Optimized protocol for soluble prokaryotic expression, purification and refolding of the human inhibin α subunit, a cysteine rich peptide chain

Kolivand, Sedighe; Nazari, Mahboobeh; Modarressi, Mohammad Hossein; Najafabadi, Mohammad Reza H.; Hemati, Atefeh; Ghafouri‐Fard, Soudeh; Motevaseli, Elahe

doi:10.3233/hab-190399

Cited by 2 publications

(2 citation statements)

References 28 publications

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“…However, the presence of cysteine residues may have an adverse effect on the expression of soluble proteins in E. coli or the refolding of denatured proteins. − This was the situation we found for apoptin. Sometimes, the C-terminal cysteine has to be removed to stabilize the protein and facilitate purification.…”

Section: Discussionmentioning

confidence: 65%

Highly Efficient Method for Intracellular Delivery of Proteins Mediated by Cholera Toxin-Induced Protein Internalization

et al. 2021

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Delivery of functional proteins into cells may help us understand how specific protein influences cell behavior as well as treat diseases caused by protein deficiency or loss-of-function mutations. However, protein cannot enter cells by diffusion. In this work, a novel cell biology tool for delivering recombinant proteins into mammalian cells was developed. We hijacked the intracellular transport routes used by the cholera toxin and took advantage of recent development on split intein that is compatible with denatured conditions and shows an exceptional splicing activity to deliver a protein of interest into mammalian cells. Here, we used green fluorescent protein and apoptin as proofs-of-concept. The results demonstrate that the cholera toxin B subunit alone could deliver other recombinant proteins into cells through either covalent conjugation or noncovalent interaction. Our method offers more than 10-fold better delivery efficiency than the tat cellpenetrating peptide and is selective for ganglioside-rich cells. This study adds a useful tool to the receptor-mediated intracellular targeting toolkit and opens possibility for the selective delivery of therapeutic proteins into ganglioside-rich cells.

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Section: Discussionmentioning

confidence: 65%

Highly Efficient Method for Intracellular Delivery of Proteins Mediated by Cholera Toxin-Induced Protein Internalization

et al. 2021

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“…The major processes in the refolding process are the purification of inclusion bodies comprising insoluble, aggregated proteins, solubilization using denaturants such as guanidinium chloride, and refolding with a redox system, cosolvents and additives to encourage correct folding [73]. The dilution approach and using detergents with cosolvents are valuable methods for refolding cysteine-rich proteins [74]. Several factors influence the efficiency of the refolding process, including protein concentration, disulfide bond formation, chaperone proteins (such as small heat shock proteins and Hsp70 chaperones), micelle size and composition, and salt concentration.…”

Section: Importance Of Proper Refoldingmentioning

confidence: 99%

Ultrasound-assisted innovations in protein processing: review

Rebezov,

Assenova,

Luneva

et al. 2024

Potr. S. J. F. Sci.

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The contemporary landscape of protein processing is witnessing a paradigm shift propelled by innovative technologies. This review unveils innovations in protein processing through the lens of an ultrasound-assisted approach. The focus was on the interplay between ultrasound waves and proteins during ultrasound extraction technology. The realm of protein extraction, where traditional methods face challenges and ultrasound emerges as a transformative force, was highlighted, as well as ultrasound's role in enhancing protein yield and quality in relationship to protein structure and function. Comparative analyses have showcased the remarkable advancements ushered in by ultrasound-assisted techniques, and this review also extends to enzymatic hydrolysis, where ultrasound catalyses reactions, unlocking new dimensions in the production of bioactive peptides and nutritionally enriched proteins. In the bio-industrial sectors, ultrasound facilitates protein refolding and revolutionises recombinant protein production, stability and bioavailability. Ultrasound has emerged as a catalyst for efficiency and bioactivity enhancement, defeating conventional limitations to the intricate optimisation strategies of refolding. This review envisages the advantages of ultrasound technology and its applications in the bio-industrial sector. The prospects of ultrasound-assisted protein processing are outlined, and roadmaps and processing techniques are offered.

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Optimized protocol for soluble prokaryotic expression, purification and refolding of the human inhibin α subunit, a cysteine rich peptide chain

Cited by 2 publications

References 28 publications

Highly Efficient Method for Intracellular Delivery of Proteins Mediated by Cholera Toxin-Induced Protein Internalization

Highly Efficient Method for Intracellular Delivery of Proteins Mediated by Cholera Toxin-Induced Protein Internalization

Ultrasound-assisted innovations in protein processing: review

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