Multiple Mutations on α, β and γ Domains of Streptokinase Lead to the Generation of Highly Efficient Cysteine Analogues with Promising Features
Copyright© Bentham Science Publishers; For any queries, please email at epubbenthamscience.net..
BACKGROUND: Streptokinase, one of the most widely used thrombolytic medicines, is a favorable protein for site-specific PEGylation as it lacks any cysteine residues in its amino acid sequence; however, any changes in the protein's structure should be carefully planned to avoid undesired changes in its function.
OBJECTIVES: This study aimed to design and produce novel di/tri-cysteine variants of streptokinase from previously developed cysteine analogues, Arg45, Glu263, and Arg319, as candidates for multiple site-specific PEGylation.
METHODS: Using bioinformatics tools and site-directed mutagenesis, we incorporated concurrent mutations at Arg45, Glu263, and Arg319 (carried out in our previous study) to create di/tri-cysteine variants of streptokinase proteins (SK45-319cys, SK263-319cys, and SK45-263-319cys) and evaluated their kinetic activity parameters by a colorimetric method, using H-D-Val-Leu-Lys-pNA.2HCl (S2251) as substrate.
RESULTS: Based on the kinetic results, SK263-319cys with 44% enzyme efficiency increment compared to wild-type SK was the superior protein in terms of activity; as well, SK45-319cys and SK45-263-319cys showed 17 and 22% activity enhancement, respectively. Docking of the mutant streptokinase proteins with μ-plasmin demonstrated that changes in intermolecular interactions caused by amino acid substitution could be the reason for activity difference.
CONCLUSION: The novel mutant proteins created in this study exhibit remarkable biological activity and may be uniquely suitable for simultaneous PEGylation on two/three domains. As well, PEGylated derivates of these variants might prove to be more proficient proteins, compared to the singlecysteine analogs of streptokinase; because of their more surface coverage and increased molecular weight.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2023 |
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| Erschienen: |
2023 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:24 |
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| Enthalten in: |
Current pharmaceutical biotechnology - 24(2023), 10 vom: 17., Seite 1326-1334 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Alinodehi, Narges Norouzzadeh [VerfasserIn] |
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| Links: |
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| Themen: |
9001-91-6 |
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| Anmerkungen: |
Date Completed 01.06.2023 Date Revised 01.06.2023 published: Print Citation Status MEDLINE |
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| doi: |
10.2174/1389201024666221124151623 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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NLM349378312 |
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| 245 | 1 | 0 | |a Multiple Mutations on α, β and γ Domains of Streptokinase Lead to the Generation of Highly Efficient Cysteine Analogues with Promising Features |
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| 500 | |a Date Revised 01.06.2023 | ||
| 500 | |a published: Print | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a Copyright© Bentham Science Publishers; For any queries, please email at epubbenthamscience.net. | ||
| 520 | |a BACKGROUND: Streptokinase, one of the most widely used thrombolytic medicines, is a favorable protein for site-specific PEGylation as it lacks any cysteine residues in its amino acid sequence; however, any changes in the protein's structure should be carefully planned to avoid undesired changes in its function | ||
| 520 | |a OBJECTIVES: This study aimed to design and produce novel di/tri-cysteine variants of streptokinase from previously developed cysteine analogues, Arg45, Glu263, and Arg319, as candidates for multiple site-specific PEGylation | ||
| 520 | |a METHODS: Using bioinformatics tools and site-directed mutagenesis, we incorporated concurrent mutations at Arg45, Glu263, and Arg319 (carried out in our previous study) to create di/tri-cysteine variants of streptokinase proteins (SK45-319cys, SK263-319cys, and SK45-263-319cys) and evaluated their kinetic activity parameters by a colorimetric method, using H-D-Val-Leu-Lys-pNA.2HCl (S2251) as substrate | ||
| 520 | |a RESULTS: Based on the kinetic results, SK263-319cys with 44% enzyme efficiency increment compared to wild-type SK was the superior protein in terms of activity; as well, SK45-319cys and SK45-263-319cys showed 17 and 22% activity enhancement, respectively. Docking of the mutant streptokinase proteins with μ-plasmin demonstrated that changes in intermolecular interactions caused by amino acid substitution could be the reason for activity difference | ||
| 520 | |a CONCLUSION: The novel mutant proteins created in this study exhibit remarkable biological activity and may be uniquely suitable for simultaneous PEGylation on two/three domains. As well, PEGylated derivates of these variants might prove to be more proficient proteins, compared to the singlecysteine analogs of streptokinase; because of their more surface coverage and increased molecular weight | ||
| 650 | 4 | |a Journal Article | |
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| 700 | 1 | |a Nezamiha, Farahnaz Khoshdel |e verfasserin |4 aut | |
| 700 | 1 | |a Mianroodi, Reza Arabi |e verfasserin |4 aut | |
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