Cloning, expression, purification and characterization of a single chain variable fragment specific to tumor necrosis factor alpha in Escherichia coli

Krishnan, Shekhar; Vijayalakshmi, M.A.; Venkataraman, Krishnan; Satheeshkumar, Padikara Kutty

doi:10.1016/j.jbiotec.2011.06.039

Cited by 23 publications

(32 citation statements)

References 51 publications

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“…In the present experiments, CIM monoliths were giving reasonably good refolding yield (65%) ( Table 1) within 50 min, where as the Ni-NTA assisted refolding needed around 6 h for the chromatographic run [17] to complete. Easy and straight forward scale up with CIM-IMAC has been reported in the previous work [11].…”

Section: Resultsmentioning

confidence: 63%

“…Soluble expression and purification of a ScFv against human TNF-␣ has been reported previously [11]. Since the expression showed nearly 60% of the protein in the inclusion bodies ( Fig.…”

Section: Resultsmentioning

confidence: 85%

“…The generation of an expression construct for anti TNF-␣ ScFv has been reported previously [11]. In short, the DNA fragment encoding anti TNF-␣ ScFv cDNA was cloned in pET28a and used in the protein expression studies.…”

Section: Cloning Of Scfv In Expression Vectormentioning

confidence: 99%

“…The protein expression and SDS PAGE analysis was performed as described elsewhere [11]. In short, Fresh competent E. coli BL21 (DE3) strain was transformed with the construct pET28a-ScFv.…”

Section: Protein Expression and Sds-page Analysismentioning

confidence: 99%

“…To refold and purify more than 60% of the ScFv detected in the inclusion bodies, a novel and efficient methodology using monolithic matrix combined with metal affinity has been developed. CIM monoliths were previously used in our study to purify the soluble form of anti TNF-␣ ScFv [11] and in this report we explored the possibility of using CIM-IDA-Ni (II) disks for concurrent refolding and purification of insoluble anti TNF-␣ ScFv.…”

Section: Introductionmentioning

confidence: 99%

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Recovery of active anti TNF-α ScFv through matrix-assisted refolding of bacterial inclusion bodies using CIM monolithic support

Krishnan

Bilgimol

Vijayalakshmi

et al. 2012

Journal of Chromatography B

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Section: Resultsmentioning

confidence: 63%

“…Soluble expression and purification of a ScFv against human TNF-␣ has been reported previously [11]. Since the expression showed nearly 60% of the protein in the inclusion bodies ( Fig.…”

Section: Resultsmentioning

confidence: 85%

Section: Cloning Of Scfv In Expression Vectormentioning

confidence: 99%

“…The protein expression and SDS PAGE analysis was performed as described elsewhere [11]. In short, Fresh competent E. coli BL21 (DE3) strain was transformed with the construct pET28a-ScFv.…”

Section: Protein Expression and Sds-page Analysismentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Recovery of active anti TNF-α ScFv through matrix-assisted refolding of bacterial inclusion bodies using CIM monolithic support

Krishnan

Bilgimol

Vijayalakshmi

et al. 2012

Journal of Chromatography B

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Expression of anti‐tumor necrosis factor alpha (TNFα) single‐chain variable fragment (scFv) in Spirodela punctata plants transformed with Agrobacterium tumefaciens

Parthasarathy

Satheeshkumar

Venkataraman

et al. 2015

Biotech and App Biochem

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Therapeutic antibodies against tumor necrosis factor alpha (TNFα) have been considered effective for some of the autoimmune diseases such as rheumatoid arthritis, Crohn's diseases, and so on. But associated limitations of the current therapeutics in terms of cost, availability, and immunogenicity have necessitated the need for alternative candidates. Single-chain variable fragment (scFv) can negate the limitations tagged with the anti-TNFα therapeutics to a greater extent. In the present study, Spirodela punctata plants were transformed with anti-TNFα through in planta transformation using Agrobacterium tumefaciens strain, EHA105. Instead of cefotaxime, garlic extract (1 mg/mL) was used to remove the agrobacterial cells after cocultivation. To the best of our knowledge, this report shows for the first time the application of plant extracts in transgenic plant development. 95% of the plants survived screening under hygromycin. ScFv cDNA integration in the plant genomic DNA was confirmed at the molecular level by PCR. The transgenic protein expression was followed up to 10 months. Expression of scFv was confirmed by immunodot blot. Protein expression levels of up to 6.3% of total soluble protein were observed. β-Glucuronidase and green fluorescent protein expressions were also detected in the antibiotic resistant plants. The paper shows the generation of transgenic Spirodela punctuata plants through in planta transformation.

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Structure‐based engineering of an antiangiogenic scFv antibody for soluble production in E. coli without loss of activity

Öncü

Balcıoğlu

Özgür

et al. 2021

Biotech and App Biochem

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Development of monoclonal antibody therapeutics against vascular endothelial growth factor receptor 2 (VEGFR‐2) protein, which is the main regulator in angiogenesis, has been a major challenge for years. In the current study, we engineer an inclusion body forming single‐chain variable fragment (scFv) against VEGFR‐2 by using complementarity determining regions (CDR) grafting technique to improve its solubility and investigate the activity of the engineered molecule. CDR sequences of the target scFv were grafted into the framework of another intrinsically soluble scFv molecule. Based on the computational results, CDR grafting has increased the solubility of the grafted scFv molecule. Results confirmed that the grafting approach increased in vivo folding properties of the target scFv molecule compared with the original scFv molecule. Similar binding affinities to the VEGFR‐2 were observed for the original and the grafted scFv by surface plasmon resonance assays. Biological activity assays, including human umbilical vein endothelial cells proliferation and wound healing assays, showed that grafted scFv molecule has an antiangiogenic property. This study suggests that an antiangiogenic scFv fully expressed as an inclusion body can be rescued by grafting its CDR regions to a scFv expressed in a soluble form without any loss in its binding property and its activity.

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Cloning, expression, purification and characterization of a single chain variable fragment specific to tumor necrosis factor alpha in Escherichia coli

Cited by 23 publications

References 51 publications

Recovery of active anti TNF-α ScFv through matrix-assisted refolding of bacterial inclusion bodies using CIM monolithic support

Recovery of active anti TNF-α ScFv through matrix-assisted refolding of bacterial inclusion bodies using CIM monolithic support

Expression of anti‐tumor necrosis factor alpha (TNFα) single‐chain variable fragment (scFv) in Spirodela punctata plants transformed with Agrobacterium tumefaciens

Structure‐based engineering of an antiangiogenic scFv antibody for soluble production in E. coli without loss of activity

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