A general platform for efficient extracellular expression and purification of Fab from Escherichia coli

Luo, Manyu; Zhao, Meiqi; Cagliero, Cédric; Jiang, Hua; Xie, Yueqing; Zhu, Jianwei; Yang, Hui; Zhang, Mengxiao; Zheng, Ying; Yuan, Yunsheng; Du, Zongliang; Lu, Huili

doi:10.1007/s00253-019-09745-8

Cited by 27 publications

(22 citation statements)

References 45 publications

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“…We consider that the high yield of the Bi-Nb may be due to its small molecular weight, high solubility, and the high expression of target protein in the prokaryotic system. 39 In addition, in the process of purification, the proteins in the periplasmic space were used as the mother liquor for gradient elution, but there were fewer miscellaneous proteins in the cell periplasmic space, so it was easier to purify the target protein. The results of SPR and fluorescence-activated cell sorting showed that the Bi-Nb could target HER2 and Treatment with Bi-Nb markedly increased apoptosis in HT29 cells from 6.5 to 55% (►Fig.…”

Section: Discussionmentioning

confidence: 99%

Generating a Novel Bispecific Nanobody to Enhance Antitumor Activity

Sun

Bian

et al. 2020

Pharmaceutical Fronts

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Tumor cells express high levels of human epidermal growth factor receptor 2 (HER2) and vascular endothelial growth factor receptor 2 (VEGFR2), which are closely related to their proliferation and survival. Cancer treatments that target a single signaling pathway may result in immune pathway escape or drug resistance. Based on the correlation between the HER2 and VEGFR2 signaling pathways, we speculated that targeting the two pathways simultaneously may produce a synergistic effect and avoid occurrence of drug resistance, resulting in improved efficacy. Anti-VEGFR2 nanobody 3VGR19–3 and anti-HER2 nanobody 2D3 were combined to construct a bispecific nanobody (Bi-Nb). They can recognize both HER2 and VEGFR2 (both highly expressed in HT-29 cells) to simultaneously block the two signaling pathways. We verified the affinity of the Bi-Nb to its targets using the surface plasmon resonance technology, and test its effects to inhibit tumor cell growth and promote cell apoptosis in vitro by the Cell Counting Kit-8 assay and apoptosis assay. In summary, we have successfully constructed a Bi-Nb, and verified its tumor-suppressing effects in vitro. Compared with a single monospecific nanobody, our Bi-Nb showed superior antitumor effect, which provides a new perspective for treatment of tumors with high HER2 and VEGFR2 expression.

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Section: Discussionmentioning

confidence: 99%

Generating a Novel Bispecific Nanobody to Enhance Antitumor Activity

Sun

Bian

et al. 2020

Pharmaceutical Fronts

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“…These results indicate that supplementing a nitrogen source at low temperature is critical for Fab productivity in E. coli fermentations [90]. Of note, the alkaline phosphatase (phoA) promoter and the heat-stable enterotoxin II (STII) leader sequence have been also described as a positive combination to facilitate the E. coli extracellular production of Fab fragments [160].…”

Section: Choice Of Peptide Signal For Antibody Fragment Expressionmentioning

confidence: 98%

“…In this way, a functional recombinant scFv has been efficiently recovered (2.86 mg/L) from the extracellular medium by adding 0.25% Triton X-100 [159]. An experimental setting for the efficient secretion of Fab fragments in E. coli in shake flasks has been recently described by Luo and coworkers [160]. They have demonstrated that the use of the alkaline phosphatase promoter (phoA) in combination with the heat-stable enterotoxin II (STII) signal peptide (phoA-STII system) is a promising strategy for the extracellular production of Fab fragments, reaching up to 10 mg/L [160].…”

Section: Extracellular Secretionmentioning

confidence: 99%

“…An experimental setting for the efficient secretion of Fab fragments in E. coli in shake flasks has been recently described by Luo and coworkers [160]. They have demonstrated that the use of the alkaline phosphatase promoter (phoA) in combination with the heat-stable enterotoxin II (STII) signal peptide (phoA-STII system) is a promising strategy for the extracellular production of Fab fragments, reaching up to 10 mg/L [160]. The authors carefully assessed and compared the effects of promoters, E. coli strains and signal peptides on the extracellular expression of a panel of five recombinant Fab fragments.…”

Section: Extracellular Secretionmentioning

confidence: 99%

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Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments

Sandomenico

Sivaccumar

Ruvo

2020

IJMS

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Antibodies and antibody-derived molecules are continuously developed as both therapeutic agents and key reagents for advanced diagnostic investigations. Their application in these fields has indeed greatly expanded the demand of these molecules and the need for their production in high yield and purity. While full-length antibodies require mammalian expression systems due to the occurrence of functionally and structurally important glycosylations, most antibody fragments and antibody-like molecules are non-glycosylated and can be more conveniently prepared in E. coli-based expression platforms. We propose here an updated survey of the most effective and appropriate methods of preparation of antibody fragments that exploit E. coli as an expression background and review the pros and cons of the different platforms available today. Around 250 references accompany and complete the review together with some lists of the most important new antibody-like molecules that are on the market or are being developed as new biotherapeutics or diagnostic agents.

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“…Deletion of the D-alanyl-D-alanine carboxypeptidase gene dacC has resulted in enhanced extracellular protein production in E. coli (Hu et al, 2019). Alkaline phosphatase (phoA) promoter and the heatstable enterotoxin II (STII) leader sequence have also facilitated extracellular production in E. coli for the manufacture of Fab fragments (Luo et al, 2019). It was established that the posttranslational targeting of single-chain variable antibody fragment (scFv) BL1 enabled its efficient production in the periplasm due to a favorable adaptation of the E. coli proteome (Ytterberg et al, 2019).…”

Section: Escherichia Colimentioning

confidence: 99%

Recent Developments in Bioprocessing of Recombinant Proteins: Expression Hosts and Process Development

Tripathi

Shrivastava

2019

Front. Bioeng. Biotechnol.

400

243

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Infectious diseases, along with cancers, are among the main causes of death among humans worldwide. The production of therapeutic proteins for treating diseases at large scale for millions of individuals is one of the essential needs of mankind. Recent progress in the area of recombinant DNA technologies has paved the way to producing recombinant proteins that can be used as therapeutics, vaccines, and diagnostic reagents. Recombinant proteins for these applications are mainly produced using prokaryotic and eukaryotic expression host systems such as mammalian cells, bacteria, yeast, insect cells, and transgenic plants at laboratory scale as well as in large-scale settings. The development of efficient bioprocessing strategies is crucial for industrial production of recombinant proteins of therapeutic and prophylactic importance. Recently, advances have been made in the various areas of bioprocessing and are being utilized to develop effective processes for producing recombinant proteins. These include the use of high-throughput devices for effective bioprocess optimization and of disposable systems, continuous upstream processing, continuous chromatography, integrated continuous bioprocessing, Quality by Design, and process analytical technologies to achieve quality product with higher yield. This review summarizes recent developments in the bioprocessing of recombinant proteins, including in various expression systems, bioprocess development, and the upstream and downstream processing of recombinant proteins.

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A general platform for efficient extracellular expression and purification of Fab from Escherichia coli

Cited by 27 publications

References 45 publications

Generating a Novel Bispecific Nanobody to Enhance Antitumor Activity

Generating a Novel Bispecific Nanobody to Enhance Antitumor Activity

Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments

Recent Developments in Bioprocessing of Recombinant Proteins: Expression Hosts and Process Development

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