Solubilization of Human Interferon β-1b Inclusion Body Proteins by Organic Solvents

Nekoufar, Samira; Fazeli, Ahmad; Fazeli, Mohammad Reza

doi:10.34172/apb.2020.027

Cited by 6 publications

(4 citation statements)

References 25 publications

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“…Thus, the concept of “mild solubilization” originated, describing the solubilization of IBs without fully denaturing the protein. Many researchers use an arbitrary maximum threshold of 2–3 M Urea to demarcate “mild” from “traditional” solubilization ( Singh et al, 2015 ; Upadhyay et al, 2016 ; Nekoufar et al, 2020 ; Maksum et al, 2022 ; Mohammadi et al, 2023 ). However, the degree of denaturation induced by these conditions is highly dependent on the respective protein of interest (PoI).…”

Section: Mild Inclusion Body Solubilizationmentioning

confidence: 99%

“…Most prominently, alkaline pH is used to increase protein solubility at lower denaturant concentrations ( Khan et al, 1998 ; Patra et al, 2000 ; Singh et al, 2008 ; Lu and Lin, 2012 ; Ishikawa et al, 2022 ). Other chemical options are organic solvents ( Upadhyay et al, 2016 ; Sarker et al, 2019 ; Nekoufar et al, 2020 ) or detergents, like N-lauroyl sarcosine, SDS, CHAPS, and Triton X-100 ( Francis et al, 2012 ; Ishikawa et al, 2022 ; López-Cano et al, 2022 ). Physical methods for mild solubilization include high pressures of up to 2.4 kbar ( Chura-Chambi et al, 2022a ; Chura-Chambi et al, 2022b ) and, most recently, freeze-thaw cycles ( Padhiar et al, 2018 ; Maksum et al, 2022 ).…”

Section: Mild Inclusion Body Solubilizationmentioning

confidence: 99%

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State-of-the-art and novel approaches to mild solubilization of inclusion bodies

Klausser,

Kopp,

Prada Brichtova

et al. 2023

Front. Bioeng. Biotechnol.

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Throughout the twenty-first century, the view on inclusion bodies (IBs) has shifted from undesired by-products towards a targeted production strategy for recombinant proteins. Inclusion bodies can easily be separated from the crude extract after cell lysis and contain the product in high purity. However, additional solubilization and refolding steps are required in the processing of IBs to recover the native protein. These unit operations remain a highly empirical field of research in which processes are developed on a case-by-case basis using elaborate screening strategies. It has been shown that a reduction in denaturant concentration during protein solubilization can increase the subsequent refolding yield due to the preservation of correctly folded protein structures. Therefore, many novel solubilization techniques have been developed in the pursuit of mild solubilization conditions that avoid total protein denaturation. In this respect, ionic liquids have been investigated as promising agents, being able to solubilize amyloid-like aggregates and stabilize correctly folded protein structures at the same time. This review briefly summarizes the state-of-the-art of mild solubilization of IBs and highlights some challenges that prevent these novel techniques from being yet adopted in industry. We suggest mechanistic models based on the thermodynamics of protein unfolding with the aid of molecular dynamics simulations as a possible approach to solve these challenges in the future.

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Section: Mild Inclusion Body Solubilizationmentioning

confidence: 99%

Section: Mild Inclusion Body Solubilizationmentioning

confidence: 99%

State-of-the-art and novel approaches to mild solubilization of inclusion bodies

Klausser,

Kopp,

Prada Brichtova

et al. 2023

Front. Bioeng. Biotechnol.

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“…Studies on the use of non-denaturing agents such as N -lauryl sarcosine, dimethyl sulfoxide (DMSO), 5% n -propanol, mild non-ionic detergents, high pH buffers, and low denaturant concentration that preserve the native-like state of the fusion proteins have been reported [ 52 – 57 ]. To solubilize IBs a combination of denaturants such as Sodium do-decyl sulphate (SDS), urea and organic solvents such as 40% ( v/v ) 1-Propanol and 20% ( v/v ) 2-Butanol in an acidic pH (range of 2–3) are used [ 58 ]. Chaperones have the ability to transiently bind to the hydrophobic region of a protein and this binding avoids IB formation.…”

Section: Discussionmentioning

confidence: 99%

Strategic optimization of conditions for the solubilization of GST-tagged amphipathic helix-containing ciliary proteins overexpressed as inclusion bodies in E. coli

Shendge

D’Souza

2022

Microb Cell Fact

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Expression of affinity-tagged recombinant proteins for crystallography, protein–protein interaction, antibody generation, therapeutic applications, etc. mandates the generation of high-yield soluble proteins. Although recent developments suggest the use of yeast, insect, and mammalian cell lines as protein expression platforms, Escherichia coli is still the most popular, due mainly to its ease of growth, feasibility in genetic manipulation and economy. However, some proteins have a spontaneous tendency to form inclusion bodies (IBs) when over-expressed in bacterial expression systems such as E. coli, thus posing a challenge in purification and yield. At times, small peptides undergo degradation during protein production and hence using suitable tags could circumvent the problem. Although several independent techniques have been used to solubilize IBs, these cannot always be applied in a generic sense. Although tagging a GST moiety is known to enhance the solubility of fusion proteins in E. coli, resulting in yields of 10–50 mg/L of the culture, the inherent nature of the protein sequence at times could lead to the formation of IBs. We have been working on a Myc Binding Protein-1 orthologue, viz. Flagellar Associated Protein 174 (FAP174) from the axoneme of Chlamydomonas reinhardtii that binds to an A-Kinase Anchoring Protein 240 (AKAP240) which has been annotated as Flagellar Associated Protein 65 (FAP65). Using an in-silico approach, we have identified two amphipathic helices on FAP65 (CrFAP65AH1 and CrFAP65AH2) that are predicted to bind to FAP174. To test this prediction, we have cloned the GST-tagged peptides, and overexpressed them in E. coli that have resulted in insoluble IBs. The yields of these over-expressed recombinant proteins dropped considerably due to IB formation, indicating aggregation. An integrated approach has been used to solubilize four highly hydrophobic polypeptides, viz. two amphipathic helices and the respective proline variants of FAP65. For solubilizing these polypeptides, variables such as non-denaturing detergents (IGEPAL CA-630), changing the ionic strength of the cell lysis and solubilization buffer, addition of BugBuster®, diluting the cell lysate and sonication were introduced. Our statistically viable results yielded highly soluble and functional polypeptides, indiscreet secondary structures, and a yield of ~ 20 mg/L of the E. coli culture. Our combinatorial strategy using chemical and physical methods to solubilize IBs could prove useful for hydrophobic peptides and proteins with amphipathic helices.

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“…Subsequently, solubilization occurs in a high concentration of denaturing agents such as urea (6–8 M) [ 92 ] or guanidine hydrochloride (6 M) [ 60 , 90 ] and can be enhanced at alkaline pH [ 5 , 49 ]. Milder solubilization methods employing 1-propanol or 2-butanol in the presence of sodium dodecyl sulfate (SDS) have also been demonstrated to be effective for IFNβ-1b solubilization [ 93 ]. During this process, dithiothreitol (DTT) is commonly added to decrease non-native disulfide bonds [ 60 , 94 ].…”

Section: Therapeutic Cloned Interferonsmentioning

confidence: 99%

Interferon-Based Biopharmaceuticals: Overview on the Production, Purification, and Formulation

et al. 2021

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The advent of biopharmaceuticals in modern medicine brought enormous benefits to the treatment of numerous human diseases and improved the well-being of many people worldwide. First introduced in the market in the early 1980s, the number of approved biopharmaceutical products has been steadily increasing, with therapeutic proteins, antibodies, and their derivatives accounting for most of the generated revenues. The success of pharmaceutical biotechnology is closely linked with remarkable developments in DNA recombinant technology, which has enabled the production of proteins with high specificity. Among promising biopharmaceuticals are interferons, first described by Isaacs and Lindenmann in 1957 and approved for clinical use in humans nearly thirty years later. Interferons are secreted autocrine and paracrine proteins, which by regulating several biochemical pathways have a spectrum of clinical effectiveness against viral infections, malignant diseases, and multiple sclerosis. Given their relevance and sustained market share, this review provides an overview on the evolution of interferon manufacture, comprising their production, purification, and formulation stages. Remarkable developments achieved in the last decades are herein discussed in three main sections: (i) an upstream stage, including genetically engineered genes, vectors, and hosts, and optimization of culture conditions (culture media, induction temperature, type and concentration of inducer, induction regimens, and scale); (ii) a downstream stage, focusing on single- and multiple-step chromatography, and emerging alternatives (e.g., aqueous two-phase systems); and (iii) formulation and delivery, providing an overview of improved bioactivities and extended half-lives and targeted delivery to the site of action. This review ends with an outlook and foreseeable prospects for underdeveloped aspects of biopharma research involving human interferons.

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Solubilization of Human Interferon β-1b Inclusion Body Proteins by Organic Solvents

Cited by 6 publications

References 25 publications

State-of-the-art and novel approaches to mild solubilization of inclusion bodies

State-of-the-art and novel approaches to mild solubilization of inclusion bodies

Strategic optimization of conditions for the solubilization of GST-tagged amphipathic helix-containing ciliary proteins overexpressed as inclusion bodies in E. coli

Interferon-Based Biopharmaceuticals: Overview on the Production, Purification, and Formulation

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