Single-chain variable fragment antibody against ginsenoside Re as an effective tool for the determination of ginsenosides in various ginsengs

The technology of recombinant single chain variable fragments (scFvs) expression has been used in research, diagnosis and treatment of diseases. In the previous study, we studied the expression of a recombinant single chain variable fragment recognizing blood A antigen (antiA-scFv) in E. coli. However, the protein was insoluble form resulting in difficulty for purification, refolding and activity assesment. Here, we present the study on fused expression of the recombinant scFv -specific blood A antigen with thioredoxin (Trx) in the expression vector pET32a (+). The results showed that the Trx/antiA-scFv fusion protein was expressed with molecular weight of 49 kDa in a soluble form reaching 40% of the total recombinant protein. This result facilitates the optimal condition of soluble protein expression, purification and bioactivity determination of the antiA-scFv recombinant antibody.

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Development of a single-chain variable fragment antibody-based enzyme-linked immunosorbent assay for determination of fumonisin B1in corn samples

Journal of the Science of Food and Agriculture ◽

10.1002/jsfa.6505 ◽

2013 ◽

Vol 94 (9) ◽

pp. 1865-1871 ◽

Cited By ~ 27

Author(s):

Long Zou ◽

Yang Xu ◽

Yanping Li ◽

Qinghua He ◽

Bo Chen ◽

...

Keyword(s):

Immunosorbent Assay ◽

Enzyme Linked Immunosorbent Assay ◽

Single Chain Variable Fragment ◽

Single Chain

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Co-expression of recombinant single chain variable fragment recognizing blood antigen fused with sumo and chaperones in Escherichia coli

ACADEMIA JOURNAL OF BIOLOGY ◽

10.15625/2615-9023/v40n4.11689 ◽

2018 ◽

Vol 40 (4) ◽

Author(s):

Dang Thi Ngoc Ha ◽

Le Thi Thu Hong ◽

Truong Nam Hai

Keyword(s):

Recombinant Antibody ◽

Blood Type ◽

Soluble Form ◽

Supernatant Fraction ◽

Blood Group Antigens ◽

Single Chain ◽

E Coli ◽

Recombinant Scfv ◽

Single Chain Variable Fragments

Single chain variable fragments (scFv) have widely been used in research, diagnosis and treatment, but the scFv is considered as difficult protein for expression in E. coli. In previous studies, we expressed a construction of recombinant single chain variable fragments again antigen specific for blood type A (antiA-scFv) individually or fused with Trx or SUMO. However, soluble fraction was low abandant and only approximately 40% when fused with Trx, the other cases were expressed in form of inclusion body. Therefore, it was difficult for purification, refolding and activity assesment. In thispaper, we demonstrated a suitable construction for soluble production of antiA-scFv fused with SUMO (SM/antiA-scFv) in presence of chaparones. Under fermentation with 0.1 mM IPTG at 20oC, the SM/antiA-scFv was entirely expressed in soluble form. Importantly, after cleavage from SUMO with SUMOprotease, antiA-scFv was still maintained in the supernatant fraction. Therefore, it can help ensure bioactivity and is useful for purification process. To the best of our knowledge, this is the first report showing soluble recombinant scFv fused with SUMO in presence of chaperone for determination of blood group antigens. Thus, this result facilitates the optimal study of soluble expression, purification and bioactivity determination of the antiA-scFv recombinant antibody.

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Procaryotic Expression of Single-Chain Variable-Fragment (scFv) Antibodies: Secretion in L-Form Cells of Proteus mirabilis Leads to Active Product and Overcomes the Limitations of Periplasmic Expression in Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.64.12.4862-4869.1998 ◽

1998 ◽

Vol 64 (12) ◽

pp. 4862-4869 ◽

Cited By ~ 39

Author(s):

Jörg F. Rippmann ◽

Michaela Klein ◽

Christian Hoischen ◽

Bodo Brocks ◽

Wolfgang J. Rettig ◽

...

Keyword(s):

Escherichia Coli ◽

Proteus Mirabilis ◽

Binding Activity ◽

Host Cells ◽

Heterologous Proteins ◽

Single Chain Variable Fragment ◽

Single Chain ◽

E Coli ◽

Active Product ◽

Periplasmic Expression

ABSTRACT Recently it has been demonstrated that L-form cells ofProteus mirabilis (L VI), which lack a periplasmic compartment, can be efficiently used in the production and secretion of heterologous proteins. In search of novel expression systems for recombinant antibodies, we compared levels of single-chain variable-fragment (scFv) production in Escherichia coliJM109 and P. mirabilis L VI, which express four distinct scFvs of potential clinical interest that show differences in levels of expression and in their tendencies to form aggregates upon periplasmic expression. Production of all analyzed scFvs in E. coli was limited by the severe toxic effect of the heterologous product as indicated by inhibition of culture growth and the formation of insoluble aggregates in the periplasmic space, limiting the yield of active product. In contrast, the L-form cells exhibited nearly unlimited growth under the tested production conditions for all scFvs examined. Moreover, expression experiments with P. mirabilis L VI led to scFv concentrations in the range of 40 to 200 mg per liter of culture medium (corresponding to volume yields 33- to 160-fold higher than those with E. coli JM109), depending on the expressed antibody. In a translocation inhibition experiment the secretion of the scFv constructs was shown to be an active transport coupled to the signal cleavage. We suppose that this direct release of the newly synthesized product into a large volume of the growth medium favors folding into the native active structure. The limited aggregation of scFv observed in the P. mirabilis L VI supernatant (occurring in a first-order-kinetics manner) was found to be due to intrinsic features of the scFv and not related to the expression process of the host cells. The P. mirabilis L VI supernatant was found to be advantageous for scFv purification. A two-step chromatography procedure led to homogeneous scFv with high antigen binding activity as revealed from binding experiments with eukaryotic cells.

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Development of a bispecific immune engager using a recombinant malaria protein

Cell Death and Disease ◽

10.1038/s41419-021-03611-0 ◽

2021 ◽

Vol 12 (4) ◽

Author(s):

Mie A. Nordmaj ◽

Morgan E. Roberts ◽

Emilie S. Sachse ◽

Robert Dagil ◽

Anne Poder Andersen ◽

...

Keyword(s):

Cancer Cells ◽

Immune Evasion ◽

Blood Cells ◽

Xenograft Model ◽

Cancer Targeting ◽

Single Chain Variable Fragment ◽

Single Chain ◽

Conjugation System ◽

Immune Mediated

AbstractAs an immune evasion and survival strategy, the Plasmodium falciparum malaria parasite has evolved a protein named VAR2CSA. This protein mediates sequestration of infected red blood cells in the placenta through the interaction with a unique carbohydrate abundantly and exclusively present in the placenta. Cancer cells were found to share the same expression of this distinct carbohydrate, termed oncofetal chondroitin sulfate on their surface. In this study we have used a protein conjugation system to produce a bispecific immune engager, V-aCD3, based on recombinant VAR2CSA as the cancer targeting moiety and an anti-CD3 single-chain variable fragment linked to a single-chain Fc as the immune engager. Conjugation of these two proteins resulted in a single functional moiety that induced immune mediated killing of a broad range of cancer cells in vitro and facilitated tumor arrest in an orthotopic bladder cancer xenograft model.

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Single Chain Variable Fragment Fused to Maltose Binding Protein: A Modular Nanocarrier Platform for the Targeted Delivery of Antitumorals

Biomaterials Science ◽

10.1039/d0bm01903h ◽

2021 ◽

Author(s):

Francisco J. Reche-Perez ◽

Simona Plesselova ◽

Eduardo De los Reyes-Berbel ◽

Mariano Ortega-Muñoz ◽

F. Javier Lopez-Jaramillo ◽

...

Keyword(s):

Monoclonal Antibody ◽

Targeted Delivery ◽

Specific Binding ◽

Protein A ◽

Antibody Fragments ◽

Single Chain Variable Fragment ◽

Single Chain ◽

Binding Properties ◽

Single Chain Variable Fragments ◽

Selective Delivery

The use of the specific binding properties of monoclonal antibody fragments such as single-chain variable fragments (ScFv) for the selective delivery of antitumor therapeutics for cancer cells is attractive due...

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