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It is increasingly evident that seed proteins exhibit specific functions in plant physiology. However, many proteins remain yet to be functionally characterized. We have screened the seed proteome of Dolichos which lead to identification and purification of a protein, DC25. The protein was monomeric and highly thermostable in extreme conditions of pH and salt. It was crystallized and structure determined at 1.28 Å resolution using x-ray crystallography. The high-resolution structure of the protein revealed a four-bladed β-propeller hemopexin-type fold containing pseudo four-fold molecular symmetry at the central channel. While the structure exhibited homology with 2S albumins, variations in the loops connecting the outermost strands and the differences in surface-charge distribution may be relevant for distinct functions. Comparative study of the protein with other seed hemopexins revealed the presence of four conserved water molecules in between the blades which cross-link them and maintain the tertiary structure. The protein exhibited intrinsic peroxidase activity, which could be inhibited by binding of a heme analog. The identification of redox-sensitive cysteine and inhibition of peroxidase activity by iodoacetamide facilitated characterization of the possible active site. The determined peroxidase activity of DC25 may be responsible for rescuing germinating seeds from oxidative stress.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.An amendment to this paper has been published and can be accessed via a link at the top of the paper.

BackgroundCytokines and growth factors are critical reagents used in cell therapy and regenerative medicine, yet their biological activity and consistency can vary significantly based on the expression system used. HEK293-expressed growth factors offer advantages in terms of authentic post-translational modifications and improved bioactivity.MethodsIn this study, we performed various cell-based assays to compare the bioactivity, cell expansion and cell differentiation potential of cytokines derived from HEK293 and E. coli.ResultsWe highlight the significant improvement in bioactivity and performance of HEK293-derived growth factors further leading to enhancing cell expansion and differentiation. Human Betacellulin (BTC) protein expressed in HEK293 shows 100 times more bioactivity than E. coli derived BTC. Brain-derived neurotrophic factor (BDNF) expressed in HEK293 cells demonstrated enhanced efficacy in promoting neuronal differentiation of SHSY5Y cells compared to E. coli derived. Similarly, HEK293-expressed interleukin-34 (IL-34) significantly improved the generation and maturation of macrophages. Furthermore, the combination of HEK293-derived IL-2, IL-7, and IL-15 resulted in robust expansion of T cells with a favorable phenotype for immunotherapeutic applications.ConclusionsThese findings not only support the use of HEK293-expressed growth factors as biologically superior reagents but also a way to reduce cost of cell expansion and differentiation workflows.