Plant produced endotoxin binding recombinant proteins effectively remove endotoxins from protein samples

Khan, Md Rezaul Islam; Muthamilselvan, Thangarasu; Kang, Hyangju; Hwang, Inhwan

doi:10.1038/s41598-022-20776-6

Cited by 4 publications

(4 citation statements)

References 44 publications

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“…Recently, a new platform to remove LPS from recombinant proteins was designed based on the N‐terminal region containing cysteine‐rich, EGF‐like, and sushi1‐3 domains (CES3) of Factor C from the horseshoe crab Carcinoscorpius rotundicauda 29 . The recombinant protein CES3:CBM3:HDEL purified from Arabidopsis plant extracts and immobilized onto microcrystalline cellulose beads efficiently removed LPS contamination from protein samples 29 . Moreover, an alternative strategy can be applied, such as an expression or recombinant protein in a detoxifying E. coli host 30 .…”

Section: Resultsmentioning

confidence: 99%

“…Recently, a new platform to remove LPS from recombinant proteins was designed based on the N-terminal region containing cysteine-rich, EGF-like, and sushi1-3 domains (CES3) of Factor C from the horseshoe crab Carcinoscorpius rotundicauda. 29 The recombinant protein CES3:CBM3:…”

Section: Scaling Up Of the Silk Post-purification Methodsmentioning

confidence: 99%

“…HDEL purified from Arabidopsis plant extracts and immobilized onto microcrystalline cellulose beads efficiently removed LPS contamination from protein samples. 29 Moreover, an alternative strategy can be applied, such as an expression or recombinant protein in a detoxifying E. coli host. 30 Mamat et al bioengineered endotoxin-free E. coli strains and demonstrated the direct production of recombinant proteins with negligible endotoxin contamination.…”

Section: Scaling Up Of the Silk Post-purification Methodsmentioning

confidence: 99%

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Endotoxin reduction from biotech silk material inhibits the production of anti‐silk antibodies in mice

Deptuch,

Kucharczyk,

Florczak

et al. 2023

J Biomedical Materials Res

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Eliminating endotoxins is a common problem in the development of biotechnologically produced pharmaceuticals or biomaterials. Residual endotoxins in the final sample may hamper the properties of the product or induce severe adverse effects. Developing an effective downstream purification protocol that ensures a lack of minimal endotoxin content in the final product can be a challenging task. In our previous studies, we developed nanospheres produced from bioengineered silks. Despite their good overall biocompatibility, in vivo characterization of spheres showed mild activation of the immune system (mainly in terms of anti‐silk antibody production). Herein, we examined, if the endotoxins delivered with the silk spheres might have contributed to activating the adaptive immune response. We investigated various commercially available methods for endotoxin removal that can be applied as an extra step in downstream endotoxin removal from MS1‐type silk proteins. We selected a method that allowed for a 10‐fold reduction of endotoxin content in soluble silk and 2‐fold in the final product (silk spheres). The reduced level of endotoxins improved the biocompatibility of the silk spheres as these particles induced negligible titers of anti‐silk antibodies in an in vivo immune study. Since endotoxins can enhance life‐threatening immune responses, it is crucial to optimize the method of their removal before clinical use not only of silk‐based products but also of other biomolecules produced biotechnologically.

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Section: Resultsmentioning

confidence: 99%

Section: Scaling Up Of the Silk Post-purification Methodsmentioning

confidence: 99%

Section: Scaling Up Of the Silk Post-purification Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Endotoxin reduction from biotech silk material inhibits the production of anti‐silk antibodies in mice

Deptuch,

Kucharczyk,

Florczak

et al. 2023

J Biomedical Materials Res

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“…Another approach is to express recombinant proteins in the chloroplast after the integration of the recombinant gene in the chloroplast genome. However, in general, using an ordinary binary vector such as pCAMBIA1300, the recombinant protein band were not detectable by the CBB staining ( Khan et al., 2022 ). Thus, our recombinant gene, BGC : Exdn-4 , used for the production of Exdn-4 is considered to be expressed at a high level.…”

Section: Discussionmentioning

confidence: 99%

Production of active Exendin-4 in Nicotiana benthamiana and its application in treatment of type-2 diabetics

Akter

Afrin²,

Kim³

et al. 2022

Front. Plant Sci.

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GLP-1 (Glucagon-like peptide-1) is a peptide that stimulates insulin secretion from the β-cell for glycemic control of the plasma blood glucose level. Its mimetic exenatide (synthetic Exendin-4) with a longer half-life of approximately 3.3–4 h is widely used in clinical application to treat diabetes. Currently, exenatide is chemically synthesized. In this study, we report that the GLP-1 analogue recombinant Exendin-4 (Exdn-4) can be produced at a high level in Nicotiana benthamiana, with an estimated yield of 50.0 µg/g fresh biomass. For high-level expression, we generated a recombinant gene, B:GB1:ddCBD1m:8xHis : Exendin-4 (BGC : Exdn-4), for the production of Exendin-4 using various domains such as the BiP signal peptide, the GB1 domain (B1 domain of streptococcal G protein), a double cellulose binding domain 1 (CBD1), and 8 His residues (8xHis) to the N-terminus of Exendin-4. GB1 was used to increase the expression, whereas double CBD1 and 8xHis were included as affinity tags for easy purification using MCC beads and Ni2+-NTA resin, respectively. BGC : Exdn-4 was purified by single-step purification to near homogeneity using both Ni2+-NTA resin and microcrystalline cellulose (MCC) beads. Moreover, Exdn-4 without any extra residues was produced from BGC : Exdn-4 bound onto MCC beads by treating with enterokinase. Plant-produced Exdn-4 (Exendin-4) was as effective as chemically synthesized Exendin-4 in glucose-induced insulin secretion (GIIS) from mouse MIN6m9 cells a pancreatic beta cell line.

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Assembly of Human Papillomavirus 16 L1 Protein in Nicotiana benthamiana Chloroplasts into Highly Immunogenic Virus-Like Particles

2023

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Plant produced endotoxin binding recombinant proteins effectively remove endotoxins from protein samples

Cited by 4 publications

References 44 publications

Endotoxin reduction from biotech silk material inhibits the production of anti‐silk antibodies in mice

Endotoxin reduction from biotech silk material inhibits the production of anti‐silk antibodies in mice

Production of active Exendin-4 in Nicotiana benthamiana and its application in treatment of type-2 diabetics

Assembly of Human Papillomavirus 16 L1 Protein in Nicotiana benthamiana Chloroplasts into Highly Immunogenic Virus-Like Particles

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