Expression and Characterization of a Single-Chain Variable Fragment against Human LOX-1 in Escherichia coli and Brevibacillus choshinensis

Hu, Wei; Xiang, Jun; Kong, Ping; Liu, Ling; Xie, Qiuhong

doi:10.4014/jmb.1702.02007

Cited by 13 publications

(10 citation statements)

References 31 publications

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“…Thus, it is important to use genetic engineering strategies to produce large amounts of recombinant TFF3 for biochemical and biomedical applications. B. choshinensis has an exceptional capacity to produce heterologous proteins (23,24,(33)(34)(35). Target proteins can be produced and secreted efficiently with a high yield by Brevibacillus expression systems (33).…”

Section: Discussionmentioning

confidence: 99%

“…Compared with traditional E. coli expression systems, the genus Brevibacillus, including thermophilic, alkalophilic, psychrophilic, acidophilic and halophilic strains, use more carbon for heterotrophic or autotrophic growth (34). Brevibacillus expression systems can produce certain secretory or cytoplasmic proteins that E. coli expression systems fail to produce (35). Brevibacillus expression systems possess a good protein folding environment, lack proteases and have a convenient downstream processing model, e.g.…”

Section: Discussionmentioning

confidence: 99%

“…To construct the sus scrofa TFF3 expression vector pNCMO2-TFF3-6xhis, the fused TFF3-6xhis combined fragment from pMD19-T-TFF3-6xhis was amplified by PCR. The following thermocycling conditions were used for the PCR: initial denaturation at 95˚C for 10 min; followed by 35…”

Section: Construction Of Pncmo2-tff3-6xhismentioning

confidence: 99%

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Extracellular production of recombinant sus scrofa trefoil factor 3 by Brevibacillus choshinensis

Wen

et al. 2020

Exp Ther Med

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Trefoil factor 3 (TFF3) is involved in cell adhesion, motility and apoptosis, regulates mucosal immunity and maintains the functional integrity of intestinal epithelia. The upregulation of TFF3 expression in the weaning rat intestine attracted our interest. The present study hypothesized that TFF3 may serve a role in preventing diarrhea in weaning piglets, which is an important consideration in the pig farming industry. Previous recombinant TFF3 protein expression yields obtained from Escherichia coli were too low and the bioactivity of the protein was poor. Hence, this expression system was unsuitable for industrial applications. The present study explored the production of recombinant sus scrofa TFF3 in a Brevibacillus choshinensis (B. choshinensis) expression system, aiming to enhance the expression level of bioactive protein. To achieve this, the sus scrofa TFF3-encoding gene fragment was fused into an E. coli-Brevibacillus shuttle vector pNCMO2. High levels of TFF3 (30 mg/l) were produced and secreted into the B. choshinensis culture medium in soluble form with a molecular mass of 13.6 kDa and high immunoreactivity in western blotting. Thus, Brevibacillus may be used to produce useful mucosal factors for biochemical analyses and mucosal protection, and in industrial applications to produce novel inhibitors of diarrhea.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Extracellular production of recombinant sus scrofa trefoil factor 3 by Brevibacillus choshinensis

Wen

et al. 2020

Exp Ther Med

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“…The anti-LOX-1 scFv with a C-terminal Myc peptide and a polyhistidine (6xHis) metal-binding tag was synthesized according to a previous study [9] and cloned into a pUC19 vector (pUC19-scFv). Three LOX-1-binding peptides LTPATAI, FQTPPQL, and LSIPPKA were designed to fuse at the N-terminus and C-terminus or to substitute the amino acids GGGGS in the middle of the linker region of the scFv.…”

Section: Methodsmentioning

confidence: 99%

“…In our previous study, an anti-LOX-1 scFv was successfully produced in the Brevibacillus choshinensis (B. choshinensis) host and showed specific affinity for the human LOX-1 protein [9]. We believe that it has a great potential for immunodiagnostics, drug delivery, and immunotherapy.…”

Section: Introductionmentioning

confidence: 99%

Improved scFv Anti-LOX-1 Binding Activity by Fusion with LOX-1-Binding Peptides

Xie

2017

BioMed Research International

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The oxidized low-density lipoprotein receptor-1 (LOX-1) targeted single-chain variable fragment (scFvs) is a promising molecule for the targeted delivery of imaging and therapeutic molecules of atherosclerotic diseases; however, its applications are limited by the inherent low antigen affinity. In this study, the three-dimensional (3D) model of the anti-LOX-1 scFv was constructed and its docking with the LOX-1 protein was developed. To improve the LOX-1-binding activity, the anti-LOX-1 scFv was designed to fuse with one of three LOX-1-binding heptapeptides, LTPATAI, FQTPPQL, and LSIPPKA, at its N-terminus and C-terminus and in the linker region, which have different LOX-1-binding interfaces with the anti-LOX-1 scFv analyzed by an array of computational approaches. These scFv/peptide fusions were constructed, successfully expressed in Brevibacillus choshinensis hosts, and purified by a two-step column purification process. The antigen binding activity, structural characteristics, thermal stability, and stability in serum of these fusion proteins were examined. Results showed that the scFv with N-terminal fusing peptides proteins demonstrated increased LOX-1-binding activity without decrease in stability. These findings will help increase the application efficacy of LOX-1 targeting scFv in LOX-1-based therapy.

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Induction of immunocontraceptive effects in both male and female mice immunized with GnRH vaccine

Ahn

Park

et al. 2021

Veterinary Medicine & Sci

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Background Gonadotropin‐releasing hormone (GnRH) plays a pivotal role in regulating the reproductive endocrine system. Objective An immunocontraception vaccine aimed at inhibiting the functions of GnRH is tested as a potential tool for controlling animal populations. Methods We developed a recombinant immunocontraceptive vaccine composed of GnRH‐I and GnRH‐II (GnRH I+II), which was conjugated with Salmonella typhimurium flagellin. Forty‐eight BALB/c mice aged 4 weeks were divided into four groups (each group had n = 12): non‐vaccinated male (NVM), non‐vaccinated female (NVF), vaccinated male (VM), and vaccinated female (VF). Mice in the vaccinated groups were vaccinated twice by intramuscular injection at 0 and 2 weeks with 300 μg of the recombinant GnRH protein complex per mouse. Mice in the non‐vaccinated groups were injected with saline and served as the unimmunized controls. Twenty‐four pairs of male and female mice were mated for 10–12 weeks after initial immunization in four groups: 6 NVF × 6 NVM, 6 VF × 6 NVM, 6 NVF × 6 VM, and 6 VF × 6 VM. Results: An increase (p < 0.001) in antibody titers in VM and VF mice was observed. The testosterone levels and the number of spermatocytes were lower (p < 0.001) in VM mice than those in the control mice. The progesterone levels and the number of corpora lutea were lower (p < 0.001) than those in the control mice. Mating results in both VM and VF mice confirmed a 60% reduction in pregnancy rates and offspring numbers. Conclusions The recombinant GnRH vaccine can be used for birth control in both male and female animals.

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Expression and Characterization of a Single-Chain Variable Fragment against Human LOX-1 in Escherichia coli and Brevibacillus choshinensis

Cited by 13 publications

References 31 publications

Extracellular production of recombinant sus scrofa trefoil factor 3 by Brevibacillus choshinensis

Extracellular production of recombinant sus scrofa trefoil factor 3 by Brevibacillus choshinensis

Improved scFv Anti-LOX-1 Binding Activity by Fusion with LOX-1-Binding Peptides

Induction of immunocontraceptive effects in both male and female mice immunized with GnRH vaccine

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