Recently, a durum wheat (Triticum durum Desf.) secretory phospholipase A2 (TdsPLA2III) was identified in leaves as potentially involved in plant responses to conditions of limiting water supply. Therefore, to allow future functional studies on TdsPLA2III and shed further light on the involvement of sPLA2 isoforms in specific plant functions, here we report a protocol for the overexpression of TdsPLA2III in Escherichia coli in the form of inclusion bodies, and for its purification and refolding. The use of the Gateway system (Invitrogen) allows the expression of a large quantity of the mature form (without the signal peptide) of TdsPLA2III with an N-terminal 6×His-tag, for purification using Ni-affinity chromatography. The purified recombinant 6×His-TdsPLA2III fusion protein is then refolded using a step-wise dialysis approach. About 40mg purified and active protein was obtained from 1L of cell culture. This recombinant 6×His-TdsPLA2III protein shows PLA2 activity, as it can hydrolyze linoleate from the sn-2 position of 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine. Moreover, it has some features that are typical of other known plant sPLA2s: Ca(2+)-dependence, inhibition by the disulfide bond reducing agent dithiothreitol, and resistance to high temperature.
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http://dx.doi.org/10.1016/j.pep.2014.05.009 | DOI Listing |
Int J Mol Sci
December 2024
Dompé Farmaceutici S.p.A., Via Campo di Pile, Nucleo Industriale Pile, 67100 L'Aquila, Italy.
Thus far, no manufacturing process able to support industrialization has been reported for the recombinant human brain-derived neurotrophic factor (rhBDNF). Here, we described the setup of a new protocol for its production in () and its purification to homogeneity. A synthetic gene, codifying for the neurotrophin precursor, was inserted into an expression vector and transformed into BL21 (DE3) strain.
View Article and Find Full Text PDFProtein Expr Purif
March 2025
Biochemical Sciences Division, CSIR-National Chemical Laboratory, Pune, 411008, India; Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India. Electronic address:
Insulin is a pivotal peptide hormone essential for regulating glucose homeostasis. It has been known for over 100 years, but its production and purification methods are still under improvement. Escherichia coli based bacterial expression system is primarily used for insulin production.
View Article and Find Full Text PDFJ Virol Methods
December 2024
Department of Biosciences and Bioengineering, Indian Institute of Technology, Roorkee, Uttarakhand, India. Electronic address:
Current research efforts are underway to create novel approaches for the efficient diagnosis, monitoring, and mitigation of Kyasanur Forest Disease Virus (KFDV) infections. Flavivirus subunit-based vaccines based on envelope glycoprotein EDIII are now in preclinical and clinical research stages. Efficient purification and isolation methods for surface immunogenic viral antigens, including the recombinant envelope immunoglobulin-like domain III (rEDIII) protein, are crucial for the production and manufacturing of promising vaccine candidates that have been extensively assessed in previous literature.
View Article and Find Full Text PDFBiochem Biophys Res Commun
December 2024
Laboratory of Structural Biology, Department of Biochemistry, School of Life Sciences, University of Hyderabad, Prof. CR Rao Road, Gachibowli, Hyderabad, Telangana, 500046, India. Electronic address:
Many pathogens establish a successful infection by evading the host complement system, an essential arm of innate immunity. Pathogenic Leptospira is reported to escape complement-mediated killing by recruiting the host complement regulators by lipoproteins or outer surface proteins. One of the outer surface proteins, Leptospiral complement regulator-acquiring protein A (LcpA), is known to recruit complement regulators, C4b-binding protein (C4BP), and Factor H (FH) on the bacterial surface.
View Article and Find Full Text PDFBiochem Biophys Res Commun
December 2024
Laboratory of Biochemistry, Faculty of Medicine, School of Health Sciences, University of Thessaly, Biopolis, 41500, Larissa, Greece. Electronic address:
Hypoxia inducible factor 2α (HIF-2α) is a member of the basic helix-loop-helix(bHLH)-Per-Arnt-Sim (PAS) family of transcription factors. It is overexpressed in several cancers, associated with poor prognosis of the patients and resistance to treatment. Here, we study the residues 366-704 of the C-terminal end of human HIF-2α, which contains the N-transcriptional activation domain (NTAD), the oxygen-dependent degradation domain (ODD), and a part of the inhibitory domain (IH).
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