Bacterial expression of the scFv fragment of a recombinant antibody specific for Burkholderia pseudomallei exotoxin
(2003) In Journal of Biochemistry and Molecular Biology 36(5). p.493-498- Abstract
- The scFv antibody towards the Burkholderia pseudomallei exotoxin was previously constructed by phage display and exhibited good specificity towards the exotoxin. We report here the optimization of the scFv expression in an E. coli expression system. Four different E. coli strains (ER2537, TG1, HB2151, and XL1-Blue) were examined for optimal expression of the scFv protein. Two types of carbon source (i.e. 0.2% glucose and 0.2% glycerol) were also tested for their ability to induce the scFv expression. Cells that carried the scFv construct were grown at 30 degrees C and induced with 0.05 mM IPTG. The expression was then monitored by SDS-PAGE, Western blotting, and indirect ELISA. The Western blot profile showed different levels of the scFv... (More)
- The scFv antibody towards the Burkholderia pseudomallei exotoxin was previously constructed by phage display and exhibited good specificity towards the exotoxin. We report here the optimization of the scFv expression in an E. coli expression system. Four different E. coli strains (ER2537, TG1, HB2151, and XL1-Blue) were examined for optimal expression of the scFv protein. Two types of carbon source (i.e. 0.2% glucose and 0.2% glycerol) were also tested for their ability to induce the scFv expression. Cells that carried the scFv construct were grown at 30 degrees C and induced with 0.05 mM IPTG. The expression was then monitored by SDS-PAGE, Western blotting, and indirect ELISA. The Western blot profile showed different levels of the scFv expression among the host strains; XL1-Blue exhibited the highest level of the scFv protein expression. Glycerol at a concentration of 0.2% (v/v) significantly increased the scFv protein expression level when compared to 0.2% (w/v) glucose. Further optimization demonstrated that the scFv protein expression in XL1-Blue was the most optimal with a glycerol concentration as low as 0.05%. However, by indirect ELISA, only the scFv protein that was expressed in 0.2% (v/v) glycerol exhibited high specificity towards the Burkholderia pseudomallei exotoxin. (Less)
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https://lup.lub.lu.se/record/bdd63729-cfe2-4825-ae2a-4091a9672069
- author
- Su, Shanice Yc LU ; Lim, KP and Nathan, Sheila
- publishing date
- 2003
- type
- Contribution to journal
- publication status
- published
- subject
- in
- Journal of Biochemistry and Molecular Biology
- volume
- 36
- issue
- 5
- pages
- 493 - 498
- external identifiers
-
- scopus:0242319782
- DOI
- 10.5483/BMBRep.2003.36.5.493
- language
- English
- LU publication?
- no
- id
- bdd63729-cfe2-4825-ae2a-4091a9672069
- date added to LUP
- 2019-06-19 14:31:54
- date last changed
- 2022-01-31 22:08:06
@article{bdd63729-cfe2-4825-ae2a-4091a9672069,
abstract = {{The scFv antibody towards the Burkholderia pseudomallei exotoxin was previously constructed by phage display and exhibited good specificity towards the exotoxin. We report here the optimization of the scFv expression in an E. coli expression system. Four different E. coli strains (ER2537, TG1, HB2151, and XL1-Blue) were examined for optimal expression of the scFv protein. Two types of carbon source (i.e. 0.2% glucose and 0.2% glycerol) were also tested for their ability to induce the scFv expression. Cells that carried the scFv construct were grown at 30 degrees C and induced with 0.05 mM IPTG. The expression was then monitored by SDS-PAGE, Western blotting, and indirect ELISA. The Western blot profile showed different levels of the scFv expression among the host strains; XL1-Blue exhibited the highest level of the scFv protein expression. Glycerol at a concentration of 0.2% (v/v) significantly increased the scFv protein expression level when compared to 0.2% (w/v) glucose. Further optimization demonstrated that the scFv protein expression in XL1-Blue was the most optimal with a glycerol concentration as low as 0.05%. However, by indirect ELISA, only the scFv protein that was expressed in 0.2% (v/v) glycerol exhibited high specificity towards the Burkholderia pseudomallei exotoxin.}},
author = {{Su, Shanice Yc and Lim, KP and Nathan, Sheila}},
language = {{eng}},
number = {{5}},
pages = {{493--498}},
series = {{Journal of Biochemistry and Molecular Biology}},
title = {{Bacterial expression of the scFv fragment of a recombinant antibody specific for Burkholderia pseudomallei exotoxin}},
url = {{http://dx.doi.org/10.5483/BMBRep.2003.36.5.493}},
doi = {{10.5483/BMBRep.2003.36.5.493}},
volume = {{36}},
year = {{2003}},
}