• Discontinuous and continuous separation of the monomeric and dimeric forms of human bone morphogenetic protein-2 from renaturation batches.

      Gueorguieva, Ludmila; Vallejo, Luis Felipe; Rinas, Ursula; Seidel-Morgenstern, Andreas; Otto-von-Guericke-Universität Magdeburg, Institut für Verfahrenstechnik, PO Box 4120, D-39106 Magdeburg, Germany. (2006-12-01)
      Bone morphogenetic protein-2 (BMP-2) is one of the most interesting of the approximately 14 BMPs which belong to the transforming-growth-factor-beta (TGF-beta) superfamily. BMP-2 induces bone formation and thus plays an important role as a pharmaceutical protein. Recently, rhBMP-2 has been produced in form of inactive inclusion bodies in Escherichia coli. After solubilization and renaturation the biologically active dimeric form of rhBMP-2 can be generated. However, inactive monomers of BMP-2 are also formed during the renaturation process which must be separated from the active dimeric BMP-2. The purpose of this paper is to present: (a) results of an experimental study of a chromatographic separation of the monomeric and dimeric forms; and (b) a concept for a continuous counter-current simulated moving bed (SMB) process. The capacity of heparin as stationary phase was estimated for different salt concentrations in the mobile phase. A simulation study of a three-zone SMB process was performed applying a two step salt gradient. The results reveal the potential of the process for the purification of the dimeric BMP-2.
    • 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.