Engineering of deglycosylated and plasmin resistant variants of recombinant streptokinase in Pichia pastoris

Adivitiya,; Babbal,; Mohanty, S. R.; Khasa, Yogender Pal

doi:10.1007/s00253-018-9402-x

Cited by 10 publications

(3 citation statements)

References 50 publications

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“…It is worth USD 40 million on the open market. Several techniques for boosting streptokinase expression in wild-type S. equisimilis strains with various growth agents [ 20 ], plasmid-based genetically engineered E. coli strain [ 21 , 22 ], and eukaryotic expression yeast [ 23 , 24 ] were already available. Although the maximum achieved production was 720 mg/L streptokinase using E. coli BL21[9DE3] strain [ 22 ].…”

Section: Discussionmentioning

confidence: 99%

CRISPR-Cas9 Mediated Knockout of SagD Gene for Overexpression of Streptokinase in Streptococcus equisimilis

et al. 2022

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Streptokinase is an enzyme that can break down the blood clots in some cases of myocardial infarction (heart attack), pulmonary embolism, and arterial thromboembolism. Demand for streptokinase is higher globally than production due to increased incidences of various heart conditions. The main source of streptokinase is various strains of Streptococci. Expression of streptokinase in native strain Streptococcus equisimilis is limited due to the SagD gene-mediated post-translational modification of streptolysin, an inhibitor of streptokinase expression through the degradation of FasX small RNA (through CoV/RS), which stabilizes streptokinase mRNA. In order to improve the stability of mRNA and increase the expression of streptokinase, which is inhibited by SagA, we used CRISPR-Cas9 to successfully knockout the SagD gene and observed a 13.58-fold increased expression of streptokinase at the transcript level and 1.48-fold higher expression at the protein level in the mutant strain compared to wild type. We have demonstrated the successful gene knockout of SagD using CRISPR-Cas9 in S. equisimilis, where an engineered strain can be further used for overexpression of streptokinase for therapeutic applications.

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Section: Discussionmentioning

confidence: 99%

CRISPR-Cas9 Mediated Knockout of SagD Gene for Overexpression of Streptokinase in Streptococcus equisimilis

et al. 2022

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“…It possesses a market value of USD40 million. Several methods were already available for increasing expression of streptokinase using wild-type S. equisimilis strain with various growth factors [37], plasmid based genetically engineered E.coli strain [38,39], and eukaryotic expression yeast [40,41]. Although maximum achieved production was 720mg/L streptokinase using E.coli BL21[9DE3] strain [39].…”

Section: Discussionmentioning

confidence: 99%

CRISPR-Cas9 mediated knockout of SagD gene for overexpression of streptokinase in Streptococcus equisimilis

Chaudhari

Vyas

Patel

et al. 2021

Preprint

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Streptokinase is an enzyme that can break down the blood clots in some cases of myocardial infarction (Heart attack), pulmonary embolism, and arterial thromboembolism. Demand for streptokinase is high globally than the production due to increased incidences of various heart conditions. The main source of streptokinase is from various strains of Streptococcus. Expression of streptokinase in native strain Streptococcus equisimilis is limited due to the SagD inhibitor gene for production of streptokinase that needs to be knocked out in order to increase it expression. However, FasX is a small RNA (sRNA) present in group A Streptococcus species which is responsible for post-transcriptional regulation of streptokinase (ska) gene by binding at the 5' end of ska mRNA. S. equisimilis is a β-hemolysin producing streptococcus bacterium (group C) containing the orthologue of FasX and natively expresses a clinically important thrombolytic streptokinase. In order to improve the stability of mRNA and increasing the expression of streptokinase which is inhibited by SagD. We used CRISPR-Cas9 to successfully knock- out of SagD gene and observed a 13.58-fold relative quantification of streptokinase expression in the mutant strain as compared to wild type. We have also demonstrated the successful target gene knockout using CRISPR-Cas9 in S. equisimilis that engineered strain can be used further for overexpression of streptokinase for therapeutic applications.

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“…Strategies were based on finding that during the process of plasminogen activation, the generated plasmin non-physiologically cleaves streptokinase into three polypeptide chains (residues 1–59, 60–386, and 387–414) leading to drop in activity [233,234]. Preventing the cleavage by mutagenesis at the identified positions (Lys59Gln/Glu, Lys386Gln) resulted in a 21-fold increased half-life without affecting activity [[235], [236], [237]]. Alternative strategies based on combinatorial mutagenesis [14,238], PEGylation [[239], [240], [241], [242]], glycosylation [235,243], or lipidification [244] yielded streptokinase variants with improved thrombolytic activity, decreased immunogenicity, and higher half-life.…”

Section: Streptokinasementioning

confidence: 99%

Structural Biology and Protein Engineering of Thrombolytics

Mičan

Toul

Bednář

et al. 2019

Computational and Structural Biotechnology Journal

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Myocardial infarction and ischemic stroke are the most frequent causes of death or disability worldwide. Due to their ability to dissolve blood clots, the thrombolytics are frequently used for their treatment. Improving the effectiveness of thrombolytics for clinical uses is of great interest. The knowledge of the multiple roles of the endogenous thrombolytics and the fibrinolytic system grows continuously. The effects of thrombolytics on the alteration of the nervous system and the regulation of the cell migration offer promising novel uses for treating neurodegenerative disorders or targeting cancer metastasis. However, secondary activities of thrombolytics may lead to life-threatening side-effects such as intracranial bleeding and neurotoxicity. Here we provide a structural biology perspective on various thrombolytic enzymes and their key properties: (i) effectiveness of clot lysis, (ii) affinity and specificity towards fibrin, (iii) biological half-life, (iv) mechanisms of activation/inhibition, and (v) risks of side effects. This information needs to be carefully considered while establishing protein engineering strategies aiming at the development of novel thrombolytics. Current trends and perspectives are discussed, including the screening for novel enzymes and small molecules, the enhancement of fibrin specificity by protein engineering, the suppression of interactions with native receptors, liposomal encapsulation and targeted release, the application of adjuvants, and the development of improved production systems.

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Engineering of deglycosylated and plasmin resistant variants of recombinant streptokinase in Pichia pastoris

Cited by 10 publications

References 50 publications

CRISPR-Cas9 Mediated Knockout of SagD Gene for Overexpression of Streptokinase in Streptococcus equisimilis

CRISPR-Cas9 Mediated Knockout of SagD Gene for Overexpression of Streptokinase in Streptococcus equisimilis

CRISPR-Cas9 mediated knockout of SagD gene for overexpression of streptokinase in Streptococcus equisimilis

Structural Biology and Protein Engineering of Thrombolytics

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