Efficient refolding and functional characterization of PfAMA1(DI+DII) expressed in E. coli
Apical membrane antigen 1 (AMA1) is a surface protein of Plasmodium sp. that plays a crucial role in forming moving junction (MJ) during the invasion of human red blood cells. The obligatory presence of AMA1 in the parasite lifecycle designates this protein as a potential vaccine candidate and an es...
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Elsevier
2021-07-01
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| Series: | Biochemistry and Biophysics Reports |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2405580821000443 |
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| author | Anamika Biswas Sreejith Raran-Kurussi Akash Narayan Abhisek Kar Purna Chandra Mashurabad Mrinal Kanti Bhattacharyya Kalyaneswar Mandal |
| author_facet | Anamika Biswas Sreejith Raran-Kurussi Akash Narayan Abhisek Kar Purna Chandra Mashurabad Mrinal Kanti Bhattacharyya Kalyaneswar Mandal |
| author_sort | Anamika Biswas |
| collection | DOAJ |
| description | Apical membrane antigen 1 (AMA1) is a surface protein of Plasmodium sp. that plays a crucial role in forming moving junction (MJ) during the invasion of human red blood cells. The obligatory presence of AMA1 in the parasite lifecycle designates this protein as a potential vaccine candidate and an essential target for the development of novel peptide or protein therapeutics. However, due to multiple cysteine residues in the protein sequence, attaining the native fold with correct disulfide linkages during the refolding process after expression in bacteria has remained challenging for years. Although several approaches to obtain the refolded protein from bacterial expression have been reported previously, achieving high yield during refolding and proper functional validation of the expressed protein was lacking. We report here an improved method of refolding to obtain higher quantity of refolded protein. We have also validated the refolded protein's functional activity by evaluating the expressed AMA1 protein binding with a known inhibitory peptide, rhoptry neck protein 2 (RON2), using surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). |
| first_indexed | 2024-12-14T15:41:30Z |
| format | Article |
| id | doaj.art-81bdd8013957499489adfa51a69a6279 |
| institution | Directory Open Access Journal |
| issn | 2405-5808 |
| language | English |
| last_indexed | 2024-12-14T15:41:30Z |
| publishDate | 2021-07-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Biochemistry and Biophysics Reports |
| spelling | doaj.art-81bdd8013957499489adfa51a69a62792022-12-21T22:55:36ZengElsevierBiochemistry and Biophysics Reports2405-58082021-07-0126100950Efficient refolding and functional characterization of PfAMA1(DI+DII) expressed in E. coliAnamika Biswas0Sreejith Raran-Kurussi1Akash Narayan2Abhisek Kar3Purna Chandra Mashurabad4Mrinal Kanti Bhattacharyya5Kalyaneswar Mandal6TIFR Centre for Interdisciplinary Sciences, Tata Institute of Fundamental Research Hyderabad, 36/p Gopanpally, Hyderabad, Telangana 500046, IndiaTIFR Centre for Interdisciplinary Sciences, Tata Institute of Fundamental Research Hyderabad, 36/p Gopanpally, Hyderabad, Telangana 500046, IndiaTIFR Centre for Interdisciplinary Sciences, Tata Institute of Fundamental Research Hyderabad, 36/p Gopanpally, Hyderabad, Telangana 500046, IndiaTIFR Centre for Interdisciplinary Sciences, Tata Institute of Fundamental Research Hyderabad, 36/p Gopanpally, Hyderabad, Telangana 500046, IndiaDepartment of Biochemistry, School of Life Sciences, University of Hyderabad, Gachibowli, Hyderabad, Telangana 500046, IndiaDepartment of Biochemistry, School of Life Sciences, University of Hyderabad, Gachibowli, Hyderabad, Telangana 500046, IndiaTIFR Centre for Interdisciplinary Sciences, Tata Institute of Fundamental Research Hyderabad, 36/p Gopanpally, Hyderabad, Telangana 500046, India; Corresponding author.Apical membrane antigen 1 (AMA1) is a surface protein of Plasmodium sp. that plays a crucial role in forming moving junction (MJ) during the invasion of human red blood cells. The obligatory presence of AMA1 in the parasite lifecycle designates this protein as a potential vaccine candidate and an essential target for the development of novel peptide or protein therapeutics. However, due to multiple cysteine residues in the protein sequence, attaining the native fold with correct disulfide linkages during the refolding process after expression in bacteria has remained challenging for years. Although several approaches to obtain the refolded protein from bacterial expression have been reported previously, achieving high yield during refolding and proper functional validation of the expressed protein was lacking. We report here an improved method of refolding to obtain higher quantity of refolded protein. We have also validated the refolded protein's functional activity by evaluating the expressed AMA1 protein binding with a known inhibitory peptide, rhoptry neck protein 2 (RON2), using surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC).http://www.sciencedirect.com/science/article/pii/S2405580821000443PfAMA1Protein expressionE. coliRefoldingProtein-protein interactionsSurface plasmon resonance |
| spellingShingle | Anamika Biswas Sreejith Raran-Kurussi Akash Narayan Abhisek Kar Purna Chandra Mashurabad Mrinal Kanti Bhattacharyya Kalyaneswar Mandal Efficient refolding and functional characterization of PfAMA1(DI+DII) expressed in E. coli Biochemistry and Biophysics Reports PfAMA1 Protein expression E. coli Refolding Protein-protein interactions Surface plasmon resonance |
| title | Efficient refolding and functional characterization of PfAMA1(DI+DII) expressed in E. coli |
| title_full | Efficient refolding and functional characterization of PfAMA1(DI+DII) expressed in E. coli |
| title_fullStr | Efficient refolding and functional characterization of PfAMA1(DI+DII) expressed in E. coli |
| title_full_unstemmed | Efficient refolding and functional characterization of PfAMA1(DI+DII) expressed in E. coli |
| title_short | Efficient refolding and functional characterization of PfAMA1(DI+DII) expressed in E. coli |
| title_sort | efficient refolding and functional characterization of pfama1 di dii expressed in e coli |
| topic | PfAMA1 Protein expression E. coli Refolding Protein-protein interactions Surface plasmon resonance |
| url | http://www.sciencedirect.com/science/article/pii/S2405580821000443 |
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