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Insights on Structure and Function of a Late Embryogenesis Abundant Protein from Amaranthus cruentus : An Intrinsically Disordered Protein Involved in Protection against Desiccation, Oxidant Conditions, and Osmotic Stress

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ABSTRACT

Late embryogenesis abundant (LEA) proteins are part of a large protein family that protect other proteins from aggregation due to desiccation or osmotic stresses. Recently, the Amaranthus cruentus seed proteome was characterized by 2D-PAGE and one highly accumulated protein spot was identified as a LEA protein and was named AcLEA. In this work, AcLEA cDNA was cloned into an expression vector and the recombinant protein was purified and characterized. AcLEA encodes a 172 amino acid polypeptide with a predicted molecular mass of 18.34 kDa and estimated pI of 8.58. Phylogenetic analysis revealed that AcLEA is evolutionarily close to the LEA3 group. Structural characteristics were revealed by nuclear magnetic resonance and circular dichroism methods. We have shown that recombinant AcLEA is an intrinsically disordered protein in solution even at high salinity and osmotic pressures, but it has a strong tendency to take a secondary structure, mainly folded as α-helix, when an inductive additive is present. Recombinant AcLEA function was evaluated using Escherichia coli as in vivo model showing the important protection role against desiccation, oxidant conditions, and osmotic stress. AcLEA recombinant protein was localized in cytoplasm of Nicotiana benthamiana protoplasts and orthologs were detected in seeds of wild and domesticated amaranth species. Interestingly AcLEA was detected in leaves, stems, and roots but only in plants subjected to salt stress. This fact could indicate the important role of AcLEA protection during plant stress in all amaranth species studied.

No MeSH data available.


(A) Analysis of His-rAcLEA expression and purification. Lane M = molecular weight marker, Lane 1 = total proteins from non-induced BL21 cells, Lane 2 = total proteins from BL21 cells after induction with IPTG, Lane 3 = soluble proteins from Lane 2, Lane 4 = fraction not retained from Ni2+-column, Lane 5 = fraction retained in Ni2+-column and eluted with 300 mM imidazole. (B) His-tag cleavage with PreScission Protease (PSP), Lane M = molecular weight marker, Lane 1 = His-Tag rAcLEA, Lane 2 = rAcLEA.
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Figure 2: (A) Analysis of His-rAcLEA expression and purification. Lane M = molecular weight marker, Lane 1 = total proteins from non-induced BL21 cells, Lane 2 = total proteins from BL21 cells after induction with IPTG, Lane 3 = soluble proteins from Lane 2, Lane 4 = fraction not retained from Ni2+-column, Lane 5 = fraction retained in Ni2+-column and eluted with 300 mM imidazole. (B) His-tag cleavage with PreScission Protease (PSP), Lane M = molecular weight marker, Lane 1 = His-Tag rAcLEA, Lane 2 = rAcLEA.

Mentions: Two distinctive bands putatively corresponding to the recombinant AcLEA were detected in SDS-PAGE, one of them was located at 21.4 kDa, correlating with the molecular weight expected for recombinant protein linked to His-tag, the second band was located at 16.0 kDa (Figure 2). The identities of these two bands were successfully identified by LC-MS/MS and bioinformatics analysis using A. hypochondriacus database (Supplementary Figure S3). Sequences of the matched peptides as well MASCOT scores are shown in Table 1. Data confirm that both the 21.4 and 16.0 kDa bands corresponded to AcLEA. Nevertheless peptides in the N-terminal region were not detected in the 16.0 kDa product, indicating that this short protein is a truncated fragment lacking the N-terminal region.


Insights on Structure and Function of a Late Embryogenesis Abundant Protein from Amaranthus cruentus : An Intrinsically Disordered Protein Involved in Protection against Desiccation, Oxidant Conditions, and Osmotic Stress
(A) Analysis of His-rAcLEA expression and purification. Lane M = molecular weight marker, Lane 1 = total proteins from non-induced BL21 cells, Lane 2 = total proteins from BL21 cells after induction with IPTG, Lane 3 = soluble proteins from Lane 2, Lane 4 = fraction not retained from Ni2+-column, Lane 5 = fraction retained in Ni2+-column and eluted with 300 mM imidazole. (B) His-tag cleavage with PreScission Protease (PSP), Lane M = molecular weight marker, Lane 1 = His-Tag rAcLEA, Lane 2 = rAcLEA.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5384071&req=5

Figure 2: (A) Analysis of His-rAcLEA expression and purification. Lane M = molecular weight marker, Lane 1 = total proteins from non-induced BL21 cells, Lane 2 = total proteins from BL21 cells after induction with IPTG, Lane 3 = soluble proteins from Lane 2, Lane 4 = fraction not retained from Ni2+-column, Lane 5 = fraction retained in Ni2+-column and eluted with 300 mM imidazole. (B) His-tag cleavage with PreScission Protease (PSP), Lane M = molecular weight marker, Lane 1 = His-Tag rAcLEA, Lane 2 = rAcLEA.
Mentions: Two distinctive bands putatively corresponding to the recombinant AcLEA were detected in SDS-PAGE, one of them was located at 21.4 kDa, correlating with the molecular weight expected for recombinant protein linked to His-tag, the second band was located at 16.0 kDa (Figure 2). The identities of these two bands were successfully identified by LC-MS/MS and bioinformatics analysis using A. hypochondriacus database (Supplementary Figure S3). Sequences of the matched peptides as well MASCOT scores are shown in Table 1. Data confirm that both the 21.4 and 16.0 kDa bands corresponded to AcLEA. Nevertheless peptides in the N-terminal region were not detected in the 16.0 kDa product, indicating that this short protein is a truncated fragment lacking the N-terminal region.

View Article: PubMed Central - PubMed

ABSTRACT

Late embryogenesis abundant (LEA) proteins are part of a large protein family that protect other proteins from aggregation due to desiccation or osmotic stresses. Recently, the Amaranthus cruentus seed proteome was characterized by 2D-PAGE and one highly accumulated protein spot was identified as a LEA protein and was named AcLEA. In this work, AcLEA cDNA was cloned into an expression vector and the recombinant protein was purified and characterized. AcLEA encodes a 172 amino acid polypeptide with a predicted molecular mass of 18.34 kDa and estimated pI of 8.58. Phylogenetic analysis revealed that AcLEA is evolutionarily close to the LEA3 group. Structural characteristics were revealed by nuclear magnetic resonance and circular dichroism methods. We have shown that recombinant AcLEA is an intrinsically disordered protein in solution even at high salinity and osmotic pressures, but it has a strong tendency to take a secondary structure, mainly folded as α-helix, when an inductive additive is present. Recombinant AcLEA function was evaluated using Escherichia coli as in vivo model showing the important protection role against desiccation, oxidant conditions, and osmotic stress. AcLEA recombinant protein was localized in cytoplasm of Nicotiana benthamiana protoplasts and orthologs were detected in seeds of wild and domesticated amaranth species. Interestingly AcLEA was detected in leaves, stems, and roots but only in plants subjected to salt stress. This fact could indicate the important role of AcLEA protection during plant stress in all amaranth species studied.

No MeSH data available.