Expression and purification of neurolin immunoglobulin domain 2 from Carrassius auratus (goldfish) in Escherichia coli.
The immunoglobulin superfamily protein neurolin plays a central role during differentiation and development of retina ganglion cells in goldfish. As shown in earlier work, blockage of the second immunoglobulin domain (Ig2) of neurolin with domain-specific antibodies causes severe pathfinding defects of growing axons in the retina. Thus Ig2 of neurolin was identified as the critical domain for axon guidance. In the present study we have developed a protocol for expression and purification of neurolin-Ig2 suitable for structure analysis, functional studies and ligand identification. Neurolin was expressed in Rosettagami and Origami strains of Escherichia coli which is deficient in glutathione- and thioredoxin reductase facilitating proper formation of the disulfide bond in the cytoplasm. The protein was purified via an N-terminal His(6)-tag by Ni(2+) affinity and size exclusion chromatography. After purification the His(6)-tag was cut-off without loss of solubility. Analytical size exclusion chromatography revealed an apparent molecular mass for neurolin-Ig2 in agreement with a non-covalent homodimer. Analysis of CD and FTIR spectra gave a secondary structure content typical for Ig domains.
Drees
Christina
C
Department of Chemistry, University of Konstanz, 78457 Konstanz, Germany.
Stürmer
Claudia A O
CA
Möller
Heiko M
HM
Fritz
Günter
G
D020410:Activated-Leukocyte Cell Adhesion Molecule / Q000096:biosynthesis* / Q000302:isolation & purification*; D000818:Animals; D000911:Antibodies, Monoclonal; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006054:Goldfish; D007136:Immunoglobulins / Q000235:genetics; D008958:Models, Molecular; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D017550:Spectroscopy, Fourier Transform Infrared
D020410:Activated-Leukocyte Cell Adhesion Molecule; D000911:Antibodies, Monoclonal; D007136:Immunoglobulins; D011994:Recombinant Proteins