RESUMO
The temperature dependence of the heat capacity function of a recombinant streptokinase (rSK) has been studied by high-sensitivity differential scanning microcalorimetry and circular dichroism as a function of pH in low- and high-ionic strength buffers. At low ionic strength it is found that this protein, between pH 7 and 10, undergoes four reversible and independent two-state transitions during its unfolding, suggesting the existence of four domains in the native structure of the protein. This result reconciles previous conflicting reports about the number of domains of this protein obtained by differential scanning calorimetry and small-angle X-ray scattering. The number of two-state transitions decreases when the pH of the medium is decreased, without noticeable changes in its circular dichroism spectrum. A plausible localization of the four domains in the streptokinase sequences is proposed and their thermodynamic parameters are given. Increase of ionic strength to 200 mM NaCl affects positively the protein stability and confirms the existence of four reversible two-state transitions. Above 200 mM NaCl the protein stability decreases, resulting in low percentage of reversibility, and even irreversible transitions.
Assuntos
Varredura Diferencial de Calorimetria , Estreptoquinase/química , Dicroísmo Circular , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Mutação , Conformação Proteica , Desnaturação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Cloreto de Sódio/química , Estreptoquinase/genética , Estreptoquinase/metabolismoRESUMO
Streptokinase (SK) is an efficacious thrombolytic drug for the treatment of myocardial infarction. Because of its immunogenicity, patients receiving SK therapy develop high anti-SK antibody (Ab) titers, which might provoke severe allergic reactions and neutralize SK activity. In this report we studied the reactivity of a synthetic 42-residue peptide resembling SKC-2 C-terminus with patient sera. SKC-2(373-414) peptide was recognized by 39 and 64% of patients, before and after SKC-2 therapy, respectively. An SKC-2 deletion mutant (mut-C42), lacking the same 42 C-terminal residues, was constructed and expressed in Escherichia coli. Recognition of mut-C42 by preexisting Abs from patient sera was 51 and 68% of reactivity to SKC-2, as assessed by direct binding and competition assays, respectively. For most of the patients, mut-C42-neutralizing activity titer (NAT) significantly decreased with respect to SKC-2-NAT. This study opens the possibility of producing a less immunogenic variant of SK, which could constitute a preferred alternative for thrombolytic therapy.
Assuntos
Anticorpos Antibacterianos/imunologia , Deleção de Sequência/genética , Estreptoquinase/química , Estreptoquinase/imunologia , Alérgenos/química , Alérgenos/imunologia , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/química , Antígenos de Bactérias/imunologia , Ligação Competitiva , Ensaio de Imunoadsorção Enzimática , Humanos , Epitopos Imunodominantes/química , Epitopos Imunodominantes/genética , Epitopos Imunodominantes/imunologia , Epitopos Imunodominantes/isolamento & purificação , Infarto do Miocárdio/tratamento farmacológico , Infarto do Miocárdio/imunologia , Testes de Neutralização , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Estreptoquinase/genética , Estreptoquinase/uso terapêutico , Terapia TrombolíticaRESUMO
Streptokinase (SK) is the most widely used compound for the treatment of myocardial infarction and the least expensive thrombolytic agent, but a drawback to its use is the widespread presence of anti-SK antibodies (Abs). Clinical failure of the activation of the fibrinolytic system by SK has been reported due to the presence of a high titer of anti-SK neutralizing Abs. Patients receiving SK therapy develop high anti-SK antibody titers, which might provoke severe allergic reactions. These Abs are sufficient to neutralize a standard dose of SK up to four years after initial SK administration. This is a clinical problem because of the increasing number of patients who have been treated once with SK for acute myocardial infarction (AMI) and are likely to require plasminogen activator treatment in the future. In previous in vitro studies, we have shown that a deletion mutant (mut-C42), lacking the 42 C-terminal residues, was significantly less antigenic when compared with the native molecule (SKC-2). In this study, 14 monkeys were subjected to treatment with SKC-2 and mut-C42 in order to compare their humoral response by determining SK neutralizing activity in monkey's sera. All monkeys developed anti-SKC-2 Ab titers, but in the case where treatment induced Abs directed against the C-terminus of SKC-2, neutralizing activity against the native protein was significantly higher than that developed against mutant SK mut-C42.
Assuntos
Fibrinolíticos/imunologia , Mutação , Fragmentos de Peptídeos/imunologia , Estreptoquinase/genética , Estreptoquinase/imunologia , Animais , Chlorocebus aethiops , Feminino , Fibrinolíticos/uso terapêutico , Masculino , Testes de Neutralização , Fragmentos de Peptídeos/uso terapêutico , Engenharia de Proteínas , Deleção de Sequência , Estreptoquinase/uso terapêutico , Trombose/tratamento farmacológicoRESUMO
We cloned the streptokinase (STK) gene of Streptococcus equisimilis in an expression vector of Escherichia coli to overexpress the profibrinolytic protein under the control of a tac promoter. Almost all the recombinant STK was exported to the periplasmic space and recovered after gentle lysozyme digestion of induced cells. The periplasmatic fraction was chromatographed on DEAE Sepharose followed by chromatography on phenyl-agarose. Active proteins eluted between 4.5 and 0 percent ammonium sulfate, when a linear grandient was applied. Theree major STK derivatives of 47.5 kDa, 45 kDa and 32 kDa were detected by Western blot analysis with a polyclonal antibody. The 32-kDa protein formed a complex with human plasminogen but did not exhibit Glu-plasminogen activator activity, as revealed by a zymographic assay, whereas the 45-kDa protein showed a Km = 0.70 muM and kcat = 0.82 s(-1), when assayed with a chromogen-coupled subtrate. These results suggest that these proteins are putative fragments of STK, possibly derived from partial degradation during the export pathway or the purification steps. The 47.5-kDa band corresponded to the native STK, as revealed by peptide sequencing.
Assuntos
Clonagem Molecular , Escherichia coli , Proteínas Recombinantes , Streptococcus/genética , Estreptoquinase/genética , Estreptoquinase/isolamento & purificação , Cromatografia em AgaroseRESUMO
We cloned the streptokinase (STK) gene of Streptococcus equisimilis in an expression vector of Escherichia coli to overexpress the profibrinolytic protein under the control of a tac promoter. Almost all the recombinant STK was exported to the periplasmic space and recovered after gentle lysozyme digestion of induced cells. The periplasmic fraction was chromatographed on DEAE Sepharose followed by chromatography on phenyl-agarose. Active proteins eluted between 4.5 and 0% ammonium sulfate, when a linear gradient was applied. Three major STK derivatives of 47.5 kDa, 45 kDa and 32 kDa were detected by Western blot analysis with a polyclonal antibody. The 32-kDa protein formed a complex with human plasminogen but did not exhibit Glu-plasminogen activator activity, as revealed by a zymographic assay, whereas the 45-kDa protein showed a K(m) = 0.70 microM and kcat = 0.82 s-1, when assayed with a chromogen-coupled substrate. These results suggest that these proteins are putative fragments of STK, possibly derived from partial degradation during the export pathway or the purification steps. The 47.5-kDa band corresponded to the native STK, as revealed by peptide sequencing.
Assuntos
Clonagem Molecular , Escherichia coli , Proteínas Recombinantes , Streptococcus/genética , Estreptoquinase/genética , Estreptoquinase/isolamento & purificaçãoRESUMO
Streptokinase (SK), which activates human plasminogen by promoting its conversion to plasmin, is normally obtained from beta-hemolytic streptococci. Treatment with SK is an effective therapy for improving survival and preserving left ventricular function after coronary thrombosis. We report the cloning, expression in E. coli to levels of 25% of the total cell protein, and characterization of a novel SK (SKC-2) gene, the product of which is functionally equivalent to the naturally-derived protein. The availability of a recombinant streptokinase (rSK) in high yield and purity offers a potentially attractive alternative source of this important therapeutic agent.