Effective Strategies to Overcome the Insolubility of Recombinant ScFv Antibody against EpCAM Extracellular Domain in E. coli

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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Solubility assessment of single-chain antibody fragment against epithelial cell adhesion molecule extracellular domain in four Escherichia coli strains

Journal of Genetic Engineering and Biotechnology ◽

10.1186/s43141-021-00126-1 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Fatemeh Sadat Javadian ◽

Majid Basafa ◽

Aidin Behravan ◽

Atieh Hashemi

Keyword(s):

Escherichia Coli ◽

Cell Adhesion ◽

Epithelial Cell ◽

Adhesion Molecule ◽

Cell Adhesion Molecule ◽

Extracellular Domain ◽

Epithelial Cell Adhesion Molecule ◽

Single Chain ◽

E Coli ◽

The Impact

Abstract Background Overexpression of the EpCAM (epithelial cell adhesion molecule) in malignancies makes it an attractive target for passive immunotherapy in a wide range of carcinomas. In comparison with full-length antibodies, due to the small size, the scFvs (single-chain variable fragments) are more suitable for recombinant expression in E. coli (Escherichia coli). However, the proteins expressed in large amounts in E. coli tend to form inclusion bodies that need to be refolded which may result in poor recovery of bioactive proteins. Various engineered strains were shown to be able to alleviate the insolubility problem. Here, we studied the impact of four E. coli strains on the soluble level of anti-EpEX-scFv (anti-EpCAM extracellular domain-scFv) protein. Results Although results showed that the amount of soluble anti-EpEX-scFv obtained in BL21TM (DE3) (114.22 ± 3.47 mg/L) was significantly higher to those produced in the same condition in E. coli RosettaTM (DE3) (71.39 ± 0.31 mg/L), and OrigamiTM T7 (58.99 ± 0.44 mg/L) strains, it was not significantly different from that produced by E. coli SHuffleTM T7 (108.87 ± 2.71 mg/L). Furthermore, the highest volumetric productivity of protein reached 318.29 ± 26.38 mg/L in BL21TM (DE3). Conclusions Although BL21TM (DE3) can be a suitable strain for high-level production of anti-EpEX-scFv protein, due to higher solubility yield (about 55%), E. coli SHuffleTM T7 seems to be better candidate for soluble production of scfv compared to BL21TM (DE3) (solubility yield of about 30%).

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Chaperone-Assisted Soluble Expression of a Humanized Anti-EGFR ScFv Antibody in E. Coli

Advanced Pharmaceutical Bulletin ◽

10.15171/apb.2015.084 ◽

2015 ◽

Vol 5 (Suppl 1) ◽

pp. 621-627 ◽

Cited By ~ 8

Author(s):

Kamal Veisi ◽

Safar Farajnia ◽

Nosratollah Zarghami ◽

Hamid Reza Khoram Khorshid ◽

Nasser Samadi ◽

...

Keyword(s):

Soluble Expression ◽

Scfv Antibody ◽

E Coli

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Expression and Purification of the Extracellular Domain of Luteinizing Hormone Receptor From Ovis Aries Testicle

10.21203/rs.3.rs-428805/v1 ◽

2021 ◽

Author(s):

Jose Luis Villalpando-Aguilar ◽

Itzel López-Rosas ◽

Arnulfo Montero-Pardo ◽

Elisa Irene Azuara-Liceaga ◽

Javier de Jesus Valencia-Méndez ◽

...

Keyword(s):

Luteinizing Hormone ◽

Hormone Receptor ◽

Extracellular Domain ◽

Insoluble Fraction ◽

Ovis Aries ◽

Luteinizing Hormone Receptor ◽

New Drugs ◽

E Coli ◽

Fertility Disorders ◽

G Protein Coupled

Abstract The luteinizing hormone receptor (LHR) is a glycoprotein member of the G protein-coupled receptor superfamily. Physiologically, this receptor participates in corpus luteum formation and ovulation in females. In males, it acts in testosterone synthesis and spermatogenesis and is involved in some fertility disorders. RNA was extracted from Ovis aries testicles, and the corresponding cDNA was synthesized to amplify the lhr gene, termed lhr-bed here, consisting of 762 bp that encodes 273 amino acids of the extracellular domain of LHR. Thus, the lhr-bed was cloned into pJET1.2/blunt, subcloned into the pCOLD II expression vector and finally transformed into E. coli BL21 cells. Since the induced rLHR-Bed protein was found in the insoluble fraction, the purification protocol was modified as follows: induction at 25°C, denaturing conditions (8 M UREA and 0.1% CHAPS) and refolding in the column to increase solubility. The rLHR-Bed expression was corroborated by western blotting and mass spectrometry (MS) analysis. This successful method to obtain the recombinant LHR extracellular domain yields 0.2 mg/L of the protein with approximately 90% purity from a single chromatographic purification step. The present approach demonstrates the feasibility of obtaining large quantities of rLHR-Bed. This might be useful to accomplish future studies regarding the structure and functional analysis of the binding interplay with its ligand luteinizing hormone and its isoforms. Additionally, this biotechnological strategy might be used to improve and to develop new drugs for the treatment of reproductive disorders and might also be applied to species reproduction in the livestock industry.

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Photocatalytic Treatments for Personal Protective Equipment: Experimental Microbiological Investigations and Perspectives for the Enhancement of Antimicrobial Activity by Micrometric TiO2

International Journal of Environmental Research and Public Health ◽

10.3390/ijerph18168662 ◽

2021 ◽

Vol 18 (16) ◽

pp. 8662

Author(s):

Lory Marika Margarucci ◽

Gianluca Gianfranceschi ◽

Vincenzo Romano Spica ◽

Giuseppe D’Ermo ◽

Cristiano Refi ◽

...

Keyword(s):

Antimicrobial Activity ◽

Personal Protective Equipment ◽

Environmental Sustainability ◽

Light Exposure ◽

Protective Equipment ◽

Light Radiation ◽

Gram Negative ◽

E Coli ◽

Effective Strategies ◽

Waste Load

The COVID-19 pandemic has led to countries enforcing the use of facial masks to prevent contagion. However, acquisition, reuse, and disposal of personal protective equipment (PPE) has generated problems, in regard to the safety of individuals and environmental sustainability. Effective strategies to reprocess and disinfect PPE are needed to improve the efficacy and durability of this equipment and to reduce waste load. Thus, the addition of photocatalytic materials to these materials, combined with light exposure at specific wavelengths, may represent promising solutions. To this aim, we prepared a series of masks by depositing micrometer-sized TiO2 on the external surfaces; the masks were then contaminated with droplets of bacteria suspensions and the coatings were activated by light radiation at different wavelengths. A significant reduction in the microbial load (over 90%, p < 0.01) was observed using both Gram negative (E. coli) and Gram positive (S. aureus) bacteria within 15 min of irradiation, with UV or visible light, including sunlight or artificial sources. Our results support the need for further investigations on self-disinfecting masks and other disposable PPE, which could positively impact (i) the safety of operators/workers, and (ii) environmental sustainability in different occupational or recreational settings.

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Selection and characterization of scFv antibody against nucleocapsid protein of Porcine reproductive and respiratory syndrome virus

Acta Veterinaria Brno ◽

10.2754/avb202089010039 ◽

2020 ◽

Vol 89 (1) ◽

pp. 39-45

Author(s):

Magdalena Krasna ◽

Vladimír Celer

Keyword(s):

Infectious Agent ◽

Scfv Antibody ◽

Respiratory Syndrome Virus ◽

Single Chain ◽

Single Chain Antibody ◽

N Protein ◽

E Coli ◽

Metal Affinity ◽

Binding Epitope

Porcine reproductive and respiratory syndrome virus (PRRSV) is a widespread infectious agent in pigs. Nucleocapsid (N) protein of PRRSV has been identified as the most immunodominant viral protein. The main goal of the work was the selection and characterization of a single-chain antibody fragments (scFv) antibody specific to the N protein. Specific scFv antibody clone D5 was selected from the Tomlinson phagemid library and purified by immobilized metal affinity chromatography from the periplasmatic space of E. coli cells. The antibody was then characterized by sequencing and the ability to recognize the native virus N protein by Western blot and competitive ELISA. Pepscan analysis identified the position of the binding epitope between amino acids 62–84 of the N protein. Our study could help to improve the diagnostics and prevention of PRRSV in Central Europe.

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Production of Stabilized scFv Antibody Fragments in the E. coli Bacterial Cytoplasm

Methods in Molecular Biology - Human Monoclonal Antibodies ◽

10.1007/978-1-62703-586-6_10 ◽

2014 ◽

pp. 171-184 ◽

Cited By ~ 10

Author(s):

Lilach Vaks ◽

Itai Benhar

Keyword(s):

Antibody Fragments ◽

Scfv Antibody ◽

E Coli ◽

Bacterial Cytoplasm

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Comparative Genome Analysis Reveals Human Pathogenic Potential of ESBL-Escherichia coli Isolated from Swine Microbiomes

10.1101/2021.07.15.452468 ◽

2021 ◽

Author(s):

Luria L Founou ◽

Raspail Carrel Founou ◽

Mushal Allam ◽

Arshad Ismael ◽

Sabiha Yusuf Essack

Keyword(s):

South Africa ◽

Virulence Genes ◽

De Novo ◽

Antibiotic Resistance Genes ◽

Whole Genome ◽

Comparative Genome Analysis ◽

Pathogenic Potential ◽

E Coli ◽

Effective Strategies ◽

Sequence Types

Background: Extended-spectrum β-lactamase-producing E. coli (ESBL-Ec) harbouring virulence genes in the microbiome of food animals could likely threaten human health but is poorly understood in Cameroon and South Africa. Here, we assessed the resistome, virulome and mobilome of ESBL-Ec isolated from swine microbiomes in these countries, using whole genome sequencing (WGS). Materials/methods: Eleven clonally-related phenotypic ESBL-Ec isolates were subjected to WGS. The isolates were de novo assembled using the CLC Genomics Workbench and SPAdes while RAST and PROKKA were used for annotation of the assembled contigs. Prediction of antibiotic resistance genes, virulence factors and plasmids was performed using ResFinder, VirulenceFinder and PlasmidFinder, respectively. Results: Diverse STs were detected with sequence types ST2144 and ST88 predominating and blaCTX-M-15 (55%) as principal ESBL genes. Although the isolates belonged mainly to commensal phylogroups A/B1 (45/28.3%) and C (18.18%), all harboured at least three extraintestinal pathogenic E. coli (ExPEC) VFs with one isolate harbouring up to 18 ExPEC VFs. Conclusion: The resistance and pathogenic potential of ESBL-Ec colonizing the gut microbiota of swine in both countries demonstrate the urgent need to implement effective strategies to contain the dissemination of virulent ESBL-Ec through the food chain in Cameroon and South Africa.

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