Optimization of Tris/EDTA/Sucrose (TES) periplasmic extraction for the recovery of functional scFv antibodies
Abstract Single-chain variable fragments (scFvs) have gained increased attention among researchers in both academic and industrial fields owing to simple production in E. coli. The E. coli periplasm has been the site of choice for the expression of scFv molecules due to its oxidizing milieu facilita...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
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SpringerOpen
2020-07-01
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| Series: | AMB Express |
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| Online Access: | http://link.springer.com/article/10.1186/s13568-020-01063-x |
| _version_ | 1818120455308967936 |
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| author | Elham Ghamghami Marjan Abri Aghdam Mohammad Reza Tohidkia Asadollah Ahmadikhah Morteza Khanmohammadi Tayebeh Mehdipour Ahad Mokhtarzadeh Behzad Baradaran |
| author_facet | Elham Ghamghami Marjan Abri Aghdam Mohammad Reza Tohidkia Asadollah Ahmadikhah Morteza Khanmohammadi Tayebeh Mehdipour Ahad Mokhtarzadeh Behzad Baradaran |
| author_sort | Elham Ghamghami |
| collection | DOAJ |
| description | Abstract Single-chain variable fragments (scFvs) have gained increased attention among researchers in both academic and industrial fields owing to simple production in E. coli. The E. coli periplasm has been the site of choice for the expression of scFv molecules due to its oxidizing milieu facilitating correctly formation of disulfide bonds. Hence, the recovery of high-yield and biologically active species from the periplasmic space is a critical step at beginning of downstream processing. TES (Tris/EDTA/Sucrose) as a simple and efficient extraction method has been frequently used but under varied extraction conditions, over literature. This study, for the first time, aimed to interrogate the effects of four independent variables (i.e., Tris–HCl concentration, buffer’s pH, EDTA concentration, and incubation time) and their potential interactions on the functional extraction yield of an scFv antibody from the periplasmic space of E. coli. The results indicated that the Tris–HCl concentration and pH are the most significant variables in the TES method and displayed a positive effect at their lower values on the functional extraction yield. Besides, the statistical analysis revealed 4 significant interactions between different variables. Here is the first report on the successful application of a design of experiment based on a central composite design to establish a generic and optimal TES extraction condition. Accordingly, an optimal condition for TES extraction of scFv molecules from the periplasm of HB2151 at the exponential phase was developed as follows: 50 mM Tris–HCl at pH 7.2, 0.53 mM EDTA, and an incubation time of 60 min. |
| first_indexed | 2024-12-11T05:26:22Z |
| format | Article |
| id | doaj.art-15b52a0bbe6847d8b2a2bc0b50e9b5ed |
| institution | Directory Open Access Journal |
| issn | 2191-0855 |
| language | English |
| last_indexed | 2024-12-11T05:26:22Z |
| publishDate | 2020-07-01 |
| publisher | SpringerOpen |
| record_format | Article |
| series | AMB Express |
| spelling | doaj.art-15b52a0bbe6847d8b2a2bc0b50e9b5ed2022-12-22T01:19:34ZengSpringerOpenAMB Express2191-08552020-07-0110111310.1186/s13568-020-01063-xOptimization of Tris/EDTA/Sucrose (TES) periplasmic extraction for the recovery of functional scFv antibodiesElham Ghamghami0Marjan Abri Aghdam1Mohammad Reza Tohidkia2Asadollah Ahmadikhah3Morteza Khanmohammadi4Tayebeh Mehdipour5Ahad Mokhtarzadeh6Behzad Baradaran7Department of Biological Science, Faculty of Basic Science, Higher Education Institute of Rab-RashidDepartment of Biological Science, Faculty of Basic Science, Higher Education Institute of Rab-RashidImmunology Research Center, Tabriz University of Medical SciencesFaculty of Life Sciences and Biotechnology, Shahid Beheshti UniversityChemical Engineering Faculty, Sahand University of Technology, Sahand New TownResearch Center for Pharmaceutical Nanotechnology, Tabriz University of Medical SciencesImmunology Research Center, Tabriz University of Medical SciencesImmunology Research Center, Tabriz University of Medical SciencesAbstract Single-chain variable fragments (scFvs) have gained increased attention among researchers in both academic and industrial fields owing to simple production in E. coli. The E. coli periplasm has been the site of choice for the expression of scFv molecules due to its oxidizing milieu facilitating correctly formation of disulfide bonds. Hence, the recovery of high-yield and biologically active species from the periplasmic space is a critical step at beginning of downstream processing. TES (Tris/EDTA/Sucrose) as a simple and efficient extraction method has been frequently used but under varied extraction conditions, over literature. This study, for the first time, aimed to interrogate the effects of four independent variables (i.e., Tris–HCl concentration, buffer’s pH, EDTA concentration, and incubation time) and their potential interactions on the functional extraction yield of an scFv antibody from the periplasmic space of E. coli. The results indicated that the Tris–HCl concentration and pH are the most significant variables in the TES method and displayed a positive effect at their lower values on the functional extraction yield. Besides, the statistical analysis revealed 4 significant interactions between different variables. Here is the first report on the successful application of a design of experiment based on a central composite design to establish a generic and optimal TES extraction condition. Accordingly, an optimal condition for TES extraction of scFv molecules from the periplasm of HB2151 at the exponential phase was developed as follows: 50 mM Tris–HCl at pH 7.2, 0.53 mM EDTA, and an incubation time of 60 min.http://link.springer.com/article/10.1186/s13568-020-01063-xTES extractionPeriplasmic extractionscFvE. coli |
| spellingShingle | Elham Ghamghami Marjan Abri Aghdam Mohammad Reza Tohidkia Asadollah Ahmadikhah Morteza Khanmohammadi Tayebeh Mehdipour Ahad Mokhtarzadeh Behzad Baradaran Optimization of Tris/EDTA/Sucrose (TES) periplasmic extraction for the recovery of functional scFv antibodies AMB Express TES extraction Periplasmic extraction scFv E. coli |
| title | Optimization of Tris/EDTA/Sucrose (TES) periplasmic extraction for the recovery of functional scFv antibodies |
| title_full | Optimization of Tris/EDTA/Sucrose (TES) periplasmic extraction for the recovery of functional scFv antibodies |
| title_fullStr | Optimization of Tris/EDTA/Sucrose (TES) periplasmic extraction for the recovery of functional scFv antibodies |
| title_full_unstemmed | Optimization of Tris/EDTA/Sucrose (TES) periplasmic extraction for the recovery of functional scFv antibodies |
| title_short | Optimization of Tris/EDTA/Sucrose (TES) periplasmic extraction for the recovery of functional scFv antibodies |
| title_sort | optimization of tris edta sucrose tes periplasmic extraction for the recovery of functional scfv antibodies |
| topic | TES extraction Periplasmic extraction scFv E. coli |
| url | http://link.springer.com/article/10.1186/s13568-020-01063-x |
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