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Expression optimization, purification and in vitro characterization of human epidermal growth factor produced in Nicotiana benthamiana.

ABSTRACT: Human epidermal growth factor (hEGF) has gained clinical importance due to its ability to promote wound healing. Due to its commercial applications and high market demand, recombinant EGF has been produced in several forms. Currently, plant expression system is considered as potential alternative for low-cost recombinant protein production. Hence, this study focused on improving the production of hEGF in plants by effective gene construct design and optimizing the Agrobacterium culture conditions for high protein production. In this context, hEGF gene was cloned into plant geminiviral expression vector pBYR2e and transformed in to N. benthamiana leaves via., agroinfiltration. The recombinant hEGF was purified from the plant crude extracts by single-step affinity chromatography. Furthermore, the plant-produced hEGF has shown to promote cell migration comparable to commercial hEGF in HaCaT cells in vitro. These results indicated the potential of plant expression system for the production of recombinant hEGF for tissue engineering applications.

SUBMITTER: Hanittinan O

PROVIDER: S-EPMC7486445 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Expression optimization, purification and in vitro characterization of human epidermal growth factor produced in Nicotiana benthamiana.

Hanittinan Oranicha O Oo Yamin Y Chaotham Chatchai C Rattanapisit Kaewta K Shanmugaraj Balamurugan B Phoolcharoen Waranyoo W

Biotechnology reports (Amsterdam, Netherlands) 20200905

Human epidermal growth factor (hEGF) has gained clinical importance due to its ability to promote wound healing. Due to its commercial applications and high market demand, recombinant EGF has been produced in several forms. Currently, plant expression system is considered as potential alternative for low-cost recombinant protein production. Hence, this study focused on improving the production of hEGF in plants by effective gene construct design and optimizing the Agrobacterium culture co ...[more]

PMID: 32953470

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Project description:The sesquiterpene β-caryophyllene is an ubiquitous component in many plants that has commercially been used as an aroma in cosmetics and perfumes. Recent studies have shown its potential use as a therapeutic agent and biofuel. Currently, β-caryophyllene is isolated from large amounts of plant material. Molecular farming based on the Nicotiana benthamiana transient expression system may be used for a more sustainable production of β-caryophyllene. In this study, a full-length cDNA of a new duplicated β-caryophyllene synthase from Artemisia annua (AaCPS1) was isolated and functionally characterized. In order to produce β-caryophyllene in vitro, the AaCPS1 was cloned into a plant viral-based vector pEAQ-HT. Subsequently, the plasmid was transferred into the Agrobacterium and agroinfiltrated into N. benthamiana leaves. The AaCPS1 expression was analyzed by quantitative PCR at different time points after agroinfiltration. The highest level of transcripts was observed at 9 days post infiltration (dpi). The AaCPS1 protein was extracted from the leaves at 9 dpi and purified by cobalt-nitrilotriacetate (Co-NTA) affinity chromatography using histidine tag with a yield of 89 mg kg-1 fresh weight of leaves. The protein expression of AaCPS1 was also confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analyses. AaCPS1 protein uses farnesyl diphosphate (FPP) as a substrate to produce β-caryophyllene. Product identification and determination of the activity of purified AaCPS1 were done by gas chromatography-mass spectrometry (GC-MS). GC-MS results revealed that the AaCPS1 produced maximum 26.5 ± 1 mg of β-caryophyllene per kilogram fresh weight of leaves after assaying with FPP for 6 h. Using AaCPS1 as a proof of concept, we demonstrate that N. benthamiana can be considered as an expression system for production of plant proteins that catalyze the formation of valuable chemicals for industrial applications.

Project description:α1-Microglobulin (A1M) is a small glycoprotein that belongs to the lipocalin protein family. A major biological role of A1M is to protect cells and tissues against oxidative damage by clearing free heme and reactive oxygen species. Because of this, the protein has attracted great interest as a potential pharmaceutical candidate for treatment of acute kidney injury and preeclampsia. The aim of this study was to explore the possibility of expressing human A1M in plants through transient gene expression, as an alternative or complement to other expression systems. E. coli, insect and mammalian cell culture have previously been used for recombinant A1M (rA1M) or A1M production, but these systems have various drawbacks, including additional complication and expense in refolding for E. coli, while insect produced rA1M is heavily modified with chromophores and mammalian cell culture has been used only in analytical scale. For that purpose, we have used a viral vector (pJL-TRBO) delivered by Agrobacterium for expression of three modified A1M gene variants in the leaves of N. benthamiana. The results showed that these modified rA1M protein variants, A1M-NB1, A1M-NB2 and A1M-NB3, targeted to the cytosol, ER and extracellular space, respectively, were successfully expressed in the leaves, which was confirmed by SDS-PAGE and Western blot analysis. The cytosol accumulated A1M-NB1 was selected for further analysis, as it appeared to have a higher yield than the other variants, and was purified with a yield of ca. 50 mg/kg leaf. The purified protein had the expected structural and functional properties, displaying heme-binding capacity and capacity of protecting red blood cells against stress-induced cell death. The protein also carried bound chromophores, a characteristic feature of A1M and an indicator of a capacity to bind small molecules. The study showed that expression of the functional protein in N. benthamiana may be an attractive alternative for production of rA1M for pharmaceutical purposes and a basis for future research on A1M structure and function.

Project description:BackgroundPlant growth-promoting rhizobacteria (PGPR) release volatile organic compounds (VOCs), which promote plant growth.ResultsA potential PGPR strain GX14001 was isolated from marine samples, and the VOCs produced by GX14001 significantly promoted tobacco (Nicotiana benthamiana) growth in a plate experiment. Based on 16S rRNA sequence alignment and physiological and biochemical characterization, GX14001 was identified as Microbacterium aurantiacum. Comparative transcriptome analysis was conducted between GX14001 VOCs-treated tobacco and the control; it was found that 1286 genes were upregulated and 1088 genes were downregulated. Gene ontology (GO) analysis showed that upregulated genes were involved in three biological processes: polysaccharide metabolic, polysaccharide catabolic and carbohydrate metabolic. The downregulated genes were involved in six biological processes, namely cell redox homeostasis, cellular homeostasis, carbohydrate metabolic process, homeostatic process, obsolete electron transport, and regulation of biological quality. Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis showed that 190 upregulated differentially expressed genes were mainly involved in plant hormone signal transduction, phenylpropyl biosynthesis, plant-pathogen interaction, and flavonoid biosynthesis. The 148 downregulated differentially expressed genes were mainly involved in plant hormone signal transduction and the metabolism of ascorbic, aldehyde, and pyruvate acids. Further analysis revealed that many genes were differentially expressed in the metabolic pathways of plant hormone signals, which were speculated to be the main reason why GX14001 VOCs promoted tobacco growth. To further study its regulatory mechanism, we found that GX14001 promoted plant growth through auxin, salicylic acid, and gibberellin in Arabidopsis mutant experiments.ConclusionThe VOCs produced by Microbacterium aurantiacum GX14001 may promote the growth of tobacco through the auxin, salicylic acid and gibberellin pathways.

Dataset Information

Expression optimization, purification and in vitro characterization of human epidermal growth factor produced in Nicotiana benthamiana.

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Expression optimization, purification and <i>in vitro</i> characterization of human epidermal growth factor produced in <i>Nicotiana benthamiana</i>.

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