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      FTIR Investigation of Secondary Structure of Reteplase Inclusion Bodies Produced in Escherichia coli in Terms of Urea Concentration

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          Abstract

          Recent studies suggest that reducing the induction temperature would improve the quality of some recombinant inclusion bodies (IB) by providing a native-like secondary structure and leading to an improvement in protein recovery. This study focused on optimizing the solubilization condition of Reteplase, a recombinant protein with 9 disulfide bonds. The influence of lowering induction temperature and urea concentration was investigated on the secondary structure of the recombinant protein through FTIR analysis. Induction temperature reduction decreased the percentage of helixes and loops from 49 to 8. In addition, FTIR spectroscopy corroborates the drastic impact of this parameter on Reteplase secondary structure. Even though lowering urea concentration tripled the solubility of IBs expressed at lower induction temperature, the final yield is still quite low to be considered as optimum. On the other hand, the percentage of beta strands and turns in secondary structure of dissolved proteins were proportional to urea concentration. Therefore, in case of Reteplase, protein expression at low temperature (25 °C) was not efficient to improve the protein recovery yield. Future studies need to focus on using other methods of solubilizing IBs to improve protein recovery.

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          A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding

          Marion Bradford (1976)
          Article 0 comments Cited 8950 times – based on 0 reviews     Review now
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            The control of protein stability and association by weak interactions with water: how do solvents affect these processes?

            S Timasheff (1992)
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              Engineering inclusion bodies for non denaturing extraction of functional proteins

              Spela Peternel, Jože Grdadolnik, Vladka Gaberc-Porekar … (2008)
              Background For a long time IBs were considered to be inactive deposits of accumulated target proteins. In our previous studies, we discovered IBs containing a high percentage of correctly folded protein that can be extracted under non-denaturing conditions in biologically active form without applying any renaturation steps. In order to widen the concept of correctly folded protein inside IBs, G-CSF (granulocyte colony stimulating factor) and three additional proteins were chosen for this study: GFP (Green fluorescent protein), His7dN6TNF-α (Truncated form of Tumor necrosis factor α with an N-terminal histidine tag) and dN19 LT-α (Truncated form of Lymphotoxin α). Results Four structurally different proteins that accumulate in the bacterial cell in the form of IBs were studied, revealing that distribution of each target protein between the soluble fraction (cytoplasm) and insoluble fraction (IBs) depends on the nature of the target protein. Irrespective of the folding pattern of each protein, spectroscopy studies have shown that proteins in IBs exhibit similar structural characteristics to the biologically active pure protein when produced at low temperature. In the case of the three studied proteins, G-CSF, His7ΔN6TNF-α, and GFP, a significant amount of protein could be extracted from IBs with 0.2% N-lauroyl sarcosine (NLS) and the proteins retained biological activity although no renaturation procedure was applied. Conclusion This study shows that the presence of biologically active proteins inside IBs is more general than usually believed. A large amount of properly folded protein is trapped inside IBs prepared at lower temperatures. This protein can be released from IBs with mild detergents under non-denaturing conditions. Therefore, the active protein can be obtained from such IBs without any renaturation procedure. This is of great importance for the biopharmaceutical industry. Furthermore, such IBs composed of active proteins could also be used as pure nanoparticles in diagnostics, as biocatalysts in enzymatic processes, or even as biopharmaceuticals.
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                Author and article information

                Journal
                Journal ID (nlm-ta): Iran J Pharm Res
                Journal ID (iso-abbrev): Iran J Pharm Res
                Journal ID (publisher-id): IJPR
                Title: Iranian Journal of Pharmaceutical Research : IJPR
                Publisher: Shaheed Beheshti University of Medical Sciences (Tehran, Iran )
                ISSN (Print): 1735-0328
                ISSN (Electronic): 1726-6890
                Publication date (Print): Spring 2020
                Volume: 19
                Issue: 2
                Pages: 175-181
                Affiliations
                [1] Biotechnology Group, Chemical Engineering Department, Tarbiat Modares University, Tehran, Iran.
                Author notes
                [* ]Corresponding author: E-mail: shoja_sa@modares.ac.ir
                Article
                DOI: 10.22037/ijpr.2020.1101092
                PMC ID: 7667556
                SO-VID: 66db27eb-ded6-406e-8237-0dd0e9aaca32
                License:

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License, ( http://creativecommons.org/licenses/by/3.0/) which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                Date received : November 2015
                Date accepted : May 2016
                Categories
                Subject: Original Article

                Keywords: fourier transformed infrared technique (ftir),non-classical inclusion body,non-denaturing solubilization,reteplase
                Data availability:
                Keywords: fourier transformed infrared technique (ftir), non-classical inclusion body, non-denaturing solubilization, reteplase

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