Recombinant human erythropoietin (rhEPO) is an important CHO cell-derived glycoprotein and the degree of sialylation of this hormone is crucial for its in vivo bioactivity. In order to improve the purification process serotonin as a potential affinity ligand was tested for preparative chromatographic separation of rhEPO glycoforms into fractions of different degrees of sialylation. Therefore, two chromatographic matrices were prepared by immobilizing serotonin on CNBr- and NHS-Sepharose™. First it was shown both matrices bind rhEPO only in its sialylated form. Results indicate that binding is pH independent between pH 3.5 to 8 suggesting it is not only based on electrostatic interactions. Second, after optimal binding conditions were identified, semi-purified rhEPO was loaded onto both matrices and eluted using a stepwise elution gradient of sodium chloride. For comparison same affinity purification experiments were performed using wheat germ agglutinin-coupled agarose, a lectin known for its affinity towards sialylated glycoproteins. To monitor changes in N-glycan fingerprint, eluate fractions were analyzed by multiplexed capillary gel electrophoresis coupled to laser-induced fluorescence (xCGE-LIF). For the serotonin matrices an increasing degree of sialylation was observed from the first to the third elution fraction while purity of rhEPO could be increased at the same time. The late elution fractions of serotonin-coupled CNBr- and NHS-Sepharose™ also showed an overall sialylation degree exceeding that of the starting material. In contrast, for rhEPO bound to wheat germ agglutinin-coupled agarose, no distinct change in the degree of sialylation could be observed after elution. Overall, these encouraging results highlight the potential of serotonin as a chromatographic ligand for the improvement of pharmaceutical purification processes of rhEPO.

Chemie Ingenieur TechnikVolume 82, Issue 9 p. 1548-1548 PosterFree Access Charakterisierung von sialinsäurespezifischen Liganden für die affinitätschromatographische Aufreinigung von Glykoproteinen M. Meininger, M. Meininger meininger@mpi-magdeburg.mpg.de Otto-von-Guericke-Universität, Institut für Verfahrenstechnik, Universitätsplatz 2, D-39106 Magdeburg, GermanySearch for more papers by this authorF. Zwanziger, F. Zwanziger Eberhard-Karls-Universität, Pharmazeutisches Institut, Auf der Morgenstelle 8, D-72076 Tübingen, GermanySearch for more papers by this authorH. Rotering Dr., H. Rotering Dr. Merckle Biotec GmbH, Dornierstraße 10, D-89079 Ulm, GermanySearch for more papers by this authorK.-H. Wiesmüller Prof. Dr., K.-H. Wiesmüller Prof. Dr. EMC microcollections GmbH, Sindelfinger Straße 3, D-72070 Tübingen, GermanySearch for more papers by this authorU Reichl Prof. Dr., U Reichl Prof. Dr. Otto-von-Guericke-Universität, Institut für Verfahrenstechnik, Universitätsplatz 2, D-39106 Magdeburg, Germany Max-Planck-Institut für Dynamik komplexer technischer Systeme, Sandtorstraße 1, D-39106 Magdeburg, GermanySearch for more papers by this authorM. Wolff Dr., M. Wolff Dr. Otto-von-Guericke-Universität, Institut für Verfahrenstechnik, Universitätsplatz 2, D-39106 Magdeburg, Germany Max-Planck-Institut für Dynamik komplexer technischer Systeme, Sandtorstraße 1, D-39106 Magdeburg, GermanySearch for more papers by this author M. Meininger, M. Meininger meininger@mpi-magdeburg.mpg.de Otto-von-Guericke-Universität, Institut für Verfahrenstechnik, Universitätsplatz 2, D-39106 Magdeburg, GermanySearch for more papers by this authorF. Zwanziger, F. Zwanziger Eberhard-Karls-Universität, Pharmazeutisches Institut, Auf der Morgenstelle 8, D-72076 Tübingen, GermanySearch for more papers by this authorH. Rotering Dr., H. Rotering Dr. Merckle Biotec GmbH, Dornierstraße 10, D-89079 Ulm, GermanySearch for more papers by this authorK.-H. Wiesmüller Prof. Dr., K.-H. Wiesmüller Prof. Dr. EMC microcollections GmbH, Sindelfinger Straße 3, D-72070 Tübingen, GermanySearch for more papers by this authorU Reichl Prof. Dr., U Reichl Prof. Dr. Otto-von-Guericke-Universität, Institut für Verfahrenstechnik, Universitätsplatz 2, D-39106 Magdeburg, Germany Max-Planck-Institut für Dynamik komplexer technischer Systeme, Sandtorstraße 1, D-39106 Magdeburg, GermanySearch for more papers by this authorM. Wolff Dr., M. Wolff Dr. Otto-von-Guericke-Universität, Institut für Verfahrenstechnik, Universitätsplatz 2, D-39106 Magdeburg, Germany Max-Planck-Institut für Dynamik komplexer technischer Systeme, Sandtorstraße 1, D-39106 Magdeburg, GermanySearch for more papers by this author First published: 27 August 2010 https://doi.org/10.1002/cite.201050597AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume82, Issue9Special Issue: ProcessNet-Jahrestagung 2010 und 28. Jahrestagung der BiotechnologenSeptember, 2010Pages 1548-1548 RelatedInformation

The immune response to the P6 protein of Haemophilus influenzae was characterized with 24 synthetic icosapeptides and 45 dodecapeptides conjugated to the immune stimulator N-palmitoyl-S-[2,3-(bispalmitoyloxy)-(2RS)-propyl]-(R)-cysteinyl-( S)-serine . The antigenicity of these lipopeptides was investigated by enzyme-linked immunosorbent assays with rabbit anti-P6 serum. The epitopes of P6 protein were identified and localized within residues 31-46 and 59-70 and in the C-terminal part of the P6 protein. Mice were immunized with lipoicosapeptides without using additional adjuvants or carriers and the antibody titers were measured with isolated P6 protein and lipopeptides in a dot blot assay. Lipopeptides containing the sequence pattern QILDAHAA (P6 47-54) and the mouse B cell epitope GEYV (P6 43-46) induced high titers of anti P6 antibodies. These murine antibodies were able to neutralize the intact bacterium Haemophilus influenzae.

Expression of the cloned lysis protein of phage MS2, which is sufficient to lyse wild type Escherichia coli, does not cause lysis of mutants lacking the osmoregulatory membrane-derived oligosaccharides (MDO). The lysis gene product normally found in the membrane fraction was not stably inserted into the membranes of a mdoA mutant; rather degradation and release from the membrane occurred. Gentle plasmolysis of the MDO-lacking mutant clearly showed an increased periplasmic space as compared to wild type cells. It is concluded that the MDOs play an important role in maintaining a proper arrangement of inner and outer membrane, a prerequisite for a functional insertion of the MS2 lysis protein.

A mutant screening procedure is described which allows the identification of mutants carrying lesions in lipoprotein, membrane-derived oligosac-charides (MDO), and other compounds of the E. coli cell envelope containing glycerol derived from phospholipid metabolism. Two mutants lacking glycerol in MDO and one mutant devoid of lipoprotein demonstrate the usefulness of the procedure.

Insight as to how iron contributes to the pathogenicity of bacteria involves understanding the iron metabolism of the host and of the bacteria. The human host overcomes the extreme insolubility of Fe3+ at pH 7 by binding iron to carrier proteins, in the serum to transferring in secretory fluids to lactoferrin, and within cells to ferritin and hemoglobin. The serum concentration of free iron which is not bound to carrier proteins is only 10-18 M, which stands in sharp contrast to the iron concentration of 10-6 M required for bacterial growth.

A procedure for the isolation of mutants affected in components containing glycerol derived from phospholipids yielded two mutant strains that contain membrane-derived oligosaccharides (MDO) devoid of glycerol (Rotering, H., Fiedler, W., Rollinger, W., and Braun, V. (1984) FEMS Microbiol. Lett. 22, 61-68). MDO are found in the periplasmic space of Escherichia coli and other Gram-negative bacteria, and they may comprise up to 7% of the cells dry weight. The biosynthesis of MDO is osmoregulated (Kennedy, E. P. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 1092-1095) and linked to the metabolism of phospholipids (van Golde, L. M. G., Schulman, H., and Kennedy, E. P. (1973) Proc. Natl. Acad. Sci. U. S. A. 70, 1368-1372). This leads to substitution of MDO with sn-1-phosphoglycerol and phosphoethanolamine (Kennedy, E. P., Rumley, M. K., Schulman, P., and van Golde, L. M. G. (1976) J. Biol. Chem. 251, 4208-4213). MDO also contain succinate in O-ester linkage. We now report that one mutant strain lacks phosphoglycerol transferase I activity and thus is unable to transfer sn-1-phosphoglycerol residues from phosphatidylglycerol to MDO. The mdoB gene affected in this mutant has been located at 99.2 min on the E. coli chromosome. The ethanolamine content of MDO isolated from the mutant strain is elevated, whereas the number of succinate residues is not affected. The only phenotype of mdoB mutants we found is a dramatic reduction of the diglyceride content observed in dgk mdoB double mutants when the beta-glucoside arbutin is present in the growth medium.

Escherichia coli, in particular E. coli K-12, and the bacteriophages that multiply in E. coli became the principal model organisms of molecular biology. Since the genetic material can be exchanged easily by conjugation or introduced by phage infection, a great deal of basic knowledge on the nature and organization of genes and their regulation and expression, and about metabolic pathways in the cytoplasm and in membranes was gained and is still obtained with this organism. It was certainly wise to concentrate on one system to unravel basic features of life on the level of single cells. The theories developed led also to fruitful concepts for eukaryotes. However, the world of microbiology does not consist solely of E. coli K-12. A wealth of properties expressed by various bacteria is not contained in E. coli. But even when we only consider E. coli there are many features that cannot be studied with the K-12 strain. The K-12 strain was isolated in 1922 and has been kept in the laboratory since then. During these six decades it has lost properties that were once essential for survival in the natural habitat. E. coli is usually a harmless inhabitant of the gut but some strains cause diarrhea, some are invasive and lead to enterocolitis, certain strains distribute in the urinary tract or the kidney, or appear in the blood and elicit severe septicemia, others invade the brain and cause meningitis. Investigations aimed at unraveling the pathogenic properties of E. coli defined up to now 164 O (lipopolysaccharide), 103 K (capsular), and 56 H (flagellai) antigens. All these different antigens are exposed at the cell surface. Therefore, E. coli is a very complex species with regard to its contact with the surroundings. The antigen armory enables the organism to adhere to certain tissues and to avoid cellular and humoral defense reactions of the host. In contrast to the situation in molecular biology where insights obtained with one organism apply in principle to most organisms, ecological parameters are tailored to a single organism in a certain environment. The question of why an E. coli strain lives in a certain environment is not only of medical but also of immense biological interest. For example, studies on resistance to antibiotics opened the field to what we now call gene technology (Cohen, 1976). Recombinant DNA techniques are presently being applied to separate the various virulence factors that contribute to an infectious disease, to study them separately, and then to put them together again in various combinations with the aim of understanding the causes of a disease.

Diglyceride kinase mutants of Escherichia coli contain about 50- to 100-fold more 1,2-diglyceride than wild type cells. We now report that monoglyceride and triglyceride also accumulate in these strains. In mutant RZ60 (dgk-6) these compounds represent about 1 and 0.2%, respectively, of the total lipid fraction, while diglyceride represents 5-8% under most conditions. Monoglyceride accumulates predominantly in the outer membrane, while triglyceride builds up together with diglyceride in the cytoplasmic membrane. Under typical growth conditions about two-thirds of the diglyceride in E. coli arises in conjunction with synthesis of the membrane-derived oligosaccharides (Raetz, C.R.H., and Newman, K.F. (1979) J. Bacteriol. 137, 860-868). Inhibition of membrane-derived oligosaccharides (MDO) synthesis also curtails the accumulation of monoglyceride and triglyceride. However, there appears to be at least one other MDO-independent source of diglyceride and related metabolites. Since MDO synthesis is suppressed by high osmolarity (Kennedy, E.P. (1982) Proc. Natl. Acad. Sci. U.S. A. 79, 1092-1095), we have examined the effects of osmolarity on diglyceride accumulation in RZ60 (dgk-6). As expected, if MDO synthesis and diglyceride formation are coupled, the diglyceride level in RZ60 is higher at low osmolarity, while at high osmolarity the level of diglyceride is reduced to that observed in double mutants defective both in MDO synthesis and diglyceride kinase. Since dgk mutants do not grow at very low osmolarity, we have isolated several spontaneous phenotypic revertants that do. One class regains diglyceride kinase and has low diglyceride levels under all conditions. The other class remains defective in diglyceride kinase but tolerates higher diglyceride levels which amount to 13% of the total lipid during maximal induction of MDO synthesis at low osmolarity.

50 representative staphylococcal strains were selected from 18 clusters of a previous numerical taxonomy study (2). The types of peptidoglycan and teichoic acid were investigated in these strains. The results of this cell wall analysis were compared with the data of the numerical taxonomy study (2) and the Kloos and Schleifer-classification system.

From the mutant bacterial strain Escherichia coli JE5511 lpp lpm, muropeptide-containing and muropeptide-free lipoproteins were prepared. By gas chromatography and by infrared spectroscopy we showed that the products were deficient in the two ester-bound N-terminal fatty acids, but still carried the amide-linked fatty acid. Mutant lipoproteins were tested for mitogenicity in lipopolysaccharide nonresponder C3H/HeJ mice by incorporation of [3H]thymidine and [3H]uridine and by hemolytic plaque assays for immunoglobulin-secreting plasma cells. Our results showed that the mutant lipoproteins still exhibited marked mitogenicity toward mouse B-lymphocytes, although the activity of the products was reduced in comparison to the wild-type lipoprotein. Thus, the presence of one fatty acid in the N-terminal part of lipoprotein is sufficient to bring about mitogenicity.

FEBS LettersVolume 83, Issue 1 p. 41-44 Full-length articleFree Access Lipid deficiency in a lipoprotein mutant of Escherichia coli Correction(s) for this article Corrigenda Volume 85Issue 2FEBS Letters pages: 373-373 First Published online: October 19, 2001 Heinz Rotering, Heinz Rotering Lehrstuhl Mikrobiologie II, Universität Tübingen, Auf der Morgenstelle 28, 7400 Tübingen, FRGSearch for more papers by this authorVolkmar Braun, Volkmar Braun Lehrstuhl Mikrobiologie II, Universität Tübingen, Auf der Morgenstelle 28, 7400 Tübingen, FRGSearch for more papers by this author Heinz Rotering, Heinz Rotering Lehrstuhl Mikrobiologie II, Universität Tübingen, Auf der Morgenstelle 28, 7400 Tübingen, FRGSearch for more papers by this authorVolkmar Braun, Volkmar Braun Lehrstuhl Mikrobiologie II, Universität Tübingen, Auf der Morgenstelle 28, 7400 Tübingen, FRGSearch for more papers by this author First published: November 01, 1977 https://doi.org/10.1016/0014-5793(77)80637-6Citations: 14AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat References 1 V. Braun, Biochim. Biophys. Acta (Review in Biomembranes), 415, (1975), 335– 377. 2 K. Hantke, V. Braun, Eur. J. Biochem., 34, (1973), 284– 296. 3 H. Suzuki, Y. Nishimura, H. Iketani, J. Campisi, A. Hirashima, M. Inouye, Y. Hirota, J. Bacteriol., 127, (1976), 1494– 1501. 4 V. Braun, K. Rehn, Eur. J. Biochem., 10, (1969), 426– 438. 5 S. Inouye, K. Takeishi, N. Lee, Martini M. De, A. Hirashima, M. Inouye, J. Bacteriol., 127, (1976), 555– 563. 6 V. Braun, H. Rotering, J.-P. Ohms, H. Hagenmaier, Eur. J. Biochem., 70, (1976), 601– 610. 7 V. Braun, V. Bosch, Eur. J. Biochem., 28, (1972), 51– 69. 8 V. Braun, K. Hantke, U. Henning, FEBS Lett., 60, (1975), 26– 28. 9 G. Papacostidis, G. Zundel, E. Mehl, Biochim. Biophys. Acta, 288, (1972), 277– 281. 10 S. Inouye, S. Wang, J. Sekizawa, S. Halegoua, M. Inouye, Proc. Natl. Acad. Sci. USA, 74, (1977), 1004– 1008. 11 J.J.-C. Lin, H.C. Wu, J. Bacteriol., 125, (1976), 892– 904. 12 H.C. Wu, C. Hou, J.J.-C. Lin, D.W. Yem, Proc. Natl. Acad. Sci. USA, 74, (1977), 1388– 1392. 13 H. Schulman, E.P. Kennedy, J. Biol. Chem., 252, (1977), 4250– 4255. Citing Literature Volume83, Issue1November 01, 1977Pages 41-44 ReferencesRelatedInformation

Conformational studies on an isolated integral membrane protein are reported. Lipoprotein of Escherichia coli outer membrane was released from murein by treatment with either lysozyme or trypsin. The isolated lysozyme-released lipoprotein (lipoprotein I) contained 2 or 3 muropeptides covalently linked at the C-terminal end, while the trypsin-released lipoprotein (lipoprotein II) was free of muropeptides and lacked the C-terminal peptide Tyr-Arg-Lys. Circular dichroism spectra of the two preparations were essentially identical, and they show an alpha-helix content of about 80%. According to calculations based on the Chou-Fasman rules for proteins of known sequence, lipoprotein is 64% alpha-helix and 15% beta-structure. Infrared spectroscopy qualitatively supports these values. The conformation was stable in the pH range of 5 - 12. Danaturation of lipoprotein by heat, 8 M urea, or sodium dodecylsulphate was a fully reversible, cooperative process. The thermal denaturation of lipoprotein occurs in two steps with transition points at 79.4 degrees C for lipoprotein I and at 85.1 degrees C for lipoprotein II. Lioprotein markedly changes conformation at dodecylsulphate concentrations where micelle formation sets in. The unusual behaviour of the lipoprotein convormation in sodium dodecylsulphate is discussed in relation to the lipoprotein conformation and aggregation within the membrane.