to prepare a kit containing SAK wherein a gene
of protein of interest can be fused with SAK and expressed for purification of protein of interest.
SUMMARY OF THE INVENTION
Although the plasminogen activators have a serine protease domain, they lack protease activity.
It was surprisingly found that the SAK has autocatalytic property, which can be used as
proteolytic tool. It was seen that a full length SAK (FL-SAK) is when expressed results into 2
fragments as mature SAK and signal peptide It was surprisingly found that when FL-SAK was
expressed in BL21-A1 cells resulted into 2 fragments as mature SAK and signal protein. This
proves that SAK has autocatalytic property.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
Figure 1. Staphylokinase PCR product
Figure 2. Nde1/Hindlll digestion of clones of pET21a-SAK
Figure 3. Expression of Staphylokinase from pET21a-SAK clones
Figure 4. FL-SAK PCR product
Figure 5. PCR screening for pET21a-FL-SAK clones
Figure 6. Release of FL-SAK insert with Ndel-EcoRI dig
Figure 7: Sup of whole cell lysates of pET21a-FL-SAK
Figure 8. Pellets of whole cell lysates of clones of pET21a- FL-SAK
Figure 9. Colony PCR screening
Figure 10. Release of GST-SAK insert with Ndel/ Xhol
Figure 11. Expression of GST-SAK protein in DH5a cells
Figure 12. GST PCR product
Figure 13. Colony PCR screening for SAK-GST clones
Figure 14. Restriction digestion of pET21a- SAK and pET21a- SAK-GST clones
Figure 15. SDS-PAGE for expression of SAK-GST protein (-42 kDa size) in DH5a cells
Figure 16. SDS-PAGE profile of samples of purification of GST-SAK
Figure 17. SDS-PAGE and silver stain gel of cleavage of purified GST-SAK protein
Figure 18. SDS-PAGE, silver stain gel of purification of SAK-GST protein
Figure 19. Profile of GST-SAK expressed in BL21 cells
Figure 20. Profile of BL21 derived SAK-GST protein
Figure 21. Effect of reduced glutathione on the cleavage property of GST-SAK fusion protein
Figure 22. Amplification of NA19SAK
Figure 23. Colony screening by PCR using gene specific primer
Figure 24. Clone confirmation by insert release by BHI-HIII digestion
Figure 25: SDS PAGE for expression of GST-NA19SAK
Figure 26: Differentiation of various SAK clones by PCR
Figure 27: SDS PAGE profile of purification of GSTA19SAK
Figure 28: Degradation of GSTA19SAK
Figure 29:Activity assay of SAK and SAK preparation
Figure 30: Expression of GST*NA19SAK
Figure 31: SDS PAGE profile of purification of GST*NA19SAK

The fusion tags may not be limited to the examples but also covers the fusion tags known in the
art.
The gene of protein of interest is the DNA sequence of the corresponding protein, which is
obtained on expression of the gene. The examples of gene of protein of interest are therapeutic
proteins, which also covers other gene of proteins known in the art.
In one of the embodiment of the present invention, -further to prove this, the mature SAK was
cloned with GST at N-terminal to obtain pure SAK funsion protein to understand its proteolytic
property. The resultant fusion peptide was called as GST-SAK. When cloned with suitable
vector and expressed in bacterial cells (DH5a) the fusion protein showed 2 fragments namely,
GST and SAK, wherein SAK and GST is not identical to the length of original ones.
In another embodiment of the present invention the mature SAK was cloned with GST peptides
at the C-terminal The resultant fusion peptide was called as SAK -GST. When cloned with
suitable vector and expressed in bacterial cells (DH5a cells) the purified protein showed only
one fragment namely SAK-GST fusion , with no fragmentation at SAK or GST.
In another embodiment to prove that the proteolytic activity of SAK is not due to the external
proteases produced by the host cells. GST-SAK was introduced into BL21, which doesn't
produce any protease enzyme like Ompt unlike DH5 alpha cells. Figure 19 and 20 confirmed
self-cleavage property of GST-SAK even in protease defecint E-coli BL21 strain. However,
SAK-GST did not show such cleavage , even if the proteins were made in protease minus E.
coli host BL21. This confirms that the cleavage property is indeed due to SAK gene and not due
to any other host factors.
In another embodiment the effect of reduced glutathione on the self cleavage property of GST-
SAK protein was checked . It is evident from Figure 21 that the GST-SAK fusion protein does
not degrade in the presence of reduced glutathione while it showed cleavage in absence of the
same.
In another embodiment of present invention to check that the autocatalytic activity of SAK is
independent of its thrombolytic activity. It has been reported that when N-11 position amino acid
(lysine) was altered, the thrombolytic activity of SAK reduced drastically. In order to prove that

similar sites of peT21a vector. The ligation mix was used to transform chemically competent
DH5 alpha cells.
b. Screening of clones of pET21a-SAK by restriction enzyme digestion
The plasmid DNA's were prepared by alkaline lysis method and were processed for
Nde1/Hindlll digestion (Figure 2). All the tested clones released the required SAK insert of ~
400 bp. A total of five clones were selected for small scale expression studies
c. Expression of pET21a-SAK in BL21(DE3) cells
BL21(DE3) cells were transformed with DNA of clone nos. 1, 2, 3, 4, 5 along with pET21a as
control. The induction was done in 50 ml LB with 1 mM IPTG for 4 hr and the induced cells after
centrifuging were lysed with glass beads and the lysed culture was spun at 13,000 rpm to
separate the supernatant and pellet fractions. The fractions were loaded onto 12% SDS-PAGE
and the results are indicated in the Figure 3. Right size Staphylokinase protein (15 kDa) was
seen in the soluble fraction of the cell.
EXAMPLE 2
a. Cloning of SAK with signal peptide (FL-SAK)
The full length SAK sequence was PCR amplified using gene specific primers and the same
synthetic gene as the template. PCR amplification was performed with Taq polymerase. The
amplification conditions were as follows: 30 cycles consisting of 94°C for 1 min, 57°C for 1 min
and 72°C for 1 min. The primers used for this are as given below.
LUBT 003: FP for full length SAK
5' CCG CCG GAA TTC CAT ATG CTC AAA CGA AGT TTA TTA TTT 3'
LUBT 004: for full length SAK
5' CCG CCG GAA TTC AAG CTT TTA TTT CTT TTC TAT AAC AAC 3'
The PCR amplified FLSAK sequences were purified (Figure 4) was digested with Nde1/EcoR1
and ligated to pET21a vector (Novagen) at Ndel-EcoRI sites. The ligation mix was introduced
into competent DH5 alpha cells and the transformants were screened by colony PCR for the FL-
SAK gene. Figure 5 shows the PCR of FL-SAK gene for several individual transformants of
pET21a-FLSAK. The PCR positive clones were inoculated for plasmid DNA preparation and
these were subjected to Ndel/ERI digestion. The results of this are presented in Figure 6,
wherein it is clear that of the clones tested except for two, all the PCR positive clones of
pET21a-FLSAK released the required size insert of FL-SAK.
b. Expression studies of FL-SAK:
PCR confirmed clones were introduced into competent BL21-A1 cells and induced with

arabinose as inducer (13mM). The induced cells showed expression of full length SAK.
However, the expression was observed in both pellet and sup fraction. However, another band
of the size of SAK at the same time was observed as shown in Figure 7 and 8 indicating the
possibility of cleavage of some fraction of the FL-SAK protein to SAK alone. These results
indicated that probably SAK protein has a self-cleavage protease activity.
To examine the self-cleavage property of Staphylokinase two types of fusions of SAK, namely
GST-SAK and SAK-GST were cloned. This work was mainly thought for achieving easy
purification of both the SAK fusions with the help of the affinity matrix of GST.
EXAMPLE 3
Cloning and expression of N terminal fusion of SAK gene (GST-SAK)
The mature peptide of the SAK gene was PCR amplified using primers given below and the
PCR amplification conditions were same as performed above and the amplified PCR product
was cloned at the C terminal end of GST gene in pGEX-4T-1 vector as EcoR1/EcoR1 fragment.
The primers used for the amplification studies are given below:
LUBT 009: FP for SAK mature peptide:
5' CCG CCG GAA TTC CAT ATG TCA AGT TCA TTC GAC AAA GGA 3'
LUBT 004: RP for SAK
5' CCG CCG GAA TTC AAG CTT TTA TTT CTT TTC TAT AAC AAC 3'
The mature peptide of the SAK gene PCR product was digested with ERI and was ligated with
pGEX-4T-1. EcoR1 dephosphorylated vector at RT for 16 hours. The ligation mix was then
introduced into chemically competent DH5 alpha cells and the amp resistant transformants were
screened by colony PCR for the SAK gene, as shown in Figure 9. The PCR positive clones
were inoculated for plasmid DNA and the plasmid DNA was digested with Ndel-Xhol to confirm
the right orientation of the SAK insert(Figure 10). The clones with rightly oriented SAK gene with
respect to the pTac promoter of pGEX-4T-1 vector were tested for the expression of the GST-
SAK protein (Figure 11) in chemically competent DH5 alpha cells for expression analysis. GST-
SAK protein was expressed in supernatant and pellet after induction with 13 mM arabinose.
EXAMPLE 4
Cloning and expression of C terminal fusion of SAKgene (SAK-GST)
GST gene was amplified using gene specific primers using pGEX-4T-1 as the DNA template.
The PCR amplification with Taq DNA polymerase was performed as follows: Initial denaturation
at 94°C for 4 minutes, followed by 30 cycles consisting of 94°C for 1 min, 55°C for 1 min and

Glutathione Sepharose 4B matrix using the protocol as illustrated in Example 5.
It is evident from the Figures 19 and 20 that only GST-SAK showed self cleavage property while
SAK-GST did not show such property even if the proteins were made in protease minus E. coli
host BL21 confirming that the cleavage property is indeed due to SAK gene and not due to any
other host factors.
Further the effect of reduced glutathione on the cleavage property of GST-SAK protein was
studied. For this purpose, the purified GST-SAK protein was taken as one aliquot dialyzed to
remove reduced glutathione while another aliquot not dialyzed and incubated at 37°C after 16
hours of dialysis at RT. Figure 21 indicated that the GST-SAK fusion protein does not degrade
in the presence of reduced glutathione while it showed cleavage in absence of the same.
EXAMPLE 6
Autocatalytic activity of SAK is independent of its Staphylokinase activity. The
staphylokinase's auto cleavage property was examined whether it is independent of its
staphylokinase activity. For this purpose, the N terminal of SAK was removed and A19SAK was
fused with GST to get GSTA19 SAK by suitable primers at the N terminus, then purified and
studies for its cleavage property.
PCR amplification of the NA19SAK was done using SAK plasmid as template and the following
primers.
LUBT221 Forward primer for A19SAK 5' CCG CCG GAA TTC GAA CCA ACA GGC CCG TAT
TTG3'
LUBT 004 Reverse primer A19SAK
5' CCG CCG GAA TTC AAG CTT TTA TTT CTT TTC TAT AAC AAC 3'
The PCR was done using Taq DNA polymerase and the cycles of amplification was as follows.
Denaturation at 94°C for 4 minutes followed by 30 cycles of 94°C for 30 sec, 57°C for 30 sec
and 72°C for 30 sec with a final extension of 7 min at 72°C.
NA19 SAK PCR product (Figure 22) was digested with EcoRI and cloned in pGEX4T1 vector at
the same site. Transformants were screened by colony PCR for deleted SAK (Figure 23) and
confirmation of the rightly orientation clones was done by BamHI -Hindlll digestion (Figure 24).
Screening and differentiation of various clones of SAK were studied using PCR. The clone
DNA's were used as template for PCR of Staphylokinase version using suitable primers and the
results indicated below show that the clone of GST-N19 SAK is indeed fine in all respects.
Confirmed clone DNA's were further used to transform DH5 alpha competent cells. The
transformed cultures were inoculated in 50 LB amp and induced with 1mM IPTG for 4 hours at

after sonication of the induced pellet (Figure 30) and then the soluble fraction loaded onto
Glutathione Sepharose 4B column and purified as described earlier (Figure 31).
The purified protein was subjected to 37°C treatment after and before dialysis. The results
indicate no cleavage of the GST-SDM A SAK indicating that the SAK proteolysis happens at the
SS site. This site could be the SS at the C terminus part of GST (Figure 32).
Example 8:
SAK activity assay:
SAK activity was quantified using plasminogen coupled chromogenic substrate assay with
following modifications. Fifty nanograms of the SAK fusion protein and 20M of hPg were
incubated in 80d reaction volume in 96 well flat bottom plates (coring at 25°C for 25 min
followed by addition of 20l from 5mM stock solution of the chromogenic substrate S-2251 (Val-
Leu-Lys-p-nitroanilide, sigma). Plates were further incubated for 5 min at 25°C. Amount of P-
nitroaniline released at the end of incubation period was monitored at 405nm in a plate reader
(Thermo, Multiskan Spectrum). (Figure 29)

purification of fusion protein of interest.
11. The kit according to claim 10 wherein SAK optionally includes other peptides.
12. The kit according to the claim 10 wherein SAK includes FL-SAK or fragments of SAK,
having autocatalytic property.
13. The kit according to claim 10 wherein the gene of protein of interest is selectively
therapeutic proteins

A proteolytic agent comprising a peptide, having autocatalytic property wherein said peptide is SAK protein. A proteolytic agent comprising an effective ingredient as a peptide consisting of DNA sequence corresponding to SAK gene, having autocatalytic property when exists as a fusion protein. A method for cloning and expressing the SAK gene with a gene of protein of interest in order to utilize the proteolytic activity of SAK. A kit comprising SAK, adapted for fusion
with a gene of protein of interest and expressed for purification of fusion protein of interest.