Quality comparison of recombinant soluble proteins and proteins solubilized from bacterial inclusion bodies

López-Cano, Adrià; Sicilia, Paula; Gaja, Clara; Arı́s, Anna; García‐Fruitós, Elena

doi:10.1016/j.nbt.2022.09.003

Cited by 8 publications

(7 citation statements)

References 36 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…While initially inclusion bodies (IBs) were thought to be aggregates of misfolded protein ( García-Fruitós et al, 2012 ), they have been shown to contain correctly folded secondary and tertiary protein structures, in some cases even with biological activity ( García-Fruitós, 2010 ; Villaverde et al, 2015 ; Belkova et al, 2022 ; López-Cano et al, 2022 ). Preserving these existing structures during the solubilization step has been correlated with an increase in refolding yield due to reduced re-aggregation ( Singh et al, 2015 ; Kachhawaha et al, 2022 ).…”

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%

See 1 more Smart Citation

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

Klausser,

Kopp,

Prada Brichtova

et al. 2023

Front. Bioeng. Biotechnol.

View full text Add to dashboard Cite

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.

show abstract

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.

View full text Add to dashboard Cite

show abstract

“…However, to increase protein yield, NLS, a mild detergent with few interferences on biological activity of recombinant proteins was used [ 34 , 35 ]. A more recent publication showed that this detergent may affect protein folding of insoluble proteins [ 36 ], then, the final impact on Blo t 2 is unknown. Despite demonstrating that rBlo t 2 is biologically actively inducing allergic reactions and also similar rates of IgE binding to other similar studies [ 6 , 16 ], physico-chemical characterization of rBlo t 2 will confirm if the recombinant has the optimum folding to represent its natural counterpart.…”

Section: Discussionmentioning

confidence: 99%

The Allergenic Activity of Blo t 2, a Blomia tropicalis IgE-Binding Molecule

Rangel

Regino

Hernandez

et al. 2023

IJMS

View full text Add to dashboard Cite

Only few allergens derived from house dust mite (HDM) species have been evaluated in terms of their potential to induce allergic inflammation. In this study, we aimed to evaluate different aspects of the allergenicity and allergenic activity of Blo t 2, a Blomia tropicalis allergen. Blo t 2 was produced as a recombinant protein in Escherichia coli. Its allergenic activity was tested in humans by skin prick test and basophil activation assays, and in mice, by passive cutaneous anaphylaxis and a model of allergic airway inflammation. Sensitization rate to Blo t 2 (54.3%) was similar to that found to Blo t 21 (57.2%) and higher than to Der p 2 (37.5%). Most Blo t 2-sensitized patients showed a low intensity response (99.5%). Blo t 2 elicited CD203c upregulation and allergen induced skin inflammation. Additionally, immunized animals produced anti-Blo t 2 IgE antibodies and passive transfer of their serum to non-immunized animals induced skin inflammation after allergen exposure. Immunized animals developed bronchial hyperreactivity and a strong inflammatory lung reaction (eosinophils and neutrophils). These results confirm the allergenic activity of Blo t 2 and supports its clinical relevance.

show abstract

“…Indeed, solubilized IB proteins show antimicrobial activity against E. coli in a dose-dependent fashion against carbapenem-resistant K. pneumoniae embedded in biofilms [ 20 ]. However, it is important to point out that the selection of an optimal mild solubilizer is especially relevant when antimicrobial proteins are purified because recently it has been reported that they can impair the antimicrobial activity [ 81 ].…”

Section: Forms Of Recombinant Hdpsmentioning

confidence: 99%

The future of recombinant host defense peptides

et al. 2022

Self Cite

View full text Add to dashboard Cite

The antimicrobial resistance crisis calls for the discovery and production of new antimicrobials. Host defense peptides (HDPs) are small proteins with potent antibacterial and immunomodulatory activities that are attractive for translational applications, with several already under clinical trials. Traditionally, antimicrobial peptides have been produced by chemical synthesis, which is expensive and requires the use of toxic reagents, hindering the large-scale development of HDPs. Alternatively, HDPs can be produced recombinantly to overcome these limitations. Their antimicrobial nature, however, can make them toxic to the hosts of recombinant production. In this review we explore the different strategies that are used to fine-tune their activities, bioengineer them, and optimize the recombinant production of HDPs in various cell factories.

show abstract

Quality comparison of recombinant soluble proteins and proteins solubilized from bacterial inclusion bodies

Cited by 8 publications

References 36 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

The Allergenic Activity of Blo t 2, a Blomia tropicalis IgE-Binding Molecule

The future of recombinant host defense peptides

Contact Info

Product

Resources

About