Production of functional human fetal hemoglobin in Nicotiana benthamiana for development of hemoglobin-based oxygen carriers

Kanagarajan, Selvaraju; Carlsson, Magnus; Chakane, Sandeep; Kettisen, Karin; Smeds, Emanuel; Kumar, Ranjeet; Ortenlöf, Niklas; Gram, Magnus; Åkerström, Bo; Bülow, Leif; Zhu, Lu

doi:10.1016/j.ijbiomac.2021.06.102

Cited by 3 publications

(1 citation statement)

References 61 publications

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“…To further address the underlying mechanism of how T57C mutation leads to thermostability increase in clMagR, far-UV CD spectroscopy (190–260 nm) was applied to follow the unfolding and folding of proteins as a function of temperature from 25 to 95°C at 1°C intervals ( Figures 5C,D ) ( Kanagarajan et al, 2021 ; Wensien et al, 2021 ). The clMagR T57C mutant showed improved thermostability compared with clMagR WT in the temperature range we recorded ( Figures 5C,D ).…”

Section: Resultsmentioning

confidence: 99%

The rational design of iron-sulfur cluster binding site for prolonged stability in magnetoreceptor MagR

Tong

Zhou²,

Fei³

et al. 2022

Front. Mol. Biosci.

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Iron-sulfur proteins play essential roles in a wide variety of cellular processes such as respiration, photosynthesis, nitrogen fixation and magnetoreception. The stability of iron-sulfur clusters varies significantly between anaerobic and aerobic conditions due to their intrinsic sensitivity to oxygen. Iron-sulfur proteins are well suited to various practical applications as molecular redox sensors or molecular “wires” for electron transfer. Various technologies have been developed recently using one particular iron-sulfur protein, MagR, as a magnetic tag. However, the limited protein stability and low magnetic sensitivity of MagR hindered its wide application. Here in this study, the iron-sulfur binding site of pigeon clMagR was rationally re-designed. One such mutation, T57C in pigeon MagR, showed improved iron-sulfur binding efficiency and higher iron content, as well as prolonged thermostability. Thus, clMagRT57C can serve as a prototype for further design of more stable and sensitive magnetic toolbox for magnetogenetics in the future.

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Section: Resultsmentioning

confidence: 99%

The rational design of iron-sulfur cluster binding site for prolonged stability in magnetoreceptor MagR

Tong

Zhou²,

Fei³

et al. 2022

Front. Mol. Biosci.

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Iron loss and hypoxia are involved in lethal Pseudomonas plecoglossicida infections in the orange-spotted grouper Epinephelus coioides

Sun

Weng

et al. 2023

Aquaculture

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Heterologous Production of Cyprosin B inNicotiana benthamiana: Unveiling the Role of the Plant-Specific Insert Domain in Protein Function and Subcellular Localization

Muthusamy,

Vetukuri,

Lundgren

et al. 2024

Preprint

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The aqueous extract of Cynara cardunculus flowers is traditionally used in cheese production across Mediterranean countries. To meet the growing industrial demand for plant-based milk-clotting enzymes and to explore potential biotechnological applications, we initiated a study to heterologously produce cyprosin B (CYPB), a key milk-clotting enzyme from C. cardunculus, in Nicotiana benthamiana. We also investigated the role of its plant-specific insert (PSI) domain in the CYPBs activity and its localization. In this study, full-length CYPB and a PSI domain deleted CYPB (CYPBΔPSI) were transiently expressed in N. benthamiana leaves using Agrobacterium-mediated infiltration. The leaves were harvested nine days post-infiltration, and proteins were purified, yielding approximately 81 mg/kg (CYPB) and 60 mg/kg (CYPBΔPSI) fresh weight. CYPBΔPSI showed significantly higher proteolytic activity (156.72 IU/mg) than CYPB (57.2 IU/mg), indicating that the PSI domain is not essential for enzymatic activity and that its removal results in enhanced enzymatic efficiency. In the milk-clotting activity assay, CYPBΔPSI demonstrated a significantly faster clotting time than full-length CYPB, indicating enhanced milk-clotting efficiency for CYPBΔPSI. Subcellular localization studies revealed that CYPB and PSI were localized in the vacuole and endocytic vesicles. In contrast, CYPBΔPSI was primarily localized in the endoplasmic reticulum (ER) and the tonoplast, suggesting that the PSI domain is critical for vacuolar targeting and membrane permeabilization that affects overall protein yield. This study demonstrates the feasibility of using N. benthamiana as a platform for the scalable production of more efficient recombinant CYPB. It highlights the multifunctional role of the PSI domain in vacuolar sorting without impairing its functionality. These results underscore the potential of plant-based expression systems as a viable alternative for the industrial production of plant milk-clotting enzymes, with significant implications for sustainable cheese production.

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Production of functional human fetal hemoglobin in Nicotiana benthamiana for development of hemoglobin-based oxygen carriers

Cited by 3 publications

References 61 publications

The rational design of iron-sulfur cluster binding site for prolonged stability in magnetoreceptor MagR

The rational design of iron-sulfur cluster binding site for prolonged stability in magnetoreceptor MagR

Iron loss and hypoxia are involved in lethal Pseudomonas plecoglossicida infections in the orange-spotted grouper Epinephelus coioides

Heterologous Production of Cyprosin B inNicotiana benthamiana: Unveiling the Role of the Plant-Specific Insert Domain in Protein Function and Subcellular Localization

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