The effects of denaturation of amaranth globulins (A-G) by urea, guanidine hydrochloride (GuHCl), and sodium dodecyl sulfate (SDS) were studied. Progressive unfolding of globulins was measured as a function of fluorescent light intensity, peak response, and shift in the maximum emission wavelength. Decreases in fluorescence intensity and shift in maximum emission wavelength (from 341.5 to 355.0 nm) were shown using the fluorescence of tryptophan at 295 nm. Kinetic measurements showed that the decrease of fluorescence intensity was faster with SDS than with GuHCl and exhibited a double exponential decay pattern. Differences in secondary and tertiary structures during denaturation were observed, and changes in R-helical content and position of aromatic groups were calculated. The A-G were compared with quinoa globulins (Q-G). Comparison of relative structural stability of native globulins by intrinsic fluorescence and circular dichroism measurements showed that A-G are relatively stable in comparison with Q-G and bovine γ-globulins.
Amaranth seed proteins were sequentially extracted and partially characterized by electrophoresis and immunological analysis using three polyclonal antibodies which were produced against an acidic subfraction of amaranth globulin, an oat globulin, and an acidic subunit of rice glutelin. Significant cross‐reactivity was observed among glutelin and globulin fractions with these antibodies. Prolamin fraction did not react. Data suggest that there is some structural homology between amaranth glutelin and globulin fractions and that they could be legumin‐like proteins because of their affinity with protein antibodies; such antibodies have been produced from legumin‐like proteins. Two‐dimensional electrophoretical analysis of a 32 kDa glutelin band clearly showed six acid components from pI 5.7 to 6.3; four of them cross‐reacted with the acidic subfraction of amaranth globulin antibody. These results confirm that there is homology between these acidic components (32 kDa) of amaranth glutelin and the amaranth acidic sub‐fraction of globulin.
The purpose of this study was to purify, crystallize, and characterize by X-ray diffraction an amaranth globulin for its subsequent structure elucidation. A 36-kDa amaranth globulin was extracted by sequential precipitation and purified by gel filtration and cationic exchange columns. It was crystallized at 18 degrees C from 4 M sodium formate. Suitable crystals for X-ray analysis were found to belong to the tetragonal crystal system with cell dimensions of a = b = 195.5 A and c = 164.14 A. Two possible tetragonal space groups P4(1)2(1)2 or P4(3)2(1)2 were determined. The crystals diffracted up to 2.5 A.
The use of antimicrobial peptides (AMPs) synthesized by bacteria (bacteriocins) is an alternative for combating multidrug resistant bacterial strains and their production by recombinant route is a viable option for their mass production. The bacteriocin E-760 isolated from the genus Enterococcus sp. has been shown to possess inhibitory activity against Gram-negative and Gram-positive bacteria. In this study, the expression of a chimeric protein coding for E-760 in the nucleus of C. reinhardtii was evaluated, as well as, its antibacterial activity. The synthetic gene E-760S was inserted into the genome of C. reinhardtii using Agrobacterium tumefaciens. A transgenic line was identified in TAP medium with hygromycin and also by PCR. The increment in the culture medium temperature of the transgenic strain at 35 °C for 10 minutes, increased the production level of the recombinant protein from 0.14 (Noninduced culture, NIC) to 0.36% (Induced culture, IC) of total soluble proteins (TSP); this was quantified by an ELISA assay. Recombinant E-760 possesses activity against Staphylococcus aureus in 0.34 U log, Streptococcus agalactiae in 0.48 U log, Enterococcus faecium in 0.36 U log, Pseudomonas aeruginosa in 2 U log and for Klebsiella pneumoniae, the activity was 0.07 U log. These results demonstrate that the nucleus transformation of C. reinhardtii can function as a stable expression platform for the production of the synthetic gene E-760 and it can potentially be used as an antibacterial agent.
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