Protein and Peptide Letters, Vol. 6, No. 1, 1999

Contents

Cytokine rhGM-CSF Interaction with Peptide Fragment of hGM-CSF Receptor a-Chain Monitored by NILIA-CD Spectroscopy. Pp. 7-14.
Rohanah Hussain, Paolo Rovero, Claudia Galoppini and Guiliano Siligardi
[Abstract]

Jack Bean Seed Coat Contains a Protein with Complete Sequence Homology to Bovine Insulin. Pp. 15-22.
Antônia Elenir A. Oliveira, Olga L. T Machado, Valdirene M. Gomes, José Xavier Neto, Alexandre C. Pereira, José Gilberto H. Vieira, Kátia V.S. Fernandes and Jose Xavier-Filho
[Abstract]

Human TNF Mutants with Selective Affinity on the R55 or R75 Receptor. Pp. 23-26.
Zhou Xiaowei, Wang Guoli and Huang Peitang
[Abstract]

Primary Structure and Kinetic Interaction with Glycoproteins of the Lectin from Seeds of Cratylia floribunda. Pp. 27-34.
Benildo S. Cavada, Nadia A.P. Nogueira, Crueza M.S.A. Farias, Thalles B. Grangeiro, Márcio V. Romas, Hubert H. Thole, Manfred Raida, Pierre Rougé and Juan J. Calvete
[Abstract]

Effect of Gene Dosage on the Expression of a Functionally Active C-Terminal Domain of Human Mucus Proteinase Inhibitor in E. coli. Pp. 35-42.
Andree Blaukat and Michael Kemme

[Abstract]

Selection of Peptide Ligands for a-1 Antitrypsin from a Phage Display Library. Pp. 43-48.
Giovanna Palombo, Maria Dani and Giorgio Fassina
[Abstract]

Enzymatic Degradation of Nociceptin Studied by LC-MS. Pp. 49-54.
Smoluch M.T., Suder P., Kotlinska J., Sällberg M. and Silberring J.
[Abstract]

Crystallisation Report

Purification and Crystallization of a Protocatechuate 4,5-Dioxygenase LigAB from Sphingomonas paucimobilis SYK-6. Pp. 55-58.
Keisuke Sugimoto, Hisae Aoshima, Toshiya Senda, Eiji Masai, Masao Fukuda and Yukio Mitsui
[Abstract]

Abstracts

[Back to top] Cytokine rhGM-CSF Interaction with Peptide Fragment of hGM-CSF Receptor a-Chain Monitored by NILIA-CD Spectroscopy. Rohanah Hussain, Paolo Rovero, Claudia Galoppini and Guiliano Siligardi.
P115 a peptide fragment of hGM-CSFR a-chain was observed to interact selectively to cytokine rhGM-CSF  (Kd=35mM) and not to IL-3 in solution by non-immobilised ligand interaction assay (NILIA) by CD spectroscopy in the spectral region characteristic of Trp aromatic side-chain and disulphide bond chromophores (290-330nm). The information provided by NILIA-CD identified two continuous peptide fragments of GM-CSF that could be used as a good candidate to bind to GM-CSF receptor a-chain. We propose this approach of combining spectroscopic and crystallographic information to guide the search for receptor ligand.

[Back to top] Jack Bean Seed Coat Contains a Protein with Complete Sequence Homology to Bovine Insulin. Antônia Elenir A. Oliveira, Olga L. T Machado, Valdirene M. Gomes, José Xavier Neto, Alexandre C. Pereira, José Gilberto H. Vieira, Kátia V.S. Fernandes and Jose Xavier-Filho.
The present study reports that a protein with complete sequence homology to bovine insulin is present in Jack bean (Canavalia ensiformis) seed coats. This protein was recognized by anti-human insulin antibodies and lowered blood glucose levels of alloxanized mice, indicating that it possesses biological potency. Our results suggest that insulin-like activities previously found in diverse plant tissues may be indeed due to insulin and indicate that insulin-dependent signaling pathways have been remarkably conserved during evolution.

[Back to top] Human TNF Mutants with Selective Affinity on the R55 or R75 Receptor. Zhou Xiaowei, Wang Guoli and Huang Peitang.
Four new-type TNF mutants, which discriminated between its two receptors R55 and R75, were designed based on the structural model of TNF-receptor complexes. All mutants were purified for competitive binding assay with ¹²⁵I wild-type hTNF to the recombinant hTNF-R55 and hTNF-R75 receptors. Two of the four mutants were found to selectively retain most of their binding capacity to one of the receptors, but decreased affinity to other one, significantly as designed.

[Back to top] Primary Structure and Kinetic Interaction with Glycoproteins of the Lectin from Seeds of Cratylia floribunda. Benildo S. Cavada, Nadia A.P. Nogueira, Crueza M.S.A. Farias, Thalles B. Grangeiro, Márcio V. Romas, Hubert H. Thole, Manfred Raida, Pierre Rougé and Juan J. Calvete.
The complete 236-amino-acid sequence of the glucose/mannose specific lectin from seeds of Cratylia floribunda (CFL) was determined by automated Edman sequencing of overlapping proteolytic peptides purified by HPLC after digestion of the lectin with endoproteinases Lys-C, Asp-N, trypsin and chymotrypsin. Mass spectrometry confirmed the sequence analysis and showed that CFL consists of a mixture of full length, single chain polypeptide (a-chain 25397 ± 3 Da) and its noncovalently associated b (residues 1-118, 12847 ± 2 Da) and g (residues 119-236, 12568 ± 1 Da) fragments. The primary structure of Cratylia floribunda lectin has extensive amino acid sequence homology with those of lectins from species of the taxonomically related genera Canavalia and Dioclea. However, using surface plasmon resonance, CFL and ConA, the seed lectin from Canavalia ensiformis, displayed distinct kinetic interactions with glycoproteins, indicating structural differences in their extended glycan binding sites.

[Back to top] Effect of Gene Dosage on the Expression of a Functionally Active C-Terminal Domain of Human Mucus Proteinase Inhibitor in E. coli. Andree Blaukat and Michael Kemme.
A gene dosage strategy was developed for efficient production of the C-terminal domain of human mucus proteinase inhibitor (cMPI) in E. coli with polycistronic expression cassettes containing one, two and four tandemly arranged cMPI genes. Upon induction, the synthesis of soluble cMPI increased proportionally to the number of genes integrated due to amplified plasmid copy numbers and enhanced protein stability, reaching a maximum up to 20 mg/l culture. Purified recombinant cMPI retained the inhibitory activity of native MPI.

[Back to top] Selection of Peptide Ligands for a-1 Antitrypsin from a Phage Display Library. Giovanna Palombo, Maria Dani and Giorgio Fassina.
An eptapeptide phage library was used to isolate peptides interacting specifically with a-l antitrypsin through a subtractive biopanning procedure. The peptide ligand identified after three selection cycles [SITPLVH] was chemically synthesized in a tetrameric form and analyzed for its capacity to recognize a-l antitrypsin by ELISA assays. Multimeric ligand immobilization on solid supports for the preparation of aiffinity columns proved useflil for the purification of a-l antitrypsin in the presence of large amounts of albumin.

[Back to top] Enzymatic Degradation of Nociceptin Studied by LC-MS. Smoluch M.T., Suder P., Kotlinska J., Sällberg M. and Silberring J.
The fate of nociceptin was investigated, using size exclusion chromatography linked to the electrospray ionization mass spectrometry. Heptadecapeptide was processed to the major metabolites: nociceptin (1-11) and (1-6). The strategy described here and involving LC-MS using a size-exclusion chromatography is convenient and rapid and minimizes identification problems often connected to the analysis of complex mixtures.

[Back to top] Purification and Crystallization of a Protocatechuate 4,5-Dioxygenase LigAB from Sphingomonas paucimobilis SYK-6. Keisuke Sugimoto, Hisae Aoshima, Toshiya Senda, Eiji Masai, Masao Fukuda and Yukio Mitsui.
A protocatechuate 4,5-dioxygenase from Sphingomonas paucimobilis SYK-6 has been crystallized by hanging drop vapor-diffusion method. The crystals were grown from a solution containing ammoniun sulfate and Tris-HCl. The crystals belonged to the monoclinic system with a space group P2₁ and unit-cell dimensions of a=65.4, b=66.5, c=119.8Å and b=92.5º. The crystals diffract to at least 2.2Å resolution.