High level production of active streptokinase in Pichia pastoris fed-batch culture

Adivitiya,; Dagar, Vikas Kumar; Devi, Nirmala; Khasa, Yogender Pal

doi:10.1016/j.ijbiomac.2015.11.062

Cited by 29 publications

(6 citation statements)

References 33 publications

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“…The methylotrophic yeast, Pichia pastoris, has been developed to be a highly successful system for large-scale production of functionally active recombinant proteins [1,2]. As a yeast, it is a unique system which has many advantages e.g., its ability to produce foreign proteins at high levels using one of the strongest and highly regulated eukaryotic promoters, alcohol oxidase I (AOX1).…”

Section: Introductionmentioning

confidence: 99%

Enhanced protein production by sorbitol co-feeding with methanol in recombinant Pichia pastoris strains

Chen

Mohsin

Chu

et al. 2017

Biotechnol Bioproc E

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Pichia pastoris strains carrying 1, 6, 12, and 18 copies of the porcine insulin precursor (PIP) gene, were employed to investigate the effects of sorbitol co-feeding with methanol on the physiology of the strains. Multicopy clones of the methylotrophic yeast were generated to vary the PIP gene dosage and recombinant proteins. Elevated gene dosage increased levels of the recombinant PIP protein when methanol served as the sole carbon and energy source i.e., an increase of 1.9% for a strain carrying 1 copy, 42.6% for a strain carrying 6 copies, 34.7% for a strain carrying 12 copies and 80.9% for a strain carrying 18 copies, respectively (using sorbitol co-feeding with methanol during the induction phase). However, it had no significant influence on a lower gene dosage strain (1 copy), but this approach affirmed enhancement in cell growth and PIP production for higher gene dosage strain (6, 12, and 18 copies) via using sorbitol co-feeding with methanol. Additionally, the co-feeding strategy could hold vital importance for recombinant protein production by a multi-copy P. pastoris system.

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Section: Introductionmentioning

confidence: 99%

Enhanced protein production by sorbitol co-feeding with methanol in recombinant Pichia pastoris strains

Chen

Mohsin

Chu

et al. 2017

Biotechnol Bioproc E

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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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“…35 Recently, 4.25 g/L of bioactive streptokinase was produced in P. pastoris with a volumetric productivity of 3727 IU/mL/h. 39 In the GISSI trial (Gruppo Italiano per la Sperimentazione della Streptochinasi nell'Infarto Miocardico) a reduction in mortality rate was seen when AMI patients were administered 1.5 million IU of streptokinase over 60 min. 40 The drug is available under the trade names Streptase Ò and Kabikinase Ò and is obtained from b-hemolytic streptococci.…”

Section: First Generation Plasminogen Activatorsmentioning

confidence: 99%

The evolution of recombinant thrombolytics: Current status and future directions

Adivitiya

Khasa

2016

Bioengineered

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Cardiovascular disorders are on the rise worldwide due to alcohol abuse, obesity, hypertension, raised blood lipids, diabetes and age-related risks. The use of classical antiplatelet and anticoagulant therapies combined with surgical intervention helped to clear blood clots during the inceptive years. However, the discovery of streptokinase and urokinase ushered the way of using these enzymes as thrombolytic agents to degrade the fibrin network with an issue of systemic hemorrhage. The development of second generation plasminogen activators like anistreplase and tissue plasminogen activator partially controlled this problem. The third generation molecules, majorly t-PA variants, showed desirable properties of improved stability, safety and efficacy with enhanced fibrin specificity. Plasmin variants are produced as direct fibrinolytic agents as a futuristic approach with targeted delivery of these drugs using liposome technlogy. The novel molecules from microbial, plant and animal origin present the future of direct thrombolytics due to their safety and ease of administration.

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High level production of active streptokinase in Pichia pastoris fed-batch culture

Cited by 29 publications

References 33 publications

Enhanced protein production by sorbitol co-feeding with methanol in recombinant Pichia pastoris strains

Enhanced protein production by sorbitol co-feeding with methanol in recombinant Pichia pastoris strains

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

The evolution of recombinant thrombolytics: Current status and future directions

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