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Title: Purification, characterization and proteomics analysis of important plant proteins from family Brassicaceae
Authors: Khaliq, Benish
Keywords: Botany
Issue Date: 2018
Publisher: Bahauddin Zakariya University Multan
Abstract: Brassicaceae or Cruciferae is one of the most wide spread economically important plant family comprising of many weed vegetables, condiments and oilseed crops. Pakistan is growing oilseed Brassicaceae species over an area of 272,100 ha with an oil production of 232,000 tons per year. Additionally, very strong protein content (3840%) is also present in different genera of this family. Brassicaceae species have a notable economic value due to their nutritionally and medicinally important seed proteins in addition to their oil content. Initially 18 different species from Brassicaceae family were screened in search of important seed proteins. Among the commercially cultivated Brassicaceae species, Brassica nigra, Eruca sativa and Sisymbrium irio have significant amounts of proteins in their seeds. Cruciferins (11/12S globulins) and Napins (2S) are the predominant storage proteins while other proteins like Ubiquitins and Thionins are also important proteins of Brassicaceae seeds that contribute to different properties and functions in comparison to storage proteins. Brassica nigra Cruciferin (BnC): Cruciferins (12S globulins) are seed storage proteins, which are getting attention due to their allergenic and pathogenicity related nature. This study describes the purification and characterization of a trimeric (~ 190 kDa) Cruciferin protein from the seeds of Brassica nigra (L.). Cruciferin was first partially purified by ammonium sulfate precipitation (30 % saturation constant) and further purified by size exclusion chromatography. The N-terminal amino-acid sequence analysis showed 82% sequence homology with Cruciferin from Arabidopsis thaliana. The 50-55 kDa monomeric Cruciferin produced multiple bands of two major molecular weight ranges (α-polypeptides of 28-32 kDa and β-polypeptides of 17-20 kDa) under reduced conditions of SDS-PAGE. The 2D gel electrophoretic analysis showed the further separation of the bands into their isoforms with major pI ranges between 5.7-8.0 (α-polypeptides) and 5.5-8.5 (β-polypeptides). The Dynamic Light Scattering (DLS) showed the monodisperse nature of the Cruciferin with hydrodynamic radius of 5.8 ± 0.1 nm confirming the trimeric nature of the protein. The Circular Dichroism (CD) spectra showed both α-helices and β-sheets in the native conformation of the trimeric protein. The pure Cruciferin protein (40 mg/ml) was successfully crystallized; however, the crystals diffracted only to low resolution data (8Å). Small-Angle X-ray Scattering (SAXS) was applied to gain insights into the three-dimensional structure in solution. XXVI SAXS showed that the radius of gyration is 4.24 ± 0.2 nm and confirmed the nearly globular shape. The SAXS based ab initio dummy model of B. nigra Cruciferin was compared with 11S globulins (PDB ID: 3KGL) of B. napus which further confirmed a highly similar molecular weight and globular shape indicating a conserved trimerization of B. nigra Cruciferin. The comparison of the scattering patterns of both proteins showed a minimized χ2-value of 1.337 confirming a similar molecular structure. This is the first report describing the purification and characterization of a Cruciferin protein from seeds of B. nigra. Eruca sativa Cruciferin (EsC): E. sativa Cruciferin (12S globulin) protein was identified and characterized from seeds in trimeric form. The protein was identified by feeding the LC-MS/MS mass spectrometric residual data in UniProtKB online server which indicated 63 % sequence identity with Cruciferins (CRU1) of Raphanus sativus and Brassica napus (CRU3). SDS-PAGE exhibited multiple isoform protein banding pattern of 50-52 kDa monomeric Cruciferin which was separated into α-polypeptides of 30 kDa and β-polypeptides of 20 kDa under reduced conditions. Secondary structure contents of EsC were analyzed by Circular Dichroism spectroscopy which indicated 7.5 % α-helix, 47.6 % β-sheet, 7 % β-turn and 37.9 % random conformation. The monodispersity and stability of EsC for Crystallization was performed through Dynamic Light Scattering (DLS) and hydrodynamic radius of EsC was calculated as 5.5 ± 0.3 nm which further confirmed the trimeric nature of the protein. The pure Cruciferin protein (41 mg/ml) produced rather tiny crystals (0.1 × 0.05 × 0.05 mm) which yielded low resolution diffraction data of 7Å only. SAXS analysis showed that the molecular shape of EsC is globular and oligomeric state of the protein revealed a gyration radius of 4.3± 0.30 nm. The ab initio dummy model of EsC with P3 symmetry was thoroughly compared with 11S globulins (PDB ID: 3KGL) of B. napus which indicated a highly similar molecular weight and more importantly globular shape corresponding to a conserve trimerization of EsC molecule in the solution form. Moreover, the scattering patterns of both proteins showed a minimized χ2-value of 2.0 which further confirms the similar molecular conformation. Sisymbrium irio Ubiquitin (SiU): Ubiquitin protein was identified and characterized from the seeds of Sisymbrium irio. Results of LC-MS/MS spectrometry revealed that Sisymbrium irio Ubiquitin (SiU) shares around 77 % sequence identity with XXVII already reported Ubiquitin proteins from Triticum aestivum, Avena sativa, Arabidopsis thaliana and others. SiU revealed a molecular weight of 17 kDa on non-reduced SDSPAGE while a reduced band of approximately 9 kDa in the presence of βmercaptoethanol. A pI of 9.2 was calculated by isoelectric focusing measurement. Circular Dichroism (CD) spectroscopic studies showed that the secondary structure of Siu comprised of about 6.5 % α-helix, 39.3 % β-sheet, 0.7 % turn, 53.5 % random coil which indicates that it is mostly β-sheeted. Similarly, a hydrodynamic radius (RH) of 2.44 nm was calculated by Dynamic Light Scattering (DLS) for monodispersive SiU which further infers the presence of a dimeric status of 17 kDa native protein in solution. Multiple sequence alignment of 20 different Ubiquitin sequences revealed that Ubiquitin sequence is well conserved in nature with very few amino acid differences, e.g., R42K, Q49D, and Q62D. Homology modeling of SiU (11-28, and 42-76) residues suggested the overall conserved secondary and tertiary conformation and other structural motifs. Brassica nigra Napin (BnN): A 16 kDa BnN was identified by N-terminal amino acid sequencing which showed 78.8 % homology to that of Napin (2S albumin) from Raphanus sativus and 71.4 % identity to Napin-3 and Napin proteins from Brassica napus and Brassica campestris respectively. BnN was made highly purified by optimizing a combination of Cation exchange and size-exclusion chromatography. Purified BnN showed a typical banding pattern on 15 % SDS-PAGE exhibiting approximately 16 kDa molecular weight under non-reduced condition while a visible band of 10 kDa in the presence of β-mercaptoethanol confirming the presence of inter-chain disulfide linkage between two chains. However, the other cleaved band of approximately 5-6 kDa is not visible on gels. The secondary structure of BnN was studied by Circular Dichroism (CD) spectroscopy and it showed that BnN consists of about 38 % α-helix, 20 % β-sheet and 11 % β-turns. DLS data indicated the monodispersity and stability of BnN in 20 mM acetate buffer of pH 5.0. DLS data recording also indicated the zero effect of two temperatures (4 and 20ºC) and BnN monomeric conformation remained stable at both temperatures. Moreover, a hydrodynamic radius (RH) of 1.99 nm was calculated which confers the monomeric nature of the 16 kDa BnN in solution form. Purified BnN (15 mg/ml) produces small size diamond shape crystals (0.035 × 0.02 × 0.02 mm) in 0.1 M Sodium citrate, pH 5.5, 20 % w/v PEG 3k condition. However, crystals diffracted to poor resolution (10Å) and as a result no structure was solved. Nevertheless, BnN exhibited strong antifungal activity against phytopathogenic Fusarium solanii and Fusarium XXVIII oxysporum at a concentration of 30 µg per disc. Similarly, BnN showed good insecticidal activity against T. castaneum larval growth at a concentration of 3 mg/ml. Similarly, total number of pupae and adults were also significantly reduced in comparison to control experiment. Indeed comparison of treatments to control; indicated the highest mortality due to BnN treatment for all three populations (average larval, pupal and adult) in comparison to control. Precursor Eruca sativa Thionin (pEsT): Another 16 kDa Precursor Eruca sativa Thionin (pEsT) was identified by MALDI-TOF/TOF mass spectrometry. The fragmented amino acid sequence was BLAST in UniProtKB online server and result showed a 100 % sequence identity with Thionin 2.4 of Arabidopsis thaliana. Purified pEsT showed a compact 16 kDa protein band on 15 % SDS-PAGE under non-reduced condition while in the presence of β-mercaptoethanol; it produces two bands of 11 and 5 kDa. Secondary structure of pEsT was calculated by using CD spectroscopy and spectra indicated the 38 % α-helix, 9 % β-sheet, 19 % turn and a higher content of 34 % which was not compactly folded (random). Monodispersity and stability of pEsT was checked by DLS for three consecutive days at two different temperatures (4 and 20°C). Poor quality diffraction data were obtained from rectangular shape pEsT crystals. An elongated shape dummy model of pEsT was calculated by SAXS scattering pattern (Rg of 1.96 ± 0.1 nm). The ab initio model calculated by DAMMIF used P1 symmetry and a volume of 31100 nm3 which corresponded to approximate 15.5 kDa molecular weight of protein. pEsT showed the cytotoxity against the hepatic cell line Huh7 and LC50 was obtained at ≥ 12.5 µM. Antifungal activity of pEsT was checked against Fusarium solanii and Fusarium oxysporum by Disc diffusion method while 96 well-microtiter plate methodology was used against Fusarium graminearum. pEsT significantly inhibited the growth of these pathogenic fungi. pEsT protein showed strong Entomotoxic activity against different life stages of T. castaneum growt
Gov't Doc #: 18740
Appears in Collections:PhD Thesis of All Public / Private Sector Universities / DAIs.

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