A toolkit for recombinant production of seven human EGF family growth factors in active conformation

Ferreira, Arthur Schveitzer; Lopacinski, Amanda; Batista, Michel; Hiraiwa, Priscila Mazzocchi; Guimarães, B.G.; Zanchin, Nilson Ivo Tonin

doi:10.1038/s41598-022-09060-9

Cited by 3 publications

(4 citation statements)

References 53 publications

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“…2E), indicating that the hNRG1α EGF domain acquires the same conformation after thermal denaturation. That result is similar to the CD spectra found for hAREG hBTC, HB-EGF and hTGFα [20] in similar conditions.…”

Section: Stability and Conformational Analysis Of The Recombinant Hnr...supporting

confidence: 89%

“…However, it seems that not all EGF domains aggregate after disulfide bond reduction. In an analysis of the hydrodynamic radius of seven different EGF domains reported previously [20], a similar behavior was observed for the recombinant EGF domain of hAREG and hHBEGF and hTGFa show only partial aggregation even after long incubation times (24 and 44 h) under reduction conditions, indicating that different EGF domains may present different solubility behavior under reducing conditions [20].…”

Section: Discussionsupporting

confidence: 71%

“…A synthetic gene encoding this 67-residue segment was acquired from GenScript (Piscataway, NJ, USA) cloned between the NcoI and XhoI restriction sites of plasmid pET32a (Novagen/Merck, Darmstadt, Germany). The expression plasmids for EGF and TGFα in fusion with TrxA (pET32a-EGF and pET32a-TGFα) were described in a previous study from our group [20]. In pET32a, the EGF domain of these growth factors are expressed in fusion with thioredoxin and a hexahistidine tag.…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

“…However, it is possible to overcome this limitation by using fusion proteins, which can function as folding and solubility chaperones or modified E. coli strains designed to favor disulfide bond formation. Indeed, several studies described the fusion of EGF domains with different expression tags, such as SUMO for hHB-EGF [16], oleosin for hEGF [17], glutathione S-transferase, B1 domain of streptococcal protein G and protein disulfide isomerase for hEGF [18] and, thioredoxin (Trx) for hEPR [19] and for seven members of the EGF family of growth factors [20]. In addition to the use of fusion proteins to improve solubility, it is also important to demonstrate that the recombinant proteins intended for biological applications display correct structural conformation and do not form aggregates.…”

Section: Introductionmentioning

confidence: 99%

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Functional EGF domain of the human neuregulin 1α produced in Escherichia coli with accurate disulfide bonds

et al. 2022

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Background Neuregulins comprise a large family of growth factors containing an epidermal growth factor (EGF) domain. NRG1 acts in signaling pathways involved in proliferation, apoptosis, migration, differentiation, and adhesion of many normal cell types and in human diseases. The EGF domain of NRG1 mediates signaling by interaction with members of the ErbB family of receptors. Easy access to correctly folded hNRG1α EGF domain can be a valuable tool to investigate its function in different cell types. Materials and methodsThe EGF domain of hNRG1α was produced in Escherichia coli in fusion with TrxA and purified after cleavage of TrxA. Conformation and stability analyses were performed by using biophysical methods and the disulfide bonds were mapped by mass spectrometry. The activity of the hNRG1α EGF domain was demonstrated in cell proliferation and migration assays. Results Approximately 3.3 mg of hNRG1α EGF domain were obtained starting from a 0.5 L of E. coli culture. Correct formation of the three disulfide bonds was demonstrated by mass spectrometry with high accuracy. Heat denaturation assays monitored by circular dichroism and dynamic light scattering revealed that it is a highly stable protein. The recombinant EGF domain of hNRG1α purified in this work is highly active, inducing cell proliferation at concentration as low as 0.05 ng/mL. It induces also cell migration as demonstrated by a gap closure assay. ConclusionThe EGF domain of hNRG1α was produced in E. coli with the correct disulfide bonds and presented high stimulation of HeLa cell proliferation and NDFH cell migration. KeywordsHuman Neuregulin-1 • Recombinant EGF domain • Disulfide bond assignment • hNRG1α EGF domain activity * Nilson I. T. Zanchin

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Section: Stability and Conformational Analysis Of The Recombinant Hnr...supporting

confidence: 89%

Section: Discussionsupporting

confidence: 71%

Section: Protein Expression and Purificationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Functional EGF domain of the human neuregulin 1α produced in Escherichia coli with accurate disulfide bonds

et al. 2022

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The systemic-level repercussions of cancer-associated inflammation mediators produced in the tumor microenvironment

et al. 2022

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The tumor microenvironment is a dynamic, complex, and redundant network of interactions between tumor, immune, and stromal cells. In this intricate environment, cells communicate through membrane–membrane, ligand–receptor, exosome, soluble factors, and transporter interactions that govern cell fate. These interactions activate the diverse and superfluous signaling pathways involved in tumor promotion and progression and induce subtle changes in the functional activity of infiltrating immune cells.The immune response participates as a selective pressure in tumor development. In the early stages of tumor development, the immune response exerts anti-tumor activity, whereas during the advanced stages, the tumor establishes mechanisms to evade the immune response, eliciting a chronic inflammation process that shows a pro-tumor effect.The deregulated inflammatory state, in addition to acting locally, also triggers systemic inflammation that has repercussions in various organs and tissues that are distant from the tumor site, causing the emergence of various symptoms designated as paraneoplastic syndromes, which compromise the response to treatment, quality of life, and survival of cancer patients. Considering the tumor–host relationship as an integral and dynamic biological system, the chronic inflammation generated by the tumor is a communication mechanism among tissues and organs that is primarily orchestrated through different signals, such as cytokines, chemokines, growth factors, and exosomes, to provide the tumor with energetic components that allow it to continue proliferating. In this review, we aim to provide a succinct overview of the involvement of cancer-related inflammation at the local and systemic level throughout tumor development and the emergence of some paraneoplastic syndromes and their main clinical manifestations. In addition, the involvement of these signals throughout tumor development will be discussed based on the physiological/biological activities of innate and adaptive immune cells. These cellular interactions require a metabolic reprogramming program for the full activation of the various cells; thus, these requirements and the by-products released into the microenvironment will be considered. In addition, the systemic impact of cancer-related proinflammatory cytokines on the liver—as a critical organ that produces the leading inflammatory markers described to date—will be summarized. Finally, the contribution of cancer-related inflammation to the development of two paraneoplastic syndromes, myelopoiesis and cachexia, will be discussed.

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Production of recombinant human epidermal growth factor fused with HaloTag protein and characterisation of its biological functions

Bai,

Liu,

Liu

et al. 2024

PeerJ

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Epidermal growth factor (EGF) protein is a crucial biomolecule involved in regulating cell growth, proliferation, migration and differentiation, which is used in various therapeutic applications, such as wound healing and tissue regeneration. The production of recombinant EGF is essential for studying its biological function and for its clinical translation. However, EGF protein expressed in prokaryotic cells often occurs in inclusion bodies, and co-expression with soluble tag protein is an effective method to prepare recombinant EGF. In this study, we expressed recombinant human EGF (rhEGF) fused to a HaloTag (Halo-rhEGF) and a large portion of Halo-rhEGF was found in the soluble fraction. Cell growth assay showed that the purified Halo-rhEGF protein could promote the proliferation of fibroblasts (NIH 3T3) and epithelial cells (HaCaT), and significantly increased their viability. Phosphorylation of the intracellular signaling proteins, ERK1/2 and c-Jun, was stimulated by treatment with Halo-rhEGF and the expression levels of proteins regulating cell proliferation were significantly increased. RNA sequencing analysis revealed that rhEGF could increase the transcription of genes enriched in ribosome generation and cell proliferation. Moreover, Halo-rhEGF can be labelled by HaloTag ligand for fluorescence imaging and can be slowly released in tissue repair by binding to anion biomaterials. In conclusion, HaloTag is an efficient fusion tag for rhEGF protein expression, purification and controlled release, and Halo-rhEGF can promote the proliferation and viability of epithelial and fibroblast cells.

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A toolkit for recombinant production of seven human EGF family growth factors in active conformation

Cited by 3 publications

References 53 publications

Functional EGF domain of the human neuregulin 1α produced in Escherichia coli with accurate disulfide bonds

Functional EGF domain of the human neuregulin 1α produced in Escherichia coli with accurate disulfide bonds

The systemic-level repercussions of cancer-associated inflammation mediators produced in the tumor microenvironment

Production of recombinant human epidermal growth factor fused with HaloTag protein and characterisation of its biological functions

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