Lipid modification of staphylokinase and its implications on stability and activity

Mannully, Sheethal Thomas; Shanthi, C.; Pulicherla, K. K.

doi:10.1016/j.ijbiomac.2018.10.134

Cited by 9 publications

(4 citation statements)

References 33 publications

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Section: Resultsmentioning

confidence: 99%

“…Apart from amino acid substitutions by site directed mutagenesis techniques, conjugation of the protein to some polymers such as polyethylene glycol (PEG) or lipids has the potential to modify the thermal stability, immunogenicity, and clearance as well. [44][45][46][47][48][49] F I G U R E 8 (a) Comparable expression level, peak time and methanol concentration dependence of SAK-2RGD-TTI in clones. SAK-2RGD-TTI production was compared by GS-PIC-SAK-2RGD-TTI-1, GS-PIC-SAK-2RGD-TTI-2, and KM-PIC-SAK-2RGD-TTI-3 clones.…”

Section: Determining Optimum Ph and Temperature For Fibrinolytic Acmentioning

confidence: 99%

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Comparison of expression optimization of new derivative of staphylokinase (SAK‐2RGD‐TTI) with the rSAK

Faraji

Ramezani

Mashkani

et al. 2019

Biotechnology Progress

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Staphylokinase (SAK) is a promising thrombolytic agent for the treatment of patients suffering from blood‐clotting disorders. To increase the potency of SAK and to minimize vessel reocclusion, a new construct bearing SAK motif fused to tsetse thrombin inhibitor (TTI) via a 20‐amino acid linker with 2 RGD (2 × arginine‐glycine‐aspartic acid inhibiting platelet aggregation via attachment to integrin receptors of platelet) was codon optimized and expressed comparatively in Pichia pastoris GS115 as a Mut+ strain and KM71H as a Muts strain. Fusion protein was optimized in terms of best expression condition and fibrinolytic activity and compared with the rSAK. Expression level of the designed construct reached up to 175 mg/L of the culture medium after 72‐hr stimulation with 2.5% methanol and remained steady for 3–4 days. The highest expression was obtained at the range of 2–3% methanol. The SAK‐2RGD‐TT (relative activity >82%) was more active at 25–37 °C than rSAK (relative activity of 93%). Further, it showed relative activity >80% at pH ranges of 7–9. Western blot analysis showed two bands of nearly 27 and 24 kDa at ratio of 5 to 3, respectively. The specific fibrinolytic activity of the SAK‐2RGD‐TTI was measured as 8,269 U/mg, and 19,616 U/mg for the nonpurified and purified proteins, respectively. Deglycosylation by using tunicamycin in culture medium resulted in higher fibrinolytic activity of SAK‐2RGD‐TTI (2.2 fold). Consequently, compared to the rSAK, at the same equimolar proportion, addition of RGD and TTI fragments could increase fibrinolytic activity. Also, P. pastoris can be considered as an efficient host for overexpression of the soluble SAK‐2RGD‐TTI with high activity without requiring a complicated purification procedure.

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Section: Resultsmentioning

confidence: 99%

Section: Determining Optimum Ph and Temperature For Fibrinolytic Acmentioning

confidence: 99%

Comparison of expression optimization of new derivative of staphylokinase (SAK‐2RGD‐TTI) with the rSAK

Faraji

Ramezani

Mashkani

et al. 2019

Biotechnology Progress

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“…Furthermore, the 3-D structure of the protein facilitated the design of PEG (polyethylene glycol) attachment sites, prolonging plasma half-life and reducing antigenicity [ 169 , 170 , 171 , 172 , 173 , 174 ]. A recent study reported that N-terminal lipid modification of SAK fosters its activity, stability, and translocation across the blood–brain barrier (BBB), and therefore, holds great promise in treating diseases like stroke [ 175 ].…”

Section: Microbial-derived Drugs In Thrombolytic Therapymentioning

confidence: 99%

Thrombolytic Enzymes of Microbial Origin: A Review

Diwan

Usmani

Sharma

et al. 2021

IJMS

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Enzyme therapies are attracting significant attention as thrombolytic drugs during the current scenario owing to their great affinity, specificity, catalytic activity, and stability. Among various sources, the application of microbial-derived thrombolytic and fibrinolytic enzymes to prevent and treat vascular occlusion is promising due to their advantageous cost–benefit ratio and large-scale production. Thrombotic complications such as stroke, myocardial infarction, pulmonary embolism, deep venous thrombosis, and peripheral occlusive diseases resulting from blood vessel blockage are the major cause of poor prognosis and mortality. Given the ability of microbial thrombolytic enzymes to dissolve blood clots and prevent any adverse effects, their use as a potential thrombolytic therapy has attracted great interest. A better understanding of the hemostasis and fibrinolytic system may aid in improving the efficacy and safety of this treatment approach over classical thrombolytic agents. Here, we concisely discuss the physiological mechanism of thrombus formation, thrombo-, and fibrinolysis, thrombolytic and fibrinolytic agents isolated from bacteria, fungi, and algae along with their mode of action and the potential application of microbial enzymes in thrombosis therapy.

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“…After disruption of the identified contacts by combinatorial mutagenesis, the immunogenicity of the best variant decreased to less than 30 % [283,[286], [287], [288], [289]]. Other engineering strategies using PEGylation [[290], [291], [292], [293], [294], [295]], glycosylation [296], protein fusion [288,[297], [298], [299], [300], [301]], and lipidification [302] provided variants of staphylokinase exhibiting higher efficiency, improved half-life, decreased immunogenicity, and a lower risk of reocclusion.…”

Section: Staphylokinasementioning

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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Lipid modification of staphylokinase and its implications on stability and activity

Cited by 9 publications

References 33 publications

Comparison of expression optimization of new derivative of staphylokinase (SAK‐2RGD‐TTI) with the rSAK

Comparison of expression optimization of new derivative of staphylokinase (SAK‐2RGD‐TTI) with the rSAK

Thrombolytic Enzymes of Microbial Origin: A Review

Structural Biology and Protein Engineering of Thrombolytics

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