Identification and modification of immunogenic regions in streptokinase

produced by Streptococcus agalactiae

By

Rabia Amin
M.Sc. Biochemistry
2015-ag-2665

A technical report submitted in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE
IN
BIOCHEMISTRY

Department of Biochemistry
Faculty of Sciences
University of Agriculture, Faisalabad
Pakistan
2017

1
TECHNICAL REPORT EVALUATION COMMITTEE

Advisor ________________________________
(Dr. Muhammad Anjum Zia)

Chairman _________________________________

(Prof. Dr. Khalil-ur-Rehman)

Dean’s Nominee _________________________________

\ Department of Biochemistry
University of Agriculture,
Faisalabad

2
Acknowledgement:
I have taken efforts in this report. However, it would not have been possible
without the kind support and help of many individuals and university. I would like to
extend my sincere thanks to all of them.

I am highly indebted to (University of Faisalabad) for their guidance and constant

supervision as well as for providing necessary information regarding the project & also
for their support in completing the project.
I would like to express my gratitude towards my parents & member of (University of
Faisalabad) for their kind co-operation and encouragement which help me in completion
of the report.
My thanks and appreciations also go to my class fellows and sisters in developing the
report and people who have willingly helped me out with their abilities.

Rabia Amin

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 TABLE OF CONTENTS

Sr. No. Contents Page No.

1 Title 1
6
2 Introduction
12
3 Review of literature
42
4 Materia and Methods
49
5 Conclusion
50
6 Refences

4
 Contents

Title……………………………………………………………………6

Abstract……………………………………………………………………………6

Introduction…………………………………………………………………….7

Review of literature…………………………………………………12

2.1. Structure of streptokinase……………………………………………….13

2.2. Streptokinase producing species…………………………………………15

2.2.1. Streptococci………………………………………………………………15

2.3. Myocardial infarction and administration of streptokinase……………………17

2.4. Risk associated with streptokinase therapy…………………………….20

2.5. Antigenic regions of streptokinase……………………………………….21

2.6. Modification of streptokinase to lessen its immunogenicity……………24

2.7. Chemical modification of streptokinase…………………………………25

2.8. Site directed mutagenesis……………………………………………...…27

2.8.1. Deletion mutation of streptokinase…………………………………….30

2.8.2. Substitutional modifications……………………………………………32

2.8.3. Insertion and deletion mutation……………………………………….34

2.9. Recombinant streptokinase…………………………………………….37

2.10. Assessment of truncated and full length purified streptokinase……39

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Material and Method……………………………………………………………41

3.1. Specie identification…………………………………………………….….41

3.1.1. Morphologic characteristics…………………………………………...…41

3.1.2. Biochemical characterization…………………………………………….41

3.1.3. Molecular methods for characterization……………………………….42

3.2. Site-directed mutagenesis of SK………………………………………...…43

3.2.1. Primer designing……………………………………………………….…43

3.2.2. Gene amplification through PCR…………………………………….…44

3.2.3. Cloning of SK gene from streptococci………………………………...…45

3.3. Expression of SK variants…………………………………………………45

3.4. Protein purification………………………………………………….….…45

3.4.1. Affinity purification of SK protein………………………………….….46

3.5. Bioassay of recombinant SK proteins………………………………….…47
3.5.1. Caseinolysis……………………………………………………………….47
3.5.2. Chromogenic assay……………………………………………………….47

3.5.3. Clot lysis assay………………………………………………………….…47

3.6. Immunoassays………………………………………………………………48

3.6.1. Direct binding assay………………………………………………………48

3.6.2. Competition assays………………………………………………….……48

Conclusion………………………………………………………………………49

6
Identification and modification of immunogenic regions in streptokinase
produced by Streptococcus agalactiae

Abstract:

Streptokinase is the potent therapeutic agent that is most extensively used for the treatment of
myocardial infarctions, regardless of its benefits it might provoke allergic reactions due to
presence of streptokinase antibodies in the body. Here I reported about the genes which are
antigenic in streptokinase structure along with previously described regions especially in
Streptococcus agalactiae.Various methods have been employed to minimize the allergic
reactions in such a way that does not change the structure or function of enzyme.Elimination of
59 N-terminal residues led to the identification of SK antigenic regions in streptokinase.A
mutated streptokinase deficient in 42 amino acid at C terminal region the is less immunogenic in
nature. Modified streptokinase is more effective in its reactivity than the native molecule.

7
 CHAPTER NO.1

Introduction:

Streptokinase enzyme is composed of 426 residues produced by one the species of

theStreptococcus aglactiaeand many more with different number of amino acid that is used as a
thrombolytic agent for the treatment of disorders. The enzyme is a single-chain polypeptide that
appliesits fibrinolytic action indirectly by activating the circulatory PNG.Jackson and Tang
(1982). Determined thewholeamino acid sequence of streptokinase. Streptokinase has a molar
mass of 47 kDa and is composed up of 414 amino acid residues. The streptokinaseshowsits
maximum activity at a pH of 7.5. Streptokinaseproduced by different streptococci can be
differentiated according to their function and structure (Huang et al., 1989).

Streptokinase comprises of three different structural domains (i.e.α, β and γ) with unique
functional properties. Calorimetric analysis acclaims that the protein is composed of two discrete
domains (Welfleet al., 1992). The N-terminal region (i.e., residue 1–59) has been found to
balance the low plasminogen activation ability of the 60–414 amino acids (Nihalani et al.,
1998).A remarkable degree of heterogeneity present among the streptokinases produced by
different species of streptococci. Biophysical techniques such as circular dichroism, nuclear
magnetic resonance spectroscopy, Fourier transform infrared (FT–IR) spectroscopy
anddifferential scanning calorimetry (DSC) have provided essential information about the
structure of streptokinase.

How streptokinase activates plasminogen has been the attention of wide research
(Zhaiet al., 2003); however, mechanisms of activation of plasminogen by streptokinase are still
investigated (Boxrudet al., 2001). Streptokinase stimulate plasminogen either by
fibrinindependent or fibrin-dependent mechanisms (Reedet al., 1998). Streptokinase combines
with plasminogen via multiple domain of structure. At least two independent binding sites of
plasminogen had been identified (Nihalani and sahni, 1995).

The C-terminal domain of streptokinase is capable of substrate recognition and

activation later on. Similarly, the Asp41–His48 residues areimportant in plasminogen binding
with streptokinase (Kim et al., 2000). The action level of adjacent region (48-50) on
plasminogen activation is also investigated (Wakehamet al., 2002). The coiled region of the

8
streptokinase γ-domain is said to be necessary for plasminogen activation (Wuet al., 2001).
Likewise, the streptokinase β domain has its action level in forming the plasminogen–
streptokinase complex that is responsible for activating the plasminogen (Robinsonet al., 2000).
Streptokinase binds favorably to the extended region of plasminogen through the lysine binding
site to trigger 1conformational activation of PNG (Boxrudet al., 2001). Streptokinase–
plasminogen activator complex combine with plasminogen through protein–protein binding to
exploit catalytic turnover (Sundramet al., 2003). The first 59 amino acids may have several
functional roles in streptokinase structure (Younget al., 1995). Without these N-terminal
residues, the structure of streptokinase will be unstable. Forfeitureof amino acids 1–59 affect the
activity of other residues (residues 60-414) (Young et al., 1995). Plasminogen activation has
been verified in different species using 56 isolates of the disease-causing group C streptococci
(McCoyet al.,1991).

There are two essential steps in the SK-catalysed plasminogen activation. In the first
step, proteolytic species formed which act as the mediatorcatalysing the cleavage of the Arg560–

Val561 peptide bond is obligatory for plasminogen activation. Investigations have shown that

mortality can be reduced using thrombolysis (particularly fibrinolysis) in the treatment of heart
attacks. Streptokinase stimulates secondary fibrinolysis by plasmin through distillation of
plasminogen activator, the protein product use to activate PN, Stroke (ischemic stroke). For the
treatment of respiratory complications streptokinase therapy is an effective and safer more
alternative to systemic thrombolysis .Acute limb ischaemia, Severe deep vein thrombosis are
some of the function of streptokinase.

Streptokinase is a complex with three domains (α, β and γ) each domain is structurally
unique and showing a similar folding pattern. Various structural examination reveals that the
determined antigenic regions can be map more accurately. The amino-terminal region (1-15), the
carboxylic-terminal regioncontaining 32 residues and the loop area contains46-70 residues are
disordered, suggesting that they areavailable andmobileeven aftercomplex formation with
plasmin. This is dependable with the discovered antigenic spots 1, 6, 7, 39 and 40. The antigenic
regions 130-149 and 260-299 contains linking domains α with β and b with γ and loop regions.
The region 350-369encompasses the available β hairpingb3 and gb4. The peptide 320-339
assume to maps a loopregion of domain complex and may be involved in activation.

9
Nevertheless, this loop must be available in the native molecule of streptokinase. The knowledge
of the immunogenic regions of streptokinase can guide us in the formulation of mutated
molecules with reduced antigenicity and perhaps with reduced lethal effects on health.

These studies recognized fourstructurally discrete regions via immunoblotting method. These
regions of streptokinaseestablishing the antigenic amino acid orders were built by amino acids 1-
13,1-253, 120-352, and amino acids 352^114. Three of the peptide classifications
weredocumented by MAbs A7.4, A4.3 andA4.5, could be considersignificant for the function of
streptokinase, so the capacity of streptokinase to formulate a functional SKPAC wasabridged
when it was incubated with 3 MAbs. The process by which each MAb hinder the
streptokinaseactionmay be different. For example, MAb A4.5, which fixes to the region of
streptokinase moleculemade up of different amino acids 120-352, a region comprising the
primary plasminogen site which plays role in binding of SK to plasminogen, may be blocked.
Moreover, the binding of SKtowardplasminogen, SK bringsa structural modification to bring the
active site at its actual position and in this way, contribute in binding to substrate plasminogen
molecules to the active site.'23'24' The formation of functional streptokinase plasminogen
complex can be inhibited by MAbs A7.4 and A4.3 or due to theinhibition of conformational
changes required for SK and plasminogen to form streptokinase plasminogen complex. Another
research reveals that steric hindrance also play role in in substrate binding at active site.

Immunogenicity of SK and its short life spanin circulation render it less efficient
therapeutic agent. Streptokinase is proteolytically tainted by plasmin for conversion into active
form. Subsequently, researches has focused on structurally modifying SK to enhance its half-life,
improve plasminogen activation, and reduce or eliminate immunogenicity. Any structural
modification requires to be informed via adetailed structural and functionalexamination of
streptokinase and this has been the theme ofrecent investigation now a day. Structurally,
modified streptokinases have been designed by using different methods includingenzymatic and
chemical modification of the native streptokinase molecule, genetic mutation or
throughrecombinant DNA technology. Mutated streptokinase with advanced stability have been
formulated after thorough investigation (Shi et al., 1998). Two important sites of proteolytic
action of plasmin on SK are Lys 59 and Lys386 (Wuet al., 1998). This detail is being used for

10
the formulation of different variants of streptokinase that are resistant to plasmin (Wu et al.,
1998) and, consequently, have an extended efficient life span. The nucleotide regions which
causes allergic reactions is determined along with the amino acid sequence from streptococci.
Similarity can be happened with other species of same group or different (Jackson and Tang,
1982). The total sequences of SK 24, SK 12, SK 1 and many more are being described.
Additionally, the sequences of amino acids from N- and C-termini of different streptokinase
producing species is defined more (Huang et al., 1989a).

streptokinase (SK) in acute myocardial infarction have potential toliquify human

thrombus. The first experimental trial of streptokinase wastestified by Fletcher and colleagues in
1958in myocardial infarction. This preliminaryuse of streptokinase was tracked by twenty
onepracticability studies of fibrinolytic agents. Optimistic results from these practicability
studies directed to the accountability of 10 industrial scale accommodating clinical trials.Initial
trials of streptokinase in severe myocardial complicationsconfirmed a significant reduction in
mortality rate of people treated with streptokinase. Later on, tests were done inendeavoring to
demonstrate the clinical efficiencyin coronary-care unit, have proven ineffective in representing
a statistically noteworthy reduction in death rate.

Parhami-Serenet al.,(1996)described that certain regions of streptokinaseseems to be

more immunodominant or antigenic. They utilize recombinant streptokinase-truncated fragments
as a molecular probe and murine anti-SK monoclonal Abs to examine human polyclonal
antibodiesretortsto SK in normal people and in patients before and after treatment with
streptokinase therapy. The informationobtained from this discovery suggests
numerousdeductions in this respect. First, there are substantialdifferences in the amount ofanti-
streptokinase antibodies before and after SK therapies. Patients treated with streptokinase
therapies, inclines to display the similar binding design after therapy as they ensured before the
administration with streptokinase, however the quantity of definite anti-streptokinase
antibodiesupsurges 7 times in the body. Third, the anti-streptokinase antibodiesin patients are
focused towards the 3 major antigenic regions in streptokinase enzyme. These 3 immunogenic
regionscontain epitopes made up of amino acids residues 120-352 and 1-253comprising 2
discrete and distinct antigenic regions. Bruserudet al., (1992) have also documented information
about the T-cell epitope in the streptokinase and made up of 238-246amino acid residues. Beside
11
these two more epitopes in streptokinase, built up by amino acids353-414 and1-13were not
antigenic in all individualsverified by Parhami-Seren et al(1996). Human antibodies from many
sources exhibit minimumor no bindingattraction towards streptokinase made by 14 to 127amino
acids.The streptokinase genetic factor from Streptococci were sequenced and narrated by
Malkeet al., (1985). The functional analysis of its promoter has been formulatedalong with
transcriptional control of this gene which was thoughtful. Significantdata is provided for the
effective use of thatgene in expressing streptokinase in non-disease causing microbes especially
in bacteria.

Various studies about streptokinase reveals that it is polymorphic and diverse in its
functions(Malke, 1993). The (skc) which represent streptokinase gene were formerly cloned in S.
equisimilis later expressed in gram positive and gram negative especially B. subtilis WB600
(Zhanget al., 1999; Pupoet al., 1999). Vectors having streptokinase gene along with
antibiotic(erythromycin) were introduced in S. equisimilis so that we can obtain efficient clone
which then use as a model (Muller and Malke, 1990). The new vector plasmid which was
designed for the expression of streptokinase gene has been effectively estimated for producing it
(Koet al., 1995). An aptitude to harvest recombinant SK greatly improves the chances for
improved production of the desired recombinant streptokinase and helpful structural changes in
the structure of streptokinase molecule.Parhami-Serenet al., (2003) studied that streptokinase is a
potent therapeutic agent produced by a various species ofstreptococci that has the capability to
make lysis of blood thrombus. Streptokinase is produced through recombinant technology in a
suitable host or can be use without ant modifications. Recombinant technology is in expensive
and most prevalent. For more than three decades, streptokinase is most effectively used in
treating various diseases like thrombosis and heart complications. Along with its benefits a
number of risks are associated with it. It was estimated that streptokinase mediates the
conversion of plasmin into plasminogen. Streptokinase therapy act as an effective method after
its inoculation (Wu et. al., 1998). The half-life of streptokinase reduced to 15 minutes due to this
reason. (Wu et. al., 1998). The persistent time of streptokinase in blood is higher than the other
drugs which used in thrombosis, but still use an effective drug in managing cure. (Wu et
al.,1998). Various studies indicate that activated plasminogen rapidly vanished so trend is
shifted towards the recombinant production of streptokinase (Wu et al., 1998). Various risks are

12
associated with the streptokinase therapy but still is used for treatments because it is cost
effective and prove to be better than other drugs

To lessen the allergic reactions of streptokinase and to make it more efficient several
strategies have been employed. One of the them is the chemical modification of streptokinase
molecule by using different chemical agents.This will not only increase its stability but also
minimize the immunogenic hetaeristic. We simply make poly ethylene glycol complex with
streptokinase molecule which proves more effective. The modified molecule formed in this way
is less immunogenic than the original form of streptokinase this will also improves the life span
of streptokinase.

Wuet al., (1998) formulate various mutated streptokinase by replacing amino acids in
the region of Pro58-Lys59-Ser60 so that they act as most efficient fibrinolytic agent and less
immunogenic. He found that, the favoured mutant attained by substituting the Lys59 residues
with glutamic acid. Plasmin or the plasminogen interaction and involvement from lysine
binding site isdependent or independent towards the stability of this complex. Thisrecommends
that the loss in function due to these modifications can’t be understood because vague
information obtained from history. Instead modified streptokinase is more efficient in
functionality than the native one.

13
 CHAPTER NO.2

Review of literature

Parhami-Serenet al., (2003) studied that streptokinase is a potent therapeutic agent

produced by a various species ofstreptococcithat has the capability to make lysis of blood
thrombus. Streptokinase is produced through recombinant technology in a suitable host or can be
use without ant modifications. Recombinant technology is in expensive and most prevalent. For
more than three decades, streptokinase is most effectively used in treating various diseases like
thrombosis and heart complications. Along with its benefits a number of risks are associated with
it. It was estimated that streptokinase mediates the conversion of plasmin into
plasminogen.Streptokinase therapy act as an effective method after its inoculation (Wu et. al.,
1998). The half-life of streptokinase reduced to 15 minutes due to this reason.(Wu et. al., 1998).
The persistent time of streptokinase in blood is higher than the other drugs which used in
thrombosis, but still use an effective drug in managing cure.(Wu et al.,1998). Various studies
indicate that activated plasminogen rapidly vanished so trend is shifted towards the recombinant
production of streptokinase (Wu et al., 1998). Various risks are associated with the streptokinase
therapy but still is used for treatments because it is cost effective and prove to be better than
other drugs

Tillet and Garner (1933) reported that streptokinase can be use to cause lysis of blood
thrombus. But the functionality of streptokinase depends upon the activation of plasminogen.
(Castellino, 1981). Streptococci especially the beta-hemolytic species that can be further
categories as A, B, C and G produced Streptokinase. Streptokinaseitself it is not a protease but
play role in activation of plasminogen, streptokinase binds with plasminogen(substrate) and
convert it into active product. As SK is injected into the body it mediates the lysis of blood clot
formed within the body due to the activation of plasminogeninto plasmin.However, it may cause
hemorrhage due to excessive blood lose, it can also dissolve cellular matrix and plasma
membranes which promote invasion of bacteria into the body which cause severe infections and

14
make feeble immune system.Therefore, there is need to minimize or lessen the side-effects of
streptokinase by designing improved products.

Streptokinase isefficient fibrin clot dissolver so it is being used as a thrombolytic drug

world widely some risks are also associated with it as appearance of allergic reactions on the
body. β hemolytic streptococci produced streptokinase so that it can be used for animal use to
cure disasters (McGrath and Patterson, 1984; Schweitzer et al., 1991).SK administration promote
production of antibody titters which can persist in the body for prolong period of time. (Lee et
al., 1993). Thus, the production of antibodies against streptokinase limits its use as an effective
drug (Jalihal and Morris, 1990) or it may cause allergic reactions in the (McGrath and Patterson,
1984).

2.1. Structure of streptokinase:

The complete amino acid sequence of SK was determined by sequential Edman

performed degradational experiments on the streptokinase during his findings he also evaluate
the number of amino acid present in streptokinase enzyme. (Jackson and Tang, 1982). His results
show that 415 amino acid combines to forms complete structure of streptokinase present in a
single long chain which folds upon itself to form secondary structure. Streptokinase form
complex with human plasminogen as revealed by the crystallographic analysis. Three structural
domains have been observed in streptokinase (α, β and γ) each domainshowing a similar folding
pattern but after its folding pattern becomes diverse. Through structural examination we can also
map or point out the antigenic regions responsible for allergy. Three most prevalent regions were
found i-e loop region containing 46-70 amino acid,the COOH-terminal 32 residues and the NH2-
terminal region containing 1-15residuesthese domains remain intact during the complex
formation. The antigenicity can be visualized at genetic level by marking spots 1-40 at different
locations of whole genome. Antigenic regions260-299 (spots 27, 28 and 29 from 130 to149 and)
contains three domains and loop region. Beta hairpin gb3 and gb4contains 350-369 (spot 36).
The amino acid residues from 320-339 related spot is 33may be involved in activation of
complex cancharts a loop region of domain of gamma. Nevertheless, the uncomplexed
streptokinase must contain amino acids which form loop region. We can formulate mutated
streptokinase molecules which proof to be more effective and beneficial than the native one just
about knowing the immunogenic regions.
15
 fig. 2.1. Structure of streptokinase along with its domains

According to the cyclographic studies the molecule of streptokinase is made up of three

structural domain α,1–146, β,147–290, and γ291–414 with each representing the location in
genome (Wang et al., 1998). Out of three domains the binding of beta domain with plasmin to
convert it into plasminogen is difficult than the other structural domains which can simply
interact and make product. Various experimental studies reveal that the alpha domain of
streptokinase has ability to recognize and bind free plasmingenic components while other can’t
do that (Loy et al., 2001). The γ -domain of streptokinase is responsible for the production of
activated complex and active enzyme by binding with plasminogen containing catalytic
apparatus. (Wang et al., 1998). The β-domain of streptokinase which create non-proteolytic
active site iteract with inactive plasmin with its special domain (kimet al., 2002). To understand
the mechanism of plasmin activation we have to first recognize the amino acid residues that form
each domain of streptokinase.
16
 Streptokinase produced by each specie varies from other on the basis of number of amino
acid residues present in it. Lancefield’s grouping help in identification of individual
streptokinase (Jackson and Tang, 1982). Amino acid sequences can also be predicted by
dividing the streptokinase as SK1, SK2 and so on. The sequence of SK 374 can be taken as a
model so that we can compare them and find better one (Ohkuniet al., 199). The nucleotide
regions which causes allergic reactions is determined along with the amino acid sequence from
streptococci. Similarity can be happened with other species of same group or different (Jackson
and Tang, 1982). The total sequences ofSK 24, SK 12, SK 1 and many more are being described.
Additionally, the sequences of amino acids from N- and C-termini of different streptokinase
producing species is defined more (Huang et al., 1989a).

Malke and co-workers, in 1985 described the nucleotide sequence of streptokinase which
first time proof as thrombolytic agent in S. equisimilis strain of streptococci. The pattern of its
functionality a transcriptional governor of this gene has been preplanned.Streptokinase can be
now produced which act as better fibrinolytic agent by using these information about gene
location ant afterwards its transcription. But this can be possible when we use effective strain of
bacteria.

2.2. Streptokinase producing species:

Streptococcireported as streptokinase producing species in 1874 by Billroth when he

conducting experiments on infested disorders. The same organism was found in the patients of
scarlet fever or hay fever.Later on, in 1919, Streptococcus sp.Were grown on agar media and
observe their activities on pottery plates. These species were classified based on hemolytic
activity into tree major groups i-e α β and γ.

2.2.1. Streptococci:

Streptococcus is aGram-positive spherical bacteria. The plane of division in these

bacteria more diverse than the other it cut the cocci into two pieces horizontally or vertically.
The name of bacteria indicate that it grows in chain-like fashion. Mostare catalase-
negative, oxidase-negative and, and several are anaerobes. Classification as described earlier
based on blood dissolving capacity. Oxidation of heme group inside the RBCs id carried

17
ooutwith the help of alpha-hemolytic. While beta-hemolytic organisms rupture the RBCs so
apperars as clear zone on the pottery plate. Gamma-hemolytic species are also termed asnon-
hemolytic speciesbecause is observed during its growth.

When beta hemolytic organisms present on blood agar area seems lightened yellow or
transparent due to the complete lysis of blood cells especially RBCs. Lysis of cells is observed in
those species which contain Streptolysinenzyme.Streptolysin can be further categorized
asstreptolysin S, Streptolysin O. The hemolytic effect may be enhanced when two species were
grown on the same agar plate nominated as the CAMP test. Most prevalent in Streptococcus
agalactia. In this way, we can easily identify our interested species by applying these tests

According to the Lancefield grouping in 1933 by Lancefield the beta

hemolyticstreptococci can be further classified into different groups from A to O. The most
prevalent type are A and C while the production of other can be ignored. No erythrogenic toxic
compounds are present in group C so it can be considered as an ideal one. Any streptokinase
inoculated to human is consider to obtain from this C group.The most effective and active
streptokinase obtained from group C species so it considers that 90% products of streptokinase
are group C producer.

Streptococcus aglactiaelikeanotherstreptococci sp.I-e spherical or rounded in shape that

can survive in pair or long chain. It is afacultative anaerobe, beta-hemolytic, andcatalase-
negative during testing . GBS inhabiting thegastrointestinal track and genitourinary tract of up
human being but cause no harm so it is considered as a friendly bacterium. The capsule of group
B streptococcus is made up of special polysaccharide known as exopolysaccharide. Serotyping is
done to reduce the ambiguity among them this is based upon the capsule composition.A cleared
zone can be observed where GBS grows.Latex agglutination tests are utilized for the detailed
identification of this specie based upon Lancefield grouping. Greater hemolysis of blood agar on
the pottery plate can be observed because staphylococcus and streptococcusgrows at same time
causing rapid lysis of RBCs by increasing their growth rate. CAMP test is used for this purpose
these are the few test which can identify the GBS from other species which prove to be more
effective in its characterization.

18
2.3. Myocardial infarction and administration of streptokinase:

Sol Sherry with his teacher William Smith Tillett in 1933, discovered that streptokinase
can be use a therapeutic agent for the treatment of heart diseases which were caused by clot
formation at different regions of many individuals.Streptokinase was first used for the treatment
of different disorders like contesting fibrinous pleural exudates and tuberculous meningitis.In
1958, Sherry with his coworkers changes the trend of streptokinase use.After his findings, the
use of streptokinase increases a lot. GruppoItalianoSperimentazionedellaStreptochinasi
nell'Infarto Miocardico (GISSI) experimented in 1986, to prove the efficacy of streptokinase as
thrombolytic drug. This was the first successful attempt in medical biochemistry. More than
hundred drugs have been discovered but the use of streptokinase not abolish because it is more
effective and cheaper drug than the others.Due to its cost, effective quality it is most often used
in developing countries.

Differed methods to intravenous injection of streptokinase was developed by Fletcher and

his coworkers.To handle myocardial disorders Fletcher used to inoculate massive amount of
streptokinase for long time. The use of urokinase or plasminogen do not reduce the mortality rate
as was reduced by streptokinase. So, it was a great discovery in order to treat infectious disease.
But the use of streptokinase is not sufficient so various other treatments also followed to increase
the efficacy of these drugs.The result of Fletcher was that streptokinase is most efficient in
treating different diseases specially related to thrombi formation. After his discovery, it was
heavily used in medical industries.

Fibrin may or may not be important in the activation of plasminogen into active plasmin
form, the usable form in the human body (Reed et al., 1993). Multiple domains of streptokinase
play role in the activation of plasminogen either alpha, beta r gamma domain prove to the
essential in the activation of plasminogen.Many of the active sites of streptokinase are self-
governing that is it does not depend upon the plasminogen or other compounds for its activation
(Nihalani and Sahni, 1998). Plasminogen which is the substrate of streptokinase is converted into
active form by enzyme streptokinase. the carboxylic terminal domain performs this activity
(Zhaiet al., 2003). Along with this c-terminal different amino acid residues are important in
substrate recognition and ultimate its binding. It was assumed that 41 amino acid and 48 that was
aspartate and histidine are necessary for binding. (Kim et al., 2000). The role of other amino acid

19
in this regard is also testify. Like the alpha domain beta domain is also important in the binding
and activation of plasminogen into plasmin so that it lyse blood clot to improve circulatory
condition.

Lysine residue is the main attachment point between the streptokinase and plasminogen
for its active conversion (Boxrud and Bock, 2001). The catalytic activity of streptokinase
increases when SKPLG interact through protein protein binding (Sundramet al., 2003). Beside
this and previously defined residues the functional property of streptokinase (Young et al.,
1995). So, all the domains with their residues are required for the active conversion of
plasminogen into plasmin. Firs fifty-nine residues are responsible for the stability of whole
structure of streptokinase elimination or modification of these residues from the streptokinase
reduces its activity and configuration (Young et al., 1995). Firs 56 residues are most important
and unique in each streptococci (McCoy et al., 1991).

20
 Fig.2.2. Mechanism of action of streptokinase

2.4. Risk related to streptokinase therapy:

As streptokinase is a bacterial product so, human body produce antibodies against

streptokinase which would be appear in the form of allergy. For the assessment of danger
associated with the use of streptokinase we divide it into cardiac and extra cardiac
risks.Intracoronary administration of streptokinase can lead to severe complications.This
administration can be visceral or not depending upon the situations. As streptokinase would not
present at the site where clot formation occurs then it would cause hemorrhage at different sites.
Fibrin of fibrinogen is essential in blood cascade mechanism because when its concentration
begins to fall bleeding risks increases. Bleeding will start when dose of streptokinase increases
from 20,000 ml and fibrin level decreases to 100 dm3.This massive quantity of streptokinase can
cause allergy. As streptokinase is protein in nature so the risk of allergic reaction becomes
doubles. Due to this the cardiac complications increases as clot does not allow the blood to pass
through veins in this way, effect tissues of the body. Arrhythmia isidioventricular, transient,and
accelerated rhythm (Stehleet al.,1986). The clinical manifestation is also available.

Bleeding risks are associated with the streptokinase therapy because it cannot recognize
normal or pathogenic thrombus formed within the human body and it may lead to hemorrhage.
To indicate whether it cause harm or not pulmonary embolism is use as a standard marker in the
blood of patients.Various methods have been developed for the inoculation of streptokinase.
Intravenous method gained most importance than the other to retain the actual position of body
that was before the clot formation. Standards are formulated to check its functionality.Clot or
thrombus lysis can be done by using streptokinase, tissue type plasminogen and urokinase. But
the mechanism of action of urokinase and tissue type plasminogen is not yet understood so
streptokinase is most widely used as thrombolytic drug.The effect of urokinase and tissue type
plasminogen remains for 2 hours.Before the inoculation of streptokinase, it was experimentally
verified which patients is capable of its inoculation.

The blood clots which are formed within the body are lysed by using intravenous
inoculation of streptokinase. Because streptokinase is associated with thrombus lysis so it was
considered a great risk of hemorrhage due to its excessive use. Experimental studies
21
demonstrated that the risk of excessive bleeding due to streptokinase therapy increases in last
three years from 0.3 to 3% so, it is considered as a real risk to life.From these findings, it does
not mean that streptokinase can’t be use for MI but it was suggested that the improved or
modified form of streptokinase can be use in future studies.

2.5. Antigenic regions of streptokinase:

Myocardial infarctionswere treated by using streptokinase. But streptokinase is a

bacterial product so it is antigenic in human beings. Human body begins to make anti body titters
against the streptokinase which would eliminate it and the function of streptokinase remains
incomplete. Because streptokinase is being neutralized by the presence of anti-bodies so no clot
lysis is observed. To point out the antigenic or allergic region we have conducted experiments on
human serum which recently receive SK therapy. Any region which was consider as cause of
allergy is mapped on the polypeptide chain so that it can be modified or eliminated in effective
way. In any sample contain streptokinase than it would form antibodies against it. The
appearance of antibodies confers that no clot lysis occurs in thrombotic disorders. Various
peptide sequences studied in order to find the allergy causing gene.

Streptokinase as a whole composed of three functional domains namely alpha, beta and
gamma domains.The structure of each domain is different but the binding pattern is similar in all
respects. If we know about the structure of streptokinase we would be able to identify antigenic
regions in polypeptide. The amino-terminal region (1-15), the carboxylic-terminal
regioncontaining 32 residues and the loop area contains46-70 residues are disordered, suggesting
that they areavailable andmobile even aftercomplex formation with plasmin. The region 350-369
encompasses the available β hairpingb3 and gb4. The peptide 320-339 assume to maps a
loopregion of domain complex and may be involved in activation. Nevertheless, this loop must
be available in the native molecule of streptokinase. The knowledge of the immunogenic regions
of streptokinase can guide us in the formulation of mutated molecules with reduced antigenicity
and perhaps with reduced lethal effects on health.

Exposure of streptokinase antibody neutralize the SK in the body of patients. This can be
physically apparent in the form of allergy. We use random peptide library to evaluate the exact
location of streptokinase. The amino acid which was present at the amino terminal region
consider to be involve in causing allergic reactions. A dominant structural motif was identified
22
through repeated experiments, this motif contains 3-7 amino acid as were found in the
original.Beside this the second amino acid sequences from 59-63 were designated as a cause of
immunogenicity not only in humans but also in another animal. These findings about the
sequences of streptokinase enables us to correctly pinpoint the nucleotide sequence which were
responsible for its expression.

The experiment was conducted on the mice in order to find the effect of antigenic regions
on the activity of streptokinase. Six epitopes were identified on the polypeptide chain of
streptokinase when viewedthrough different binding pattern. The results of these experiments
which was conducted on two types of mouse were compared to find any resemblance.The
functional activity of streptokinase halted due to the presence of any kind of antibody in the
mice. The studies which was conducted to find the antigenic regions in streptokinase revealed
that most of the antigenic sequences in streptokinase play role in the activation of plasminogen
so that it can perform lysis of blood clot. So, the elimination or replacement method not only
changes the immunogenicity but also interferes with its function.

By using human serum and different strains of mouse we identified three immunogenic
regions. Half of these immunodominant sequences take part in the activation of plasminogen into
active form that is plasmin which then dissolve the blood clots.The identification of these
immunogenic regions help in bringing the mutation either through chemicals or through
radiations it depends upon that which method improves the quality. Mass spectroscopic
techniques were used to clearly nominate the antigenic regions in streptokinase due to which the
use is limited. Antibodies formed due to this activity appears as allergic reactions. We can also
find the epitopes which would be essential for the proper functioning of streptokinase enzyme.
After the recognition, these regions were plotted this includes 96 to 99 and 323 to328 residues of
streptokinase. At 4-8 position, 171-177, and 334-338 regions were previously identified. Here we
explore them and find its connection point with recently discovered antigenic epitopes.

Polypeptide chain of streptokinase is broken down into 4 components, the breaking point
present at 347,470 and237 (Bruserdet al., 1992). HLA-class II restriction enzyme was use to cut
it into different fragmentsAA 348-369, AA 238-346, AA 371415 andAA l-236 regions then find
as antigenic epitopes. The evaluation ofT cell clones demonstrates that more than one
immunodominant regions would be present. This elaboration was done to estimate the

23
consequences of mutation in these regions.The whole T cell clones of human can identify the
antigenic regions if we have a short fragment then it would be insufficient in its performance.
Methionine residue which act as antigenic marker was identified by using T cell clone. Similarly,
this methionine residue was formerly used for the cleavage of molecules. We conclude that 5
antigenic regions present at the single polypeptide chain inhibits the proliferation of defensive
immune T cells in the body.That’s why it become the major issue which can be resolve by
mutating the native molecule of streptokinase.

It was suggested that Protein, or parts of proteins, may be reduced non- immunogenic, or
less immunogenic, to a given species by identifying in them more potential epitopes for T-cells
of the given species or amino acid sequences then modifying the amino acid sequence to
eliminate at least one of the T-cell epitopes. This reduces or eliminates the immunogenicity of
the protein when exposed to the immune system. Monoclonal antibodies and other
immunoglobulin-like molecules can particularly gain benefit from being de-immunized in this
way

2.6. Modification of streptokinase to lessen its immunogenicity:

The ability of streptokinase to cure thrombolytic disorder is halted due to the drawbacks
associated with its utilization. It is also avoided because the life span in circulating blood is less
than other therapeutic agents. Streptokinase cleaves the plasminogen into plasmin so, that it
could catalyze the dissolution of blood clot. Due to this ability of streptokinase to lyse blood clot
it was considered that ant structural change in the streptokinase would make it ideal drug for
daily consumption. These structural changes not only improve the functional activity of
streptokinase but also eliminate the antigenicity. Structural modifications can be carried out by
various techniques like recombinant technology, chemical mutation and site directed
mutagenesis. Antigenic regions in streptokinase were identified in the 1930s(Tillett and Garner,
1933).If a person is subjected to streptococcal infection, antibodies begin to appear in the body
in this way deactivated the streptokinase so it can’t perform any lysis of clot and patients starved
to death.Almost in 80% of the people anti bodies against streptokinase persist because
streptococcalinfections are prevalent (Kazmiet al., 2002).

the immunogenicity potential of each domain of streptokinase is different with respect to

each other (Reed et al., 1993). After the appearance of allergic reactions due to streptokinase
24
therapy it becomes the topic of interest to find out the antigenic or immunogenic regions of in
SK (Coffey et al., 2001). In2001, the regions in the streptokinase were identified which were
consider the cause of allergic reactions (Coffey etal., 2001). After its discovery, the various
methods were employed to lessen the antigenicity either through the recombinant technology or
through chemical or genetic modifications which would prove to be effective. Site directed
mutagenesis were done to eliminate the 42-amino acid at the carboxylic terminal of
streptokinase, results matches to our goal (Torrens et al.,1999b). Along these sites directed
mutagenesis one PEG complex is also utilized to check whether it prove affective or not.
Stability and activity of streptokinase can also be improved through substitution of various
amino acid in this way a decrease in immunogenicity was observed. In these substitution
reactions one amino acid say glutamic acid is replaced by the lysine residue at 59 position of
polypeptide chain. The activity was observed along with its immunogenicity.

2.7. Chemical modification of streptokinase:

Specific activity of streptokinase was reduced by Vober Med after incubation in

the system containing I-ethyl-3(3-dimethyl aminopropyl) carbodiimide (EDC) ethylene diamine.
Under these conditions 8 free amino groups and 38 free carboxylic groups were modified in
the streptokinase molecule. The protein conformation and the state of tryptophan residues were
changed. Thestreptokinase modified by means of EDCethylene diamine treatment vanished
itstability to form a stable complex with human plasminogen.

25
 Fig.2.3. I-ethyl-3(3-dimethyl aminopropyl) carbodiimide (EDC) ethylene diamine

Mutagens act to change the nucleotide sequence within the strands these changes can be
brought about the addition, deletion or substitution of base pairs formulating the DNA strands.
Because these mutations occur at sudden without any control measure so, two type of mutations
can occur either deleterious or beneficial. Here we carry out those mutations that does not
change the structure or function of streptokinase such mutations are called as silent mutations.
In silent mutations mutation occur at non-coding region or where it could cause less damage to
protein structure.Acyl-plasmin-streptokinase is one of the mutant which is functional more
active and exposed active site so that substrate can bind it easily. Anti SK Abs changes the
functional potential of this mutated streptokinase.Acyl-plasmin-streptokinase interacts with
polyethylene glycol in order to make it better therapeutic agent via chemical modifications, the
mutated streptokinase produced in this way has capacity to persist in the body for longer period
of time i-e antibodies don’t interact with it (Pratap et al., 2000).

After the successful discovery of chemically modified streptokinase attention was

focused towards the production of more chemically modified streptokinase by using poly
ethylene glycol. It forms complex with streptokinase in order to make it less antigenic. PEGs
with average molecular weights of 2,000, 4,000, and 5,000 were activated with Carbonyl
diimidazole form complex with streptokinase, molecular weight of these streptokinase ranges
from 2000-5000 or in some cases more than that. Kinetic analysis established differences in the
Km values of modified and native streptokinase. When chemically modified plasminogen
combines with streptokinase it lowers thekcat values.So, it does not follow any Michaelis-
Menten relation.After that the functional activity of streptokinase and plasminogen complex
were determined.As we had use two reaction group one containing mutation at 5 regions of
streptokinase while other at 2 and 4 position, the activity graph shows a rapid reduction in the
first case. Streptokinase both mutated and native were further tested for the activity it was
shown that the reactivity of plasminogen to streptokinase increases in those species where
mutation occur at 2 and 4 portion.A plastic bottle was taken coated with anti-SK and incubate
the streptokinase with plasminogen.

Any mutation introduced in this way lowered the activity of streptokinase along the
alteration in structure. Plasminogen react with streptokinase to forms activated complex which

26
would promote catalysis of blood clot both in vitro and in vivo. The activity of mutated
streptokinase is also less than the native which was produced through radiation method. The
minute capacity of streptokinase to catalyze reaction depend upon the alpha domain of
streptokinase which help in complex formation and later on the catalysis. PEG-2-streptokinase
the mutated form retain activity just due to the presence of alpha domain. Alpha domain
considered to be essential for the proper functioning of streptokinase. This was the first
mutation in the structure of streptokinase which was carried out through chemicals such as poly
ethylene glycol. The adduct which formed from the PEG streptokinase and plasminogen less
immunogenic than the native molecule and can be efficiently used to cure myocardial
infarctions and other thrombolytic diseases.

2.8. Site directed mutagenesis:

Various selective or non-selective mutagenesis were used to explore the role of each
domain along with the linkers in the activation of plasminogen, the alpha domain is the most
prevalent of them. The sequences which was thought to be form beta domain is used for the
construction of specific loop region.The mutant formed in this manner showed a six fold
decrease in activity and is insufficient to carry out the binding of substrate to streptokinase so
that it could form active plasmin. the kcat level of mutated molecule is much less than the
unchanged streptokinase there also exist change in the Km value used for indicating the binding
of streptokinase with plasminogen.Surface Plasmon Resonance were used this time to find the
binary and ternary complex formation. The changes which was done by different method has
prevalent effect on the ternary complex but at the same time it does not alter the binary complex
formation through deletion, addition and substitution mutations. Kd value can be obtained when
ternary complex binds with kringle unit of plasminogen.The significance of this loop could be
observed through taking view of deletion mutation at various sites of streptokinase, complete
deletion of loop region made up of 88 to 97 residues and also on the ability to bind with plasmin.
The loop region in the alpha domain of streptokinase is thought to be essential for the activation
of human plasminogen.

27
 Fig.2.4. Depiction of 88-97 loops of streptokinase alpha domain

The above figure demonstrates about the alpha domain of streptokinase with surface
exposed amino acids involve in the loop formation.We alter the configuration of loop region in
order to find its role in activation through mutations especially site directed mutagenesis. Bigger
change in the number of amino acids that is complete deletion of loop region make difficult
binding nd catalysis of substrate.Site directed mutagenesis was done to induce changes in the
structure of streptokinase especially the loop region.Accordingly, we change the amino acid
residues which would assume for the beta turn production in the loop region to observe the rate
of mutation along with its activity.
28
 The newly introduced mutant carrying beta turns formed by altering four amino acid all
at sudden. Val-89, being altered to Pro and Ala were changed with Ile-88 respectively, Ser and
Lys to Asp-95 and Asp-96, can be changed. This is called control beta turn mutation.
SKDD95,96SK andSKIV88,91PA were also changed in the native structure. Along with them
one more mutant was produced by substituting the alanine residues at 92 position.After the
formation of mutations in the native structure of streptokinase we evaluate the impact of each
mutant on the activation of plasminogen and complex formation is also observed.The mutations
that were carried out in different streptokinase may or may not prove to be beneficial in every
respect. Most significant mutation was in the beta turn at 88 position.

The structural arrangements in alanine substituted mutant and the mutant produced in the
loop region were similar as observed in the original structure of streptokinase. These mutants
have specific protruding site which was involve in the activation of PNG.Artificial minimizing
effect did not accept as standard. Overlapping regions of loop does not show any structural
variation. Instead the modified streptokinase consider as better fibrinolytic agent and the
antigenicity reduces many folds.

Fig.2.5. Substitution mutation in streptokinase

29
2.8.1. Deletion mutation of streptokinase:

Streptokinase can be used to cure myocardial diseases but its utilization is a risk because
body form antibodies against streptokinase.The antibodies formed against it limit the activation
of fibrinolytic system which otherwise be the basic role of streptokinase. Allergic reactions are
promoted due the presence of antibodies.Streptokinase antibodies can be used to eliminate the
SK drug.Patients treated with streptokinase to dissolve the blood clot require plasmin activator
later on.For making the streptokinase less immunogenic we eliminate 42 amino acid at C
terminal. This was done by using animals especially monkey and then compared with native
molecule of streptokinase. Monkeys were given antibody which neutralize the antigens or
streptokinase (Torrens et al., 1999).

Neutralizing ability of streptokinase antibody is observed world widely when patients

were exposed to streptokinase. As we continue to use streptokinase therapy the effect of
streptokinase on the clot lysis reduces gradually and eventually becomes functionless.The
present study was conducted to modify the antigenic regions of streptokinase without affecting
the functional activity of it.The streptokinase molecule which lack specific amino acids at 42
regions act as less immunogenic in nature than the native when activity is compared the mutated
act as best choice. On the basis of anti-streptokinase antibodies two groups of monkeys were
organized containing 7 in each group. One group is subjected to normal streptokinase while other
with mutated form. Treatment show extraordinary results regarding the use of mutant
streptokinase. Antigenicity were obvious by using this mutated form of SK. Both groups of
monkeys develop antibodies revealing allergic reactions. monkeys which receive streptokinase
developed anti body titer in the blood, no matter how many times it would exposed to
streptokinase.

The organisms treated with mutated streptokinase produced antibodies but in lesser
amount than the group B monkeys. This mutated streptokinase doesn’t contain 42 amino acid at
the carboxylic terminal.Conformational topographies have shown that the antinational response
of each streptokinase is unique and don’t depends upon the other.The mutated streptokinase
lacking 42 amino acid in their structure reveal hidden active sites and in this way altering its
condition towards the immunogenicity.Other functional regions may be present in truncated

30
streptokinase. This mutated molecule is different in its action as compared to the native (Torrens
et al., 1999).

The alpha domain to streptokinase is involve in the recognition of complex plasminogen

especially the N-terminal domain. This ability of streptokinase can be explored on the basis of
amino acid residues usually 45 to 70 which might be the unique characteristic of streptococcus
involve in the activation of plasminogen into plasmin. The alpha domain linker can identify
substrate only but don’t play any role in the formation of activated complex by providing active
site for reaction to take place. Theprotease domain of plasminogen was gritty along with 2
mutated structure of streptokinase.In mutated form, the mechanism of plasminogen and
streptokinase is different from the unchanged SK however, no structural change is observed in it.
The mutated alpha streptokinase readily changes its confirmation. Activation of plasminogen is
more efficiently occur in alpha streptokinases than the other so this mutation prove to be helpful
in the identification of immunogenic regions. All this information obtained from kinetics of
reaction.

The method followed by the streptokinase to make active plasmin is discovered by

crystal structure of streptokinase. The binding pattern of native and mutated streptokinase was
similar. The activation pattern is clearer in truncated streptokinase than the native one. A he
differences can be visualized in the activity of both alpha and native streptokinase.Different
loops represent different amino acids the 760 loop and the 737 residues. The second loop which
named as autolysis loop present where streptokinase interacts with plasmin (Wang et al., 1998)
and the complex appears between streptokinase and plasminogen.The oxyanion stabilizing loop
is also reported in this complex. Because this loop is connected through di-sulfide bond with
other loops.

2.8.2. Substitutional modifications:

For the determination of antigenic regions in the streptokinase structure we carry out
substitutional or replacement modifications in native structure. Due to these modifications, the
activity of both anti SK and native one also changes. Special modification is the insertion of
serine residue at 138 position is carried out as a result loss of activity is observed so we can say
that serine 138 is essential for antigenicity in human beings. This was later confirmed by

31
changing the serine residue with lysine. Monoclonal antibody that was A.43 did not bind to
streptokinase in which serine is replaced with lysine amino acid. Later on, activity is checked.

In our precedingstudies, we mapped the antigenic regions on the basis of monoclonal

antibody. (Parhami-Seren et al., 1996). Both streptokinase variant and native used for the
identification of antigenic regions in the streptokinase.Residue 138 in streptokinase is basic part
of the monoclonal antibody A.43which was revealed from the substitution of different residues
in native and modified form of streptokinase. If we test the activity by using monoclonal
antibody A.43 there will be no reaction in those streptokinases which have altered gene sequence
that is serine 138 replaced by the lysine residue. Both the altered streptokinases that is G and
130. Streptokinase showed a loss in reaction with the antibodies that means it does not produce
any allergic reactions in the body. Site directed mutagenesis is also carried out for its
confirmation whether it is true or not. In streptokinase that has lysine residue instead of serine at
138 position don’t have clear information about the formulation of active site so that it can
catalyze reaction. The kinetics of this reaction does not change significantly.

Leucine residue at 42 position is essential for the formation of complex with plasminogen
reported by the site-directed mutagenesis in streptokinase (Kim et al., 2000). For the proper
complex formation between streptokinase and plasminogen Val 19 of streptokinase bind with
leucine 137 residues by forming hydrogen bond, in order to perform function (Kim et al., 2000).
Trans version of Alawith Leucine mainly at 42 position (Liu et al., 2000) eradicates breakdown
any connection point betweenThreonine and Leucine. These amino acids are responsible for the
compact structure of streptokinase. Glutamate amino acid at 134 position and Tyrosine at
position 713 connected with each other via hydrogen bonds formation in the loop region of
streptokinase.

32
 Fig.2.6. Important residues of alpha domain of streptokinase

Mechanism of streptokinase activation can be revealed from the genetic information but the
identification of allergic or disease causing genes is the major task. If we know the gene
sequence of allergy causing potion it will be helpful in the formulation of vacancies which
provides beneficial effects. (Nordstrandet al., 2000). In this way we can obtain information about
the structural modification of streptokinase specially the modification of antigenic regions.

2.8.3. Insertion and deletion mutation:

The streptokinase of streptococcus bacteria is composed of three domains namely alpha

beta and gamma that are inter linked with special amino acid sequence called as linker or binder.
To explain the need of linker in its structure substitution take place between the linker 1 and
linker 2. The resulting specie with altered sequence of amino acid loss its activity beacause
essential site present at active site moves towards the center of molecule. The mutated
streptokinase did not perform its function as normally. So we can say that replacement of linkers
is not suitable for the proper functioning of streptokinase.In linker 1 substitute the serine residue
with nine glycine molecules the catalytic activity of streptokinase persisted whereas the mutation
in the 2 linkers of streptokinase which is present between the beta and gamma domain losses it
33
proteolytic activity all at sudden. The deletion mutation which was done in linkers of
streptokinase don’t appear to be beneficial instead the eliminated amino acid residues were
essential for its proper functionality. Formerly, it was considered that that the length of linker is
main point for its activity to prove this some amino acids were introduced externally which has
the capacity to change into rigid or loss configuration. Reduction in activity were observed with
the addition of five amino acid in linker 2 activity were reduced to ten folds in each case whereas
it doesn’t exert ant deleterious effect on the linker 2.However, it was observed that mutated
linker form stable 3D structure and react actively with plasminogen. From these findings, we
conclude that proper length of linker is essential for its function, linker 1 does not affected by the
modifications rather linker 2 is most susceptible to the changes so linker 1 which is present
between the alpha and beta domain is considered as covalent linker. Most of the functionality
depends upon the linker 1. The distance of these linkers from all three domains is similar in all
respects. One interesting thing is that the removal of these linker does not affect the activity of
streptokinase.

The linker 1 which were present between the alpha and beta domain of streptokinase is
replaced by the liker 2, present between the beta and gamma domain. This replacement does not
carry any ideal change in the structure rather reduces its functional activity. One hypothesis that
the length of these linkers is essential for proper functioning proves to be wrong because the
linker 1 does not losses its ability whereas linker 2 show a huge damage not to its structure but
also functionality of streptokinase enzymes.

34
 Fig.2.7. Linkers of streptokinase

To construct the SKlin2–lin1 we exchange the liker position between the domains or
streptokinase that is the 1 linker which is present between the alpha and beta domain would be
replaced at 2 position between the beta and gamma domain and the second linker would be
treated in a same manner by means of extension strategies. By altering the position of these two
linkers a mutated streptokinase will produced whose ability to activate the human plasminogen
will be tested by various methods. New proteolytic sites would expose on the surface of active
site which could not perform function in a better way or the activity of streptokinase reduced by
two hundred times so it indicates that cross substitution of these linkers is not suitable for the
functionality of streptokinase.

35
36
2.9. Recombinant streptokinase:

Streptococcuswas obtained from a clinical sample collected at the São Lucas Hospital
(PUCRS), and DNA haul out using a Proteinase K-Phenol-Chlorophorm method with few
modifications (Sambrook JG, 2001).BamHI andNdeIrestriction sites were present in the primers
which were amplified through PCR technology. These amplicons inserted into blunt end vectors
and then subjected to cloning These plasmids were designated as pET30a (+)-SPskapy (ska
including the periplasmatic signalpeptide) and pET30a (+)-skapy (ska codes for the mature
protein). E. coli BL21 (DE3) and Rosetta (DE3) cells were eletroporated with the pET30a (+)-
SPskapy construction and cultivated at 37°C in liquid LB medium during 24 hours. Protein
analysis was carried out by SDS-PAGE electrophoresis after sonication of the cells(Galler, 200).

To confirm the presence of streptokinase in the culture medium and /or periplasmatic
fraction, BL21(DE3) cells were transformed with the identical construction, and cultivated in
liquid LB or YT media at 37°C, using IPTG to induce protein expression. The media and
periplasmatic fractions from osmotic shock were used for total protein analysis in SDS-PAGE
electrophoresis. The plasmid containing the ska which codifies for the mature protein was
transformed into BL21(DE3) and Rosetta(DE3), the cells were cultivated in LB at 37°C during
24 hours using IPTG for the expression of truncated gene. Cells were collected by centrifugation,
sonicated, and total protein content analyzed by SDS-PAGE electrophoresis. A 47kDa band
(streptokinase expected size) was detected in the insoluble fraction of all strains containing the
pET30a(+)-SPskapy construction. We have not observed expression of streptokinase either in the
soluble fraction or in the culture media or periplasmatic fraction. There was no SK expression in
none of the strains transformed by the vector pET30a (+)-skapy (mature SK). Additional
experiments should be performed to achieve soluble expression of SK, using diverse growing
conditions by combining distinct E. coli strains, culture media and temperature. Modifications in
streptokinase gene is done at a site where it does not affect the activity of streptokinase molecule
these changes may be based on the trans version of serine residue with other amino acid or
deletion of that to make it effective. (zhanget al.,1999). This replacement of serine residue did
not alter the structure or function of streptokinase.

37
 Fig.2.8. Truncated form of streptokinase enzyme

Streptokinase gene were introduced inPst1 and BamH1 of pET41a at multiclonal site so that we
can observe differences in mutated and unchanged form of streptokinase practically. Usually
three different genes of streptokinase were inserted into the vector to visualize the effect of these
mutations.Different genes along with streptokinase is inserted into the vector such as antibiotic
resistant gene, different tags for indication and codons that stop or initiate a process.

The gene product obtained from this method run on PCR for its amplification, as we use
2 mutated genes having mutation at 60 to 386 residues and other at 143 to 386 and one native as
a marker or standard which were amplified from the template strand. (Estrada et al., 1992).
Substituent expression of these genes along with the tags and required sites at N terminal region.
Transformation occur in the plasmid of E. coli BL21 and produced protein bands which were
truncated earlier to get better results. SDS PAGE and western blotting system is also use for this
purpose.

The truncated or mutated streptokinase obtained through cloning in E. coli BL21 were used
excessively because they are more disease curing and resistant to any structural change. Due to
the expression of tags it become more easier to identify the truncated regions which was done by
using different methods as described earlier.

38
2.10. Assessment of truncated and full length purified streptokinase:

Inadequacies of streptokinase is reported world widely but still it used a drug to cure
hearth diseases or thrombolytic problems (Banerjee et al., 2004). So, truncated molecules of
streptokinase are being constructed which may lack different amino acid residues at specific
positions like 59 amino acid is eliminated at N region or 42 at the same point along with the
removal of 28 amino acid at carboxylic terminal based on the previous studies. Thus, it
waspainstaking for the elaboration of antigenic regions present at specific regions of
streptokinase. If view towards the native and mutated streptokinase there exist a significant
change among them which is beneficial for the treatment of different diseases. By using these
findings, we can also fulfill the British pharmacological standards because no allergic reactions
associated with the truncated or mutated streptokinase and no adverse effects have been found
during its administration.

The potential of its activity can be determined by various method one of them is the
caseinolysis method (Saksella, 1981).According to British Pharmacopoeia the activity of
truncated streptokinase can be measured or estimated either through fibrin clot lysis method or
through chromogenic assay. In this method, we load our two samples mutated and native and
observed that which fragment cause more lysis of blood cells forming clear zone in Petri
plate.This will be the physical estimation we can also estimate the biological activity by
measuring its productivity. The native molecule of streptokinase having 414 amino acids show
different reactivity than the truncated or mutated streptokinase molecules either having 417 or
413 amino acid in its structure.The activity is measured in term of fibrin dependent activity that
is can streptokinase effectively produced plasmin from the plasminogen (Reed et al., 1999).
Thestreptokinase considers to be effective if it catalyzed the lysis of blood thrombus within
twenty minutes or less than this time. If catalysis done within this time period then it considers
that it has acceptable therapeutic potential as compared to the others.

39
Table No.1 Biological activity of full length and truncated streptokinase

Biological activity (IU ml-1 ) SK1-414 SK60-386 SK143-

386
3758 634
Specific activity (IU mg-1 ) 10659 2094 1356
Specific activity (IU nMol-1 ) 956 142 97

The activity of mutated streptokinase altered as compare to the native molecules that means it
does no approve as effective in functionality as unchanged.By taking an over view it becomes
easier to design mutated streptokinase molecules with higher efficiency and greater functional
probability. To formulate least antigenic and more effective streptokinase in its activity
elimination of eithercarboxylic terminal or amino terminal region or both at a time.The mutated
streptokinase proves to be perfect for its fibrinolytic activity and has less antigenic impacts when
being utilized. After various researches on mutated streptokinase molecule it is accepted by
major clinics for the treatment of heart diseases and other thrombotic issues.

40
 CHAPTER NO.3

Material and Method

3.1. Specie identification:

There are various methods to identify streptococcus aglactiae but here we mention only
few which are basic and effective in its characterization. Phenotypic, biochemical and molecular
methods are routinely used.

3.1.1. Morphologic characteristics:

Streptococcus aglactiae is a Gram-positive bacterium with a tendency to form chain thus

giving name streptococcus is surrounded by a bacterial capsule composed
of polysaccharides (exopolysaccharide). After incubation on BHI agar (MERCK, Germany) at
30°C for 24 h, the colonies appeared to be glossy and raised, with a diameter of 1.5-2.0 mm. it
also has the capability to hemolyze blood agar.

3.1.2. Biochemical characterization:

GBS grows on blood agar plates as colonies surrounded by a narrow zone of β hemolysis.
GBS is characterized by the presence in the cell wall of the antigen group B of Lancefield
classification also known as Lancefield grouping that can be observed directly in intact bacteria
using latex agglutination tests. The CAMP test is also an important test for identification of GBS.
The CAMP factor produced by GBS acts synergistically with the staphylococcal β-hemolysin
persuading enhanced hemolysis of bovine or sheep erythrocytes. GBS can also
hydrolyze Hippurate and this test can also be used to identify presumptively GBS. Hemolytic
GBS strains produce an non-isoprenoid polyene pigment (grenadine) orange-brick-red in color
when cultivated on Granada medium that allows its identification. Biochemical characteristics of
the isolates were inveterate by microbial biochemical identification which are the basis of
standard phenotypic testing criteria, Gram stain, motility, oxidase activity, growth
characteristics, and hemolysis test.

41
Table No.2 Characteristics of Streptococcus aglactiae

Characteristics Results
Gram-stain Positive
Shape Coccus
Motility Negative
Oxidase Negative
Catalase Negative
Starch Negative
Lactose Negative
Glucose Positive
Blood hemolysis Β-hemolytic

3.1.3. Molecular methods for characterization:

16S rRNA gene sequence analysis: The PCR product of the isolate was analyzed at
the amplified fragments of approximately 1500 bp in size. The 16S rRNA sequence of
the isolate was examined via BLAST network services. Sequence alignments with known
sequences in the Gene Bank database showed that the brain isolate had high resemblance (99%)
to S. agalactiaeisolated from China. The sequencing result of the isolate showed the sequence
length of the PCR product was at 1201 bpand was deposited in Gene Bank. Additionally, using
PCR methods have shown to be a convenient in the detection of S. agalactiaeas well as a vast
reduction in time in comparison with culture methods. Most new researches depend on
biochemical and molecular methods for identifying and characterizing bacteria. This study was
done using standardized set of biochemical documentations and confirmed by molecular
approaches (16S rRNA gene PCR) to the isolated bacteria. Henceforth, we don’t focus on the
pathogenicity of these bacteria.

Molecular approaches: The identified S. agalactia from the eye was exposed to 16S
rRNA gene polymerase chain reaction (PCR) amplification by universal primers for
confirmation of S. aglactiaestrains.

42
3.2. Site-directed mutagenesis of SK:

3.2.1. Primer designing:

The megaprimer method was utilized for the introduction of mutations in SK gene
(Parhami-Seren et al., 2001). Normally, two primers were used to generate the megaprimer.The
sense primer 5VGCTGACTTACTAAAGGC 3Vhybridized to nucleotides encoding amino acids
sequence 77–82 in SK and the anti-sense mutagenic primer
5VGCACATGTCCTTTTAGCAAAAATTC 3Vhybridized to the nucleotide sequences encoding
amino acids 130–142 and contained the mutation from Ser to Lys at position 138. Polymerase
chain reaction (PCR) was used to create a 201-bp fragment using these two primers (Parhami-
Seren et al., 1996). The megaprimers or PCR fragments produced by this method comprises a
naturally occurring MunI site at amino acid position 86–87 of SK. A second PCR fragment was
generated using a second anti-sense primer and that hybridized to the C terminus of SK gene and
contains the PstI site in the PCR fragment. The second PCR fragment (986 bp) which contained
truncated SK gene borderedbyPstI andMunIwas digested with the two restriction enzymes and
was cloned into the PstI–MunI site of a digested pMAL-c expression vector having the
complementary SK N-terminal sequence.

The truncated SK gene in pMAL-c was electroporated in E. coli BB4 cells. Nucleotide
sequences of DNA made from these clones chosen at random confirms mutation. Single colonies
were grown overnight and soluble MBP-SK was purified by using different techniques.

43
Table No.3 Nucleotide sequences selected for mutagenesis

Pf60(Forward) 5’-CGAGGATCCAGTCCAAAATCAAAACC-3’

Pf143(Forward) 5’-ATAGGATCCCATGTGCGCGTTAGAC-3’

Pr386(reverse) 5’-CCGCTGCAGTTACTAGGCTAAATGATAGCTAG-3’

Pf1(Forward) 5’-GAAGGATCCATTGCTGGACCTGAGTG-3’

Pr414(reverse) 5’-ATCTGCAGTTATTTGTCGTTAGGGTTATCAGG-3’

3.2.2. Gene amplification through PCR:

The PCR reaction mixture of 16s rRNA was complete in 25 µl total reaction using ×2
MyTaq mix (Bioline, UK) with 10 µM of each primer. Negative control se act as non-template
mixture. The gDNA of the isolate was amplified for 16S rRNA by bacterial primers1492R (5’-
AGAGTTTGATCCTGGATGCTCAG-3’ and 8F (5’-GTTTACCTTGTTACGACTT-3’).
The PCR reaction was performed in a Thermal cycler with an initial denaturing step at 95°C for
5 min;55°C for 1 min and 72°C for 2 min, 26 cycles of 95°C for 30 s; followed by 72°C for 10
min. 6 µl of the amplified products were electrophoresed by 1.2% (w/v) agarose gel in ×1 TBE
electrophoresis buffer. Standard DNA ladder, 1 and 100bp were used to confirm the size of the
amplified PCR products at 1500 bp. The gel was stained with ethidium bromide and documented
by UV-transilluminator. Results obtained were analyzed and compared with
sequences from Gene Bank using BLAST NCBI. Streptokinase gene from a standard
streptococcus strain (S. equisimilis, ATCC 9542) which is also called as skc2 (Estrada et al.,
1992), was PCR amplified by Pfu DNA polymerase reaction. VariousPstI-tailed reverse primers
and BamHI-tailed forward were used to amplify the nucleotide sequences corresponding to full
length streptokinase genei.eSK1-414 and truncated SKs having sequences SK60-386, SK143-
386. In this regard Pf1 and Pf414 were used as primers for amplification of skc1-414 and pairs
ofPf386 and Pf60, Pf143, Pf386 were practical for amplification of skc143–386 and skc60-386.
Thermal program was set as 30 cycles for 60 sec, at 72°C for 90 sec, 95°C, 58°C for 45 sec
which was followed by a final extension at 72°C for 5 min. The amplicons, after digestion

44
withorBamHI orPstIrestriction enzymes, were discretely cloned into the similar sites of pET41a
vector downstream and in frame of the vector-derived tags, under the control of a T7 promoter to
construct these recombinant plasmids ofpETSK1, pETSK143and pETSK60. The constructs
which encodedSK143-386, SK60-386 and SK1-414, were determined by sequencing and
subsequently transformed into the E. coli BL21 cells. Single colony transformants were
transferred into TY2X medium (OD 600 = 0.6) to express the recombinant protein for 3–5 hours
after induction by 0.1 mM IPTG (Isopropyl-thiogalactoside).

3.2.3. Cloning of SK gene from streptococci:

Genomic DNA from each streptococcus strain, was secluded from 5 ml bacterial
cultures grown at 37 ◦Covernight (Huang et al., 1989b). The full-length SK sequence (1–414 aa)
from genomic DNA of each streptococcusstrain was amplified through PCR by using two
primers which were designated based upon the nucleotide sequences of the streptokinase gene
from S. equisimilisLancefield’s group (Malke et al., 1985). The Nterminal primer contains an
EcoRI site and hybridized to the nucleotide arrangements encoding the leader peptide (26 aa).
The second primer hybridized to the C-terminus of the streptokinase gene and have a PstI
restriction site and two stop codons within it. All streptokinase genes were cloned in the EcoRI–
PstI sites of PKK- 223-3 expression vector.

3.3. Expression of SK variants:

TG1 cells containing the SK/PKK plasmids were grown in 5 ml of either super medium
(15 g Bacto-Yeast extract, 25 g Bacto-Tryptone, 5 g NaCl in 1 l SM) at 37-degree grade
temperature overnight. In the following days, bacterial cultures were diluted 100-fold and were
grown at 37 degree calcioustemperature in SM medium containing 25 Ag/ml carbenicillin for 7–
8 hours (absorbance at 600 nm will be 2.0). The recombinant proteins were collected from
culture supernatants by centrifugation method. Expression was confirmed by testing the ability
of the culture supernatants to form usefulSKPAC in chromogenic assay

3.4. Protein purification:

Pellets of induced bacteria (5 ml) were lysed in 50 μl of lysis buffer (8 M Urea,100

mMNaH 2Po4, 10 mMTris.Cl), resuspended in 50 μlLaemmli buffer (0.09 M Tris-HCl, 20%
(v/v) 0.02% (v/v) bromophenol blue, 2% (v/v) SDS,Glycerol and 2% (v/v) β-ME) and heated at
45
80°C for 5 minutes. The supernatants were electrophoresed in 12% SDSPAGE gel and separated
proteins were either transferred to nitrocellulose membrane or stained by coommasie Brilliant
Blue. After blocking the membrane in 3% (w/v) bovine serum albumin (BSA) and application of
proper dilution of mouse anti-His antibody subsequent washing steps, HRP-conjugated goat anti-
mouse IgG was utilized as tracking antibody. The bands related to the recombinant SK proteins
were visualized by application of DAB (3.3’ Diaminobenzidine) substrate. Recombinant proteins
protecting the N-terminally tagged 6xHis amino acids were purified in denaturing condition by
the application of Nickel-TitriloTriaceticAcid (Ni-NTA) agarose columnsaccording to
manufacturer protocol. Briery, the pellets of 200 ml of induced bacterial culture were lysed in 5
ml of lysis buffer (10 mMTris.Cl,100 mM NaH2Po4, and 8 M Urea) for 15 min. After
centrifugation for 20 minutes at 10000 rpm, the upper lysate was mixed with 2 ml of Ni-NTA
resin for 30 min in a Falcon tube. The mixture was then loaded into appropriate column washed
with 4 ml of washing buffer (containing the same lysis buffer havingpH 6.3) twice and finally
the streptokinase proteins were eluted by elution buffer (containing the same lysis buffer having
pH 4.5) in justfour fractions. Concentration and purity of protein fractions were analyzed by
SDS-PAGE and Bradford methods.

3.4.1. Affinity purification of SK protein:

Affinity purified anti-SK mAbs were mixed withSepharose 4B using cyanogen bromide
(Parhami-Seren et al., 1997). Bacterial culture supernatants were incubated with 1 ml of
Sepharose bound mAbs overnight at 4-degree temperature with end-to-end shaking. The
Sepharose was then transferred into a column and washed thoroughly with phosphate buffered-
saline (0.15 M NaCl containing 0.02% sodium azide, pH 7.2, 0.1 M sodium phosphate, PBSA).
Proteins were eluted from column using 100 mM triethylamine, pH 11.9 and instantly
neutralizing it in 1.5 M Tris having pH near 4.5. The absorbance of each fraction was determined
at 280 nm absorbance. The streptokinase concentration was evaluated by comparison to a
standard curve of commercially available streptokinase with known concentration. Fractions
with OD405>1.0 in the chromogenic assay were joint and aliquots kept at 20 °C.

46
3.5. Bioassay of recombinant SK proteins

Streptokinase activity was determined by three methods:

3.5.1. Caseinolysis:

In this semiquantitative standard method (Saksella, 1981) a plate containing 5% (w/v)

skim-milk, 10 mMNaCl,1% (w/v) agarose, 50 mMTris base was prepared and different dilutions
of standard streptokinase (B. Braun, Germany) and the same amounts of eluted proteins were
applied in the 5-mm wells previously prepared in the plate. All the test wells and standard were
filled with 1 mg ml–1 concentration of plasminogen solution to have the plasminogen in an extra
molarity. Two other wells containing standard SK alone or plasminogen alone were also
prepared as negative controls. After 24 hours incubation atroom temperature the caseinolysis
diameter surrounding the wells, rejected the functional activity of streptokinasewas measured
accurately.

3.5.2. Chromogenic assay:

This method which monitors the amount of formed plasmin by an end point assessment
of a synthetic substrate (Couto et al., 2004) was showed with slight modifications. In brief,
recombinant streptokinasemoleculesPlg (1 nM) and (0.4 nM) were mixed together in 96-well
plates and incubated at 37°C for 10 minutes to make activator complexes. 0.75 mM S2251 (H-D-
valyl-L-leucyl-Llysine-ρ-nitroanalide) was added to the enzymatic complex as substrate and the
mixture was incubated for 20 min at 37-degree grade temperature. The reaction was stopped with
20% (v/v) acetic acid and absorbance was reached to 405 nm. Specific activity (IU mg–1)
biological activity (IU ml–1) of the samples were calculated according to OD 405 of reference
streptokinase compared with known biological activity.

3.5.3. Clot lysis assay

This method measures the SK activity in the presence of fibrin (British Pharmacopoeia,
1998). Briery, 0.2 ml of SK (0.8 nM) was mixed with0.1 ml of thrombin (20 IU ml–1), 0.2 ml of
citric-phosphate buyerand, 0.5 ml of euglobulin (10 mg ml–1), which was purified by following
the method used by Couto et al. (Couto et al., 2004), in a test tube. After the formation of clots,
the required times for complete lysis of clots was noted. Biological activity of the

47
unknownpurified samples was measured based on the plotted curve showing the clot lysis time
against the activity of deferent concentrations of SK. Biological activity and specific activity of
samples were calculated by using reference streptokinase plot with known biological activities
(B. Braun, Germany).

3.6. Immunoassays

3.6.1. Direct binding assay:

The direct binding of mAb A4.3 to SK proteins was understood by solid-phase assay
asreported earlier (Parhami-Seren et al., 2002). Wells of microtiter plates (Becton Dickinson,
Oxnard, CA) were coated by adding 50 Al of 10 Ag/ml solution of affinity purified streptokinase
in PBS for 24 hours. Afterblocking and washing the wells with 50 Al of mAb A4.3 (10 Ag/ml in
1% BSA–PBS) and1% BSA–PBS was added to the coated wells. Binding was determined using
HRPanti-mouse Ig.

3.6.2. Competition assays:

Binding of immobilized murine anti-SK mAbs to 125 I-n SK in the presence of different
concentrations of SK variants was studied. Wells of microtiter plates were coated by adding 50
Al of 10 Ag/ml of anti-SK mAbs in PBS. Binding of 25 Al 125I-SK (40,000 cpm,
specific activity 10 Ag/ACi) was determined in the presence or absence of various amount (16–
0.5 Ag/ ml, 2-fold dilutions) of SK, recombinant SK, SK variants or SK Ser138Lys.
Pattern of the cross-reactivity of SK Ser138Lyswith anti-SK mAbs was determined using
competitive inhibition assay in which binding of biotinylated SK to immobilized anti-SK mAbs
was determined in the absence or presence of recombinant SK proteins. Binding was detected
using HRP-streptavidin.

48
Conclusion:

Streptokinase is the most efficient drug used in myocardial infarctions but some
drawbacks is also associated with its administration so it must be modified to lessen the
immunogenic impact on individuals. Usually two methods are employed in this regard. Chemical
modification of streptokinase with ethyl glycol lessen its immunogenic effect but it is not more
efficient.Cloning of streptokinase genes provides recombinant product that abolishes any risk
associated with it. By using these recombinant products patients will be safe. Non-pathogenic
microbes especially streptococcus species are being used for the effective production of
streptokinase.Along these chemical modifications of antigenic regions in streptokinase various
methods also formulated to enhance its activity, stability and half-life. The antigenic regions
present in streptokinase are first identified by various practicable techniques and then modified
to eradicate immunogenicity. Substitution, deletion and addition of amino acid residues present
at antigenic site is the most effective method. The most efficient streptokinase is produced by
eliminating 42 amino acid residues at the c terminal region of streptokinase. This modified
streptokinaseis more effective in its function and are less immunogenic in nature.The objectives
have been accomplished to various degrees also by producing mutated and engineered
streptokinases which less immunogenic and most effective in proteolytic activity.

49
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