Refolding of bioactive human epidermal growth factor from E. coli BL21(DE3) inclusion bodies &amp; evaluations on its in vitro &amp; in vivo bioactivity

Maksum, Iman Permana; Yosua, Yosua; Nabiel, Ahmad; Pratiwi, Riyona Desvy; Sriwidodo, Sriwidodo; Soedjanaatmadja, Ukun M.S.

doi:10.1016/j.heliyon.2022.e09306

Cited by 4 publications

(2 citation statements)

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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%

“…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 ). Finally, ionic liquids (ILs) are novel solvents able to dissolve IBs whilst potentially retaining secondary or even tertiary protein structure.…”

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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