Chaperone-Assisted Soluble Expression of a Humanized Anti-EGFR ScFv Antibody in E. Coli
Purpose: Formation of inclusion bodies is a considerable obstacle threatening the advantages of E. coli expression system to serve as the most common and easiest system in recombinant protein production. To solve this problem, several strategies have been proposed among which application of molecula...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Tabriz University of Medical Sciences
2015-12-01
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| Series: | Advanced Pharmaceutical Bulletin |
| Subjects: | |
| Online Access: | http://journals.tbzmed.ac.ir/APB/Manuscript/APB-5-621.pdf |
| _version_ | 1819037820433465344 |
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| author | Kamal Veisi Safar Farajnia Nosratollah Zarghami Hamid Reza Khoram Khorshid Nasser Samadi Shiva Ahdi Khosroshahi Hossein Zarei Jaliani |
| author_facet | Kamal Veisi Safar Farajnia Nosratollah Zarghami Hamid Reza Khoram Khorshid Nasser Samadi Shiva Ahdi Khosroshahi Hossein Zarei Jaliani |
| author_sort | Kamal Veisi |
| collection | DOAJ |
| description | Purpose: Formation of inclusion bodies is a
considerable obstacle threatening the advantages of E. coli expression
system to serve as the most common and easiest system in recombinant protein
production. To solve this problem, several strategies have been proposed among
which application of molecular chaperones is of remarkable consideration. The
aim of this study was to evaluate the effects of molecular chaperones on
soluble expression of aggregation-prone humanized single chain antibody.
Methods: To increase the solubility of a humanized
single chain antibody (hscFv), different chaperone plasmids including PG-tf2
(GroES- GroEL- tig), ptf16 (tig) and pGro7 (GroES- GroEL) were co-expressed in
BL21 cells containing pET-22b- hscFv construct. The solubility of recombinant
hscFv was analyzed by SDS-PAGE. After purification of soluble hscFv by Ni-NTA
column, the biological activity and cytotoxicity of the recombinant protein
were tested by ELISA and MTT assay, respectively.
Results: SDS-PAGE analysis of the hscFv revealed that
chaperone utility remarkably increased (up to 50%) the solubility of the
protein. ELISA test and MTT assay analyses also confirmed the biological
activity of the gained hscFv in reaction with A431 cells (OD value: 2.6) and
inhibition of their proliferation, respectively.
Conclusion: The
results of this study revealed that co-expression of chaperones with hscFv
leads to remarkable increase in the solubility of the recombinant hscFv, which
could be of great consideration for large scale production of recombinant
single chain antibodies. |
| first_indexed | 2024-12-21T08:27:29Z |
| format | Article |
| id | doaj.art-6315ce6f82ef4c6ca5a9a9300107dc41 |
| institution | Directory Open Access Journal |
| issn | 2228-5881 2251-7308 |
| language | English |
| last_indexed | 2024-12-21T08:27:29Z |
| publishDate | 2015-12-01 |
| publisher | Tabriz University of Medical Sciences |
| record_format | Article |
| series | Advanced Pharmaceutical Bulletin |
| spelling | doaj.art-6315ce6f82ef4c6ca5a9a9300107dc412022-12-21T19:10:18ZengTabriz University of Medical SciencesAdvanced Pharmaceutical Bulletin2228-58812251-73082015-12-015562162710.15171/apb.2015.084APB_2173_20140821193802Chaperone-Assisted Soluble Expression of a Humanized Anti-EGFR ScFv Antibody in E. ColiKamal Veisi0Safar Farajnia1Nosratollah Zarghami2Hamid Reza Khoram Khorshid3Nasser Samadi4Shiva Ahdi Khosroshahi5Hossein Zarei Jaliani6 Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. Department of Medical Biotechnologies, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran. University of Social Welfare and Rehabilitation Sciences, Tehran, Iran. Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. Department of Medical Genetics, School of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran. Purpose: Formation of inclusion bodies is a considerable obstacle threatening the advantages of E. coli expression system to serve as the most common and easiest system in recombinant protein production. To solve this problem, several strategies have been proposed among which application of molecular chaperones is of remarkable consideration. The aim of this study was to evaluate the effects of molecular chaperones on soluble expression of aggregation-prone humanized single chain antibody. Methods: To increase the solubility of a humanized single chain antibody (hscFv), different chaperone plasmids including PG-tf2 (GroES- GroEL- tig), ptf16 (tig) and pGro7 (GroES- GroEL) were co-expressed in BL21 cells containing pET-22b- hscFv construct. The solubility of recombinant hscFv was analyzed by SDS-PAGE. After purification of soluble hscFv by Ni-NTA column, the biological activity and cytotoxicity of the recombinant protein were tested by ELISA and MTT assay, respectively. Results: SDS-PAGE analysis of the hscFv revealed that chaperone utility remarkably increased (up to 50%) the solubility of the protein. ELISA test and MTT assay analyses also confirmed the biological activity of the gained hscFv in reaction with A431 cells (OD value: 2.6) and inhibition of their proliferation, respectively. Conclusion: The results of this study revealed that co-expression of chaperones with hscFv leads to remarkable increase in the solubility of the recombinant hscFv, which could be of great consideration for large scale production of recombinant single chain antibodies.http://journals.tbzmed.ac.ir/APB/Manuscript/APB-5-621.pdfSoluble expressionMolecular chaperonesScFvEGFR |
| spellingShingle | Kamal Veisi Safar Farajnia Nosratollah Zarghami Hamid Reza Khoram Khorshid Nasser Samadi Shiva Ahdi Khosroshahi Hossein Zarei Jaliani Chaperone-Assisted Soluble Expression of a Humanized Anti-EGFR ScFv Antibody in E. Coli Advanced Pharmaceutical Bulletin Soluble expression Molecular chaperones ScFv EGFR |
| title | Chaperone-Assisted Soluble Expression of a Humanized
Anti-EGFR ScFv Antibody in E. Coli |
| title_full | Chaperone-Assisted Soluble Expression of a Humanized
Anti-EGFR ScFv Antibody in E. Coli |
| title_fullStr | Chaperone-Assisted Soluble Expression of a Humanized
Anti-EGFR ScFv Antibody in E. Coli |
| title_full_unstemmed | Chaperone-Assisted Soluble Expression of a Humanized
Anti-EGFR ScFv Antibody in E. Coli |
| title_short | Chaperone-Assisted Soluble Expression of a Humanized
Anti-EGFR ScFv Antibody in E. Coli |
| title_sort | chaperone assisted soluble expression of a humanized anti egfr scfv antibody in e coli |
| topic | Soluble expression Molecular chaperones ScFv EGFR |
| url | http://journals.tbzmed.ac.ir/APB/Manuscript/APB-5-621.pdf |
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