skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Expression, purification, crystallization and preliminary X-ray analysis of the olfactomedin domain from the sea urchin cell-adhesion protein amassin

Abstract

The olfactomedin (OLF) domain from the sea urchin cell-adhesion protein amassin has been crystallized. A native data set extending to 2.7 Å has been collected using an in-house X-ray source. A family of animal proteins is emerging which contain a conserved protein motif known as an olfactomedin (OLF) domain. Novel extracellular protein–protein interactions occur through this domain. The OLF-family member amassin, from the sea urchin Strongylocentrotus purpuratus, has previously been identified to mediate a rapid cell-adhesion event resulting in a large aggregation of coelomocytes, the circulating immune cells. In this work, heterologous expression and purification of the OLF domain from amassin was carried out and initial crystallization trials were performed. A native data set has been collected, extending to 2.7 Å under preliminary cryoconditions, using an in-house generator. This work leads the way to the determination of the first structure of an OLF domain.

Authors:
 [1]; ;  [2];  [1]
  1. Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0202 (United States)
  2. Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037 (United States)
Publication Date:
OSTI Identifier:
22356253
Resource Type:
Journal Article
Resource Relation:
Journal Name: Acta Crystallographica. Section F; Journal Volume: 62; Journal Issue: Pt 1; Other Information: PMCID: PMC2150939; PMID: 16511251; PUBLISHER-ID: za5123; OAI: oai:pubmedcentral.nih.gov:2150939; Copyright (c) International Union of Crystallography 2006; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United Kingdom
Language:
English
Subject:
75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND SUPERFLUIDITY; ADHESION; AGGLOMERATION; CRYSTALLIZATION; DISULFIDES; INTERACTIONS; PROTEINS; X-RAY SOURCES

Citation Formats

Hillier, Brian J., E-mail: bhillier@ucsd.edu, Sundaresan, Vidyasankar, Stout, C. David, and Vacquier, Victor D. Expression, purification, crystallization and preliminary X-ray analysis of the olfactomedin domain from the sea urchin cell-adhesion protein amassin. United Kingdom: N. p., 2006. Web. doi:10.1107/S1744309105038996.
Hillier, Brian J., E-mail: bhillier@ucsd.edu, Sundaresan, Vidyasankar, Stout, C. David, & Vacquier, Victor D. Expression, purification, crystallization and preliminary X-ray analysis of the olfactomedin domain from the sea urchin cell-adhesion protein amassin. United Kingdom. doi:10.1107/S1744309105038996.
Hillier, Brian J., E-mail: bhillier@ucsd.edu, Sundaresan, Vidyasankar, Stout, C. David, and Vacquier, Victor D. Sun . "Expression, purification, crystallization and preliminary X-ray analysis of the olfactomedin domain from the sea urchin cell-adhesion protein amassin". United Kingdom. doi:10.1107/S1744309105038996.
@article{osti_22356253,
title = {Expression, purification, crystallization and preliminary X-ray analysis of the olfactomedin domain from the sea urchin cell-adhesion protein amassin},
author = {Hillier, Brian J., E-mail: bhillier@ucsd.edu and Sundaresan, Vidyasankar and Stout, C. David and Vacquier, Victor D.},
abstractNote = {The olfactomedin (OLF) domain from the sea urchin cell-adhesion protein amassin has been crystallized. A native data set extending to 2.7 Å has been collected using an in-house X-ray source. A family of animal proteins is emerging which contain a conserved protein motif known as an olfactomedin (OLF) domain. Novel extracellular protein–protein interactions occur through this domain. The OLF-family member amassin, from the sea urchin Strongylocentrotus purpuratus, has previously been identified to mediate a rapid cell-adhesion event resulting in a large aggregation of coelomocytes, the circulating immune cells. In this work, heterologous expression and purification of the OLF domain from amassin was carried out and initial crystallization trials were performed. A native data set has been collected, extending to 2.7 Å under preliminary cryoconditions, using an in-house generator. This work leads the way to the determination of the first structure of an OLF domain.},
doi = {10.1107/S1744309105038996},
journal = {Acta Crystallographica. Section F},
number = Pt 1,
volume = 62,
place = {United Kingdom},
year = {Sun Jan 01 00:00:00 EST 2006},
month = {Sun Jan 01 00:00:00 EST 2006}
}
  • Recombinant SOS3 and SOS2 regulatory domain from A. thaliana have been coexpressed in E. coli, purified and crystallized by the hanging-drop vapour-diffusion method. An X-ray data set has been collected at 2.0 Å resolution. The salt-tolerance genes SOS3 (salt overly sensitive 3) and SOS2 (salt overly sensitive 2) regulatory domain of Arabidopsis thaliana were cloned into a polycistronic plasmid and the protein complex was expressed in Escherichia coli, allowing purification to homogeneity in three chromatographic steps. Crystals were grown using vapour-diffusion techniques. The crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 44.14, b =more » 57.39, c = 141.90 Å.« less
  • Francisella tularensis, a potential bioweapon, causes a rare infectious disease called tularemia in humans and animals. The macrophage growth locus A (MglA) protein from F. tularensis associates with RNA polymerase to positively regulate the expression of multiple virulence factors that are required for its survival and replication within macrophages. The MglA protein was overproduced in Escherichia coli, purified and crystallized. The crystals diffracted to 7.5 {angstrom} resolution at the Advanced Photon Source, Argonne National Laboratory and belonged to the hexagonal space group P6{sub 1} or P6{sub 5}, with unit-cell parameters a = b = 125, c = 54 {angstrom}.
  • The cloning, purification and crystallization of YnaF from S. typhimurium are reported along with preliminary X-ray crystallographic studies. The universal stress protein UspF (YnaF) is a small cytoplasmic bacterial protein. The expression of stress proteins is enhanced when cells are exposed to heat shock, nutrition starvation and certain other stress-inducing agents. YnaF promotes cell survival during prolonged exposure to stress and may activate a general mechanism for stress endurance. This manuscript reports preliminary crystallographic studies on YnaF from Salmonella typhimurium. The gene coding for YnaF was cloned and overexpressed and the protein was purified by Ni–NTA affinity chromatography. Purified YnaFmore » was crystallized using vapour-diffusion and microbatch methods. The crystals belong to space group P2{sub 1}, with unit-cell parameters a = 37.51, b = 77.18, c = 56.34 Å, β = 101.8°. A data set was collected to 2.5 Å resolution with 94.6% completeness using an image-plate detector system mounted on a rotating-anode X-ray generator. Attempts to determine the structure are in progress.« less
  • Crystals of the peroxiredoxin domain of a larger natural hybrid protein from T. maritima were obtained which diffracted to 2.9 Å resolution on a synchrotron source. Thermotoga maritima contains a natural hybrid protein constituted of two moieties: a peroxiredoxin domain at the N-terminus and a nitroreductase domain at the C-terminus. The peroxiredoxin (Prx) domain has been overproduced and purified from Escherichia coli cells. The recombinant Prx domain, which is homologous to bacterial Prx BCP and plant Prx Q, folds properly into a stable protein that possesses biological activity. The recombinant protein was crystallized and synchrotron data were collected to 2.9more » Å resolution. The crystals belonged to the tetragonal space group I422, with unit-cell parameters a = b = 176.67, c = 141.20 Å.« less
  • The gene product of an open reading frame Rv1657 from Mycobacterium tuberculosis is a putative arginine repressor protein (ArgR), a transcriptional factor that regulates the expression of arginine-biosynthetic enzymes. Rv1657 was expressed and purified and a C-terminal domain was crystallized using the hanging-drop vapour-diffusion method. Diffraction data were collected and processed to a resolution of 2.15 {angstrom}. The crystals belong to space group P1 and the Matthews coefficient suggests that the crystals contain six C-terminal domain molecules per unit cell. Previous structural and biochemical studies on the arginine repressor proteins from other organisms have likewise shown the presence of sixmore » molecules per unit cell.« less