• Isolation, characterisation and molecular imaging of a high-molecular-weight insect biliprotein, a member of the hexameric arylphorin protein family.

      Kayser, Hartmut; Mann, Karlheinz; Machaidze, Gia; Nimtz, Manfred; Ringler, Philippe; Müller, Shirley A; Aebi, Ueli; Institut für Allgemeine Zoologie und Endokrinologie, Universität Ulm, Germany. hartmut.kayser@uni-ulm.de (2009-05-29)
      The abundant blue hemolymph protein of the last instar larvae of the moth Cerura vinula was purified and characterized by protein-analytical, spectroscopic and electron microscopic methods. Amino acid sequences obtained from a large number of cleavage peptides revealed a high level of similarity of the blue protein with arylphorins from a number of other moth species. In particular, there is a high abundance of the aromatic amino acids tyrosine and phenylalanine amounting to about 19% of total amino acids and a low content of methionine (0.8%) in the Cerura protein. The mass of the native protein complex was studied by size-exclusion chromatography, analytical ultracentrifugation, dynamic light scattering and scanning transmission electron microscopy and found to be around 500 kDa. Denaturating gel electrophoresis and mass spectrometry suggested the presence of two proteins with masses of about 85 kDa. The native Cerura protein is, therefore, a hexameric complex of two different subunits of similar size, as is known for arylphorins. The protein was further characterized as a weakly acidic (pI approximately 5.5) glycoprotein containing mannose, glucose and N-acetylglucosamine in an approximate ratio of 10:1:1. The structure proposed for the most abundant oligosaccharide of the Cerura arylphorin was the same as already identified in arylphorins from other moths. The intense blue colour of the Cerura protein is due to non-covalent association with a bilin of novel structure at an estimated protein subunit-to-ligand ratio of 3:1. Transmission electron microscopy of the biliprotein showed single particles of cylindrical shape measuring about 13 nm in diameter and 9 nm in height. A small fraction of particles of the same diameter but half the height was likely a trimeric arylphorin dissociation intermediate. Preliminary three-dimensional reconstruction based on averaged transmission electron microscopy projections of the individual particles revealed a double-trimeric structure for the hexameric Cerura biliprotein complex, suggesting it to be a dimer of trimers.
    • Purification, crystallization and preliminary X-ray crystallographic analysis of MIL, a glycosylated jacalin-related lectin from mulberry (Morus indica) latex.

      Patel, Ashok K; Singh, Vijay K; Bergmann, Ulrich; Jagannadham, Medicherla V; Kursula, Petri (2011-05-01)
      A quantitatively major protein has been purified from the latex of Morus indica. The purified previously uncharacterized protein, M. indica lectin (MIL), was further shown to be a glycosylated tetramer and belongs to the family of jacalin-related lectins. Crystallization of MIL was also accomplished and the tetragonal crystals diffracted synchrotron X-rays to a resolution of 2.8 Å.
    • Streamlining homogeneous glycoprotein production for biophysical and structural applications by targeted cell line development.

      Wilke, Sonja; Groebe, Lothar; Maffenbeier, Vitali; Jäger, Volker; Gossen, Manfred; Josewski, Jörn; Duda, Agathe; Polle, Lilia; Owens, Raymond J; Wirth, Dagmar; et al. (2011)
      Studying the biophysical characteristics of glycosylated proteins and solving their three-dimensional structures requires homogeneous recombinant protein of high quality.We introduce here a new approach to produce glycoproteins in homogenous form with the well-established, glycosylation mutant CHO Lec3.2.8.1 cells. Using preparative cell sorting, stable, high-expressing GFP 'master' cell lines were generated that can be converted fast and reliably by targeted integration via Flp recombinase-mediated cassette exchange (RMCE) to produce any glycoprotein. Small-scale transient transfection of HEK293 cells was used to identify genetically engineered constructs suitable for constructing stable cell lines. Stable cell lines expressing 10 different proteins were established. The system was validated by expression, purification, deglycosylation and crystallization of the heavily glycosylated luminal domains of lysosome-associated membrane proteins (LAMP).