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1.  Purification and crystallization of Cor a 9, a major hazelnut allergen 
The major hazelnut allergen Cor a 9 was purified from the natural source and crystallized. Diffraction data were collected to 1.9 Å resolution using a synchrotron-radiation source.
Hazelnut (Corylus avellana) is one of the food sources that induce allergic reaction in a subpopulation of people with food allergy. The 11S legumin-like seed-storage protein from hazelnut has been identified as one of the major hazelnut allergens and named Cor a 9. In this study, Cor a 9 was extracted from hazelnut kernels using a high-salt solution and was purified by desalting out and FPLC to a highly purified state. Diffraction-quality single crystals were obtained using the hanging-drop vapour-diffusion method. Diffraction data were collected and a structure solution has been obtained by molecular-replacement calculations. Further refinement of the structure is currently in progress.
doi:10.1107/S1744309108039894
PMCID: PMC2628846  PMID: 19153454
hazelnuts; 11S seed-storage proteins; food allergies; tree-nut allergens
2.  Purification, crystallization and initial crystallographic characterization of the Ginkgo biloba 11S seed globulin ginnacin 
The crystallization of ginnacin, the 11S seed storage protein from G. biloba, is reported.
Ginkgo biloba, a well known ‘living fossil’ native to China, is grown worldwide as an ornamental shade plant. Medicinal and nutritional uses of G. biloba in Asia have a long history. However, ginkgo seed proteins have not been well studied at the biochemical and molecular level. In this study, the G. biloba 11S seed storage protein ginnacin was purified by sequential anion-exchange and gel-filtration chromatography. A crystallization screen was performed and well diffracting single crystals were obtained by the vapor-diffusion method. A molecular-replacement structural solution has been obtained. There are six protomers in an asymmetric unit. Structure refinement is currently in progress.
doi:10.1107/S1744309108016242
PMCID: PMC2443976  PMID: 18607097
cupins; ginnacin; allergens; seed storage proteins; herbal medicine
3.  Crystallization and initial crystallographic characterization of a vicilin-type seed storage protein from Pinus koraiensis  
In this study, the Korean pine (Pinus koraiensis) vicilin-type 7S seed storage protein was isolated from defatted pine-nut extract and purified by sequential gel-filtration and anion-exchange chromatography. Well diffracting single crystals were obtained by the vapour-diffusion method in hanging drops.
The cupin superfamily of proteins includes the 7S and 11S seed storage proteins. Many members of this family of proteins are known allergens. In this study, the Korean pine (Pinus koraiensis) vicilin-type 7S seed storage protein was isolated from defatted pine-nut extract and purified by sequential gel-filtration and anion-exchange chromatography. Well diffracting single crystals were obtained by the vapor-diffusion method in hanging drops. The crystals belong to the primitive cubic space group P213, with unit-cell parameters a = b = c = 148.174 Å. Two vicilin molecules were present in the asymmetric unit and the Matthews coefficient was determined to be 2.90 Å3 Da−1, with a corresponding solvent content of ∼58%. A molecular-replacement structural solution has been obtained using the program Phaser. Refinement of the structure is currently under way.
doi:10.1107/S1744309107054310
PMCID: PMC2344101  PMID: 18084088
allergens; pine nuts; seed storage protein
4.  Purification, crystallization and initial crystallographic characterization of brazil-nut allergen Ber e 2 
The crystallization of the brazil nut allergen Ber e 2 is reported.
Peanut and tree-nut allergies have attracted considerable attention because of their frequency and their lifelong persistence. Brazil-nut (Bertholletia excelsa) allergies have been well documented and the 11S legumin-like seed storage protein Ber e 2 (excelsin) is one of the two known brazil-nut allergens. In this study, Ber e 2 was extracted from brazil-nut kernels and purified to high purity by crystalline precipitation and gel-filtration chromatography. Well diffracting single crystals were obtained using the hanging-drop vapour-diffusion method. A molecular-replacement structural solution has been obtained. Refinement of the structure is currently under way.
doi:10.1107/S1744309107051445
PMCID: PMC2339761  PMID: 18007055
brazil nut allergy; Ber e 2; cupin superfamily; allergenicity
5.  Purification, crystallization and initial crystallographic characterization of peanut major allergen Ara h 3 
The crystallization of peanut allergen Ara h 3 is reported.
The peanut is a significant food source, but is responsible for many cases of anaphylaxis. The peanut 11S legumin-like seed storage protein Ara h 3 is one of the best characterized allergens. In this study, Ara h 3 was extracted from peanut kernels and purified by sequential anion-exchange, hydrophobic interaction and gel-filtration chromatography to very high purity to facilitate crystallization and structural studies. Well diffracting single crystals were obtained by the vapor-diffusion method. A molecular-replacement structural solution has been obtained and refinement of the structure is currently under way.
doi:10.1107/S1744309107041176
PMCID: PMC2339721  PMID: 17909286
food safety; allergy; cupin; glycinin
6.  Overproduction, purification, crystallization and preliminary X-ray diffraction studies of the human transcription repressor ERH 
Crystallization of human enhancer of rudimentary homologue protein.
The human gene coding for the enhancer of rudimentary homologue (ERH) protein was overexpressed in Escherichia coli. The ERH protein was purified by anion-exchange, hydrophobic interaction and gel-filtration chromatography. Well diffracting single crystals were obtained by the vapour-diffusion method in hanging drops. The crystals belong to the trigonal space group P3121 or its enantiomorph P3221, with unit-cell parameters a = b = 53.74, c = 67.45 Å, α = β = 90, γ = 120°. They diffract to at least 1.75 Å. A selenomethionine derivative of the protein was prepared and crystallized for multiwavelength anomalous diffraction (MAD) phasing.
doi:10.1107/S1744309105012388
PMCID: PMC1952306  PMID: 16511088
transcription; pyrimidine biosynthesis; HNF1; PCD; DCoH
7.  Overproduction, purification, crystallization and preliminary X-ray diffraction studies of the human spliceosomal protein TXNL4B 
Human spliceosomal protein TXNL4B was purified and crystallized.
The human gene coding for the spliceosomal protein thioredoxin-like 4B (TXNL4B) was overexpressed in Escherichia coli and the encoded protein was purified and crystallized. Well diffracting single crystals were obtained by the vapor-diffusion method in hanging drops. The crystals belong to the primitive monoclinic space group P2, with unit-cell parameters a = 39.0, b = 63.6, c = 51.0 Å, β = 92.484°, and diffract to at least 1.50 Å. A SeMet derivative of the protein was prepared and crystallized for MAD phasing.
doi:10.1107/S1744309105002861
PMCID: PMC1952293  PMID: 16511018
spliceosomal proteins; thioredoxin-like 4B; Dim1; dim2; DLP
8.  Cell Cycle-Regulated Processing of HEF1 to Multiple Protein Forms Differentially Targeted to Multiple Subcellular Compartments 
Molecular and Cellular Biology  1998;18(6):3540-3551.
HEF1, p130Cas, and Efs/Sin constitute a family of multidomain docking proteins that have been implicated in coordinating the regulation of cell adhesion. Each of these proteins contains an SH3 domain, conferring association with focal adhesion kinase; a domain rich in SH2-binding sites, phosphorylated by or associating with a number of oncoproteins, including Abl, Crk, Fyn, and others; and a highly conserved carboxy-terminal domain. In this report, we show that the HEF1 protein is processed in a complex manner, with transfection of a single cDNA resulting in the generation of at least four protein species, p115HEF1, p105HEF1, p65HEF1, and p55HEF1. We show that p115HEF1 and p105HEF1 are different phosphorylation states of the full-length HEF1. p55HEF1, however, encompasses only the amino-terminal end of the HEF1 coding sequence and arises via cleavage of full-length HEF1 at a caspase consensus site. We find that HEF1 proteins are abundantly expressed in epithelial cells derived from breast and lung tissue in addition to the lymphoid cells in which they have been predominantly studied to date. In MCF-7 cells, we find that expression of the endogenous HEF1 proteins is cell cycle regulated, with p105HEF1 and p115HEF1 being rapidly upregulated upon induction of cell growth, whereas p55HEF1 is produced specifically at mitosis. While p105HEF1 and p115HEF1 are predominantly cytoplasmic and localize to focal adhesions, p55HEF1 unexpectedly is shown to associate with the mitotic spindle. In support of a role at the spindle, two-hybrid library screening with HEF1 identifies the human homolog of the G2/M spindle-regulatory protein Dim1p as a specific interactor with a region of HEF1 encompassed in p55HEF1. In sum, these data suggest that HEF1 may directly connect morphological control-related signals with cell cycle regulation and thus play a role in pathways leading to the progression of cancer.
PMCID: PMC108935  PMID: 9584194

Results 1-8 (8)