scholarly journals A microbial expression system for high-level production of scFv HIV-neutralizing antibody fragments in Escherichia coli

2019 ◽  
Vol 103 (21-22) ◽  
pp. 8875-8888 ◽  
Author(s):  
Marloes L. C. Petrus ◽  
Lukas A. Kiefer ◽  
Pranav Puri ◽  
Evert Heemskerk ◽  
Michael S. Seaman ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Neutralizing Antibodies ◽  
Expression System ◽  
Antibody Fragment ◽  
Green Fluorescence Protein ◽  
Antibody Fragments ◽  
Single Chain ◽  
Mammalian Expression ◽  
Anti Hiv

Abstract Monoclonal antibodies (mABs) are of great biopharmaceutical importance for the diagnosis and treatment of diseases. However, their production in mammalian expression hosts usually requires extensive production times and is expensive. Escherichia coli has become a new platform for production of functional small antibody fragment variants. In this study, we have used a rhamnose-inducible expression system that allows precise control of protein expression levels. The system was first evaluated for the cytoplasmic production of super folder green fluorescence protein (sfGFP) in various production platforms and then for the periplasmic production of the anti-HIV single-chain variable antibody fragment (scFv) of PGT135. Anti-HIV broadly neutralizing antibodies, like PGT135, have potential for clinical use to prevent HIV transmission, to promote immune responses and to eradicate infected cells. Different concentrations of L-rhamnose resulted in the controlled production of both sfGFP and scFv PGT135 antibody. In addition, by optimizing the culture conditions, the amount of scFv PGT135 antibody that was expressed soluble or as inclusions bodies could be modulated. The proteins were produced in batch bioreactors, with yields of 4.9 g/L for sfGFP and 0.8 g/L for scFv. The functionality of the purified antibodies was demonstrated by their ability to neutralize a panel of different HIV variants in vitro. We expect that this expression system will prove very useful for the development of a more cost-effective production process for proteins and antibody fragments in microbial cells.

scholarly journals Studies of Single-Chain Antibody Expression in Quiescent Escherichia coli

2004 ◽  
Vol 70 (5) ◽  
pp. 3005-3012 ◽  
Author(s):  
K. J. Mukherjee ◽  
D. C. D. Rowe ◽  
N. A. Watkins ◽  
D. K. Summers
Keyword(s):  
Escherichia Coli ◽  
Expression System ◽  
Formation Rate ◽  
Antibody Fragment ◽  
Control Culture ◽  
Temperature Sensitive ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Specific Product ◽  
Quiescent Cells

ABSTRACT Quiescent Escherichia coli cells are generated by overexpressing the Rcd transcript in an hns-205 mutant host. The resulting nongrowing, metabolically active cells were used here to express a single-chain antibody fragment (scFv) in shake flask and fermentor cultures. The expression system is based on two plasmids; one carries the product gene expressed from λPL under the control of the cI857 temperature-sensitive repressor, while the second expresses Rcd from λPR. Shifting the culture from 30 to 42°C induces Rcd expression and product expression simultaneously. Our scFv carried a PelB leader, and 90% of the protein was secreted into the culture supernatant. In a batch culture, the supernatant concentration of scFv in the quiescent-cell culture (optical density at 600 nm [OD600] of 3.5) was 37 mg liter−1, compared to a maximum of 13 mg liter−1 in the control culture (final OD600 of 20). In a fed-batch fermentor culture, quiescent cells were held at an OD600 of 20 for 24 h and the extracellular scFv concentration reached a maximum of 150 mg liter−1. A control culture with a similar feed reached an OD600 of 80, but despite the higher density, the extracellular scFv concentration did not exceed 35 mg liter−1. Quiescent cells at an OD600 of 50 exhibited a small decline in the specific product formation rate, but nevertheless, an extracellular scFv concentration of 160 mg liter−1 was achieved in 8 h. The rate of extracellular accumulation was 10-fold greater in the quiescent culture than in the control culture. This study demonstrates that it is possible to establish high-density quiescent E. coli cultures that are capable of efficient synthesis, folding, and export of proteins.

scholarly journals Two Antibody-Guided Lactic-co-Glycolic Acid-Polyethylenimine (LGA-PEI) Nanoparticle Delivery Systems for Therapeutic Nucleic Acids

2021 ◽  
Vol 14 (9) ◽  
pp. 841
Author(s):  
Jian-Ming Lü ◽  
Zhengdong Liang ◽  
Dongliang Liu ◽  
Bin Zhan ◽  
Qizhi Yao ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Plasmid Dna ◽  
Glycolic Acid ◽  
Growth Factor Receptor ◽  
Green Fluorescence Protein ◽  
Active Targeting ◽  
Single Chain ◽  
Targeting Delivery

We previously reported a new polymer, lactic-co-glycolic acid-polyethylenimine (LGA-PEI), as an improved nanoparticle (NP) delivery for therapeutic nucleic acids (TNAs). Here, we further developed two antibody (Ab)-conjugated LGA-PEI NP technologies for active-targeting delivery of TNAs. LGA-PEI was covalently conjugated with a single-chain variable fragment antibody (scFv) against mesothelin (MSLN), a biomarker for pancreatic cancer (PC), or a special Ab fragment crystallizable region-binding peptide (FcBP), which binds to any full Ab (IgG). TNAs used in the current study included tumor suppressor microRNA mimics (miR-198 and miR-520h) and non-coding RNA X-inactive specific transcript (XIST) fragments; green fluorescence protein gene (GFP plasmid DNA) was also used as an example of plasmid DNA. MSLN scFv-LGA-PEI NPs with TNAs significantly improved their binding and internalization in PC cells with high expression of MSLN in vitro and in vivo. Anti-epidermal growth factor receptor (EGFR) monoclonal Ab (Cetuximab) binding to FcBP-LGA-PEI showed active-targeting delivery of TNAs to EGFR-expressing PC cells.

scholarly journals Optimization of the crystallizability of a single-chain antibody fragment

2014 ◽  
Vol 70 (12) ◽  
pp. 1701-1706 ◽  
Author(s):  
Jana Škerlová ◽  
Vlastimil Král ◽  
Milan Fábry ◽  
Juraj Sedláček ◽  
Václav Veverka ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Antibody Fragment ◽  
Binding Mode ◽  
Antibody Fragments ◽  
Dimensional Structure ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Diagnostic Potential ◽  
Storage Buffer ◽  
Single Chain Antibody Fragment

Single-chain variable antibody fragments (scFvs) are molecules with immense therapeutic and diagnostic potential. Knowledge of their three-dimensional structure is important for understanding their antigen-binding mode as well as for protein-engineering approaches such as antibody humanization. A major obstacle to the crystallization of single-chain variable antibody fragments is their relatively poor homogeneity caused by spontaneous oligomerization. A new approach to optimization of the crystallizability of single-chain variable antibody fragments is demonstrated using a representative single-chain variable fragment derived from the anti-CD3 antibody MEM-57. A Thermofluor-based assay was utilized to screen for optimal conditions for antibody-fragment stability and homogeneity. Such an optimization of the protein storage buffer led to a significantly improved ability of the scFv MEM-57 to yield crystals.

scholarly journals Llama-Derived Single-Chain Antibody Fragments Directed to Rotavirus VP6 Protein Possess Broad Neutralizing Activity In Vitro and Confer Protection against Diarrhea in Mice

2008 ◽  
Vol 82 (19) ◽  
pp. 9753-9764 ◽  
Author(s):  
Lorena Garaicoechea ◽  
Aurelien Olichon ◽  
Gisela Marcoppido ◽  
Andrés Wigdorovitz ◽  
Marina Mozgovoj ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Binding Capacity ◽  
Antibody Fragments ◽  
Target Antigen ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Group A Rotavirus ◽  
Group A

ABSTRACT Group A rotavirus is one of the most common causes of severe diarrhea in human infants and newborn animals. Rotavirus virions are triple-layered particles. The outer capsid proteins VP4 and VP7 are highly variable and represent the major neutralizing antigens. The inner capsid protein VP6 is conserved among group A rotaviruses, is highly immunogenic, and is the target antigen of most immunodiagnosis tests. Llama-derived single-chain antibody fragments (VHH) are the smallest molecules with antigen-binding capacity and can therefore be expected to have properties different from conventional antibodies. In this study a library containing the VHH genes of a llama immunized with recombinant inner capsid protein VP6 was generated. Binders directed to VP6, in its native conformation within the viral particle, were selected and characterized. Four selected VHH directed to conformational epitopes of VP6 recognized all human and animal rotavirus strains tested and could be engineered for their use in immunodiagnostic tests for group A rotavirus detection. Three of the four VHH neutralized rotavirus in vivo independently of the strain serotype. Furthermore, this result was confirmed by in vivo partial protection against rotavirus challenge in a neonatal mouse model. The present study demonstrates for the first time a broad neutralization activity of VP6 specific VHH in vitro and in vivo. Neutralizing VHH directed to VP6 promise to become an essential tool for the prevention and treatment of rotavirus diarrhea.

scholarly journals Therapeutic Activity of an Engineered Synthetic Killer Antiidiotypic Antibody Fragment against Experimental Mucosal and Systemic Candidiasis

2003 ◽  
Vol 71 (11) ◽  
pp. 6205-6212 ◽  
Author(s):  
Luciano Polonelli ◽  
Walter Magliani ◽  
Stefania Conti ◽  
Luisa Bracci ◽  
Luisa Lozzi ◽  
...  
Keyword(s):  
Wide Spectrum ◽  
Model Organism ◽  
Killer Toxin ◽  
Antibody Fragment ◽  
Systemic Infection ◽  
Vaginal Candidiasis ◽  
Single Chain ◽  
Significant Prolongation ◽  
Antiidiotypic Antibody

ABSTRACT Peptides derived from the sequence of a single-chain, recombinant, antiidiotypic antibody (IdAb; KT-scFv) acting as a functional internal image of a microbicidal, wide-spectrum yeast killer toxin (KT) were synthesized and studied for their antimicrobial activity by using the KT-susceptible Candida albicans as model organism. A decapeptide containing the first three amino acids (SAS) of the light chain CDR1 was selected and optimized by alanine replacement of a single residue. This peptide exerted a strong candidacidal activity in vitro, with a 50% inhibitory concentration of 0.056 μM, and was therefore designated killer peptide (KP). Its activity was neutralized by laminarin, a β1-3 glucan molecule, but not by pustulan, a β1-6 glucan molecule. KP also competed with the binding of a KT-like monoclonal IdAb to germinating cells of the fungus. In a rat model of vaginal candidiasis, local, postchallenge administration of KP was efficacious in rapidly abating infections caused by fluconazole-susceptible or -resistant C. albicans strains. In systemic infection of BALB/c or SCID mice preinfected intravenously with a lethal fungal load, KP caused a highly significant prolongation of the median survival time, with >80% of the animals still surviving after >60 days, whereas >90% of control mice died within 3 to 5 days. KP is therefore the first engineered peptide derived from a recombinant IdAb retaining KT microbicidal activity, probably through the interaction with the β-glucan KT receptor on target microbial cells.

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