A human tumor necrosis factor p75 receptor agonist stimulates in vitro T cell proliferation but does not produce inflammation or shock in the baboon.

Tumor necrosis factor (TNF) is a potentially useful adjunct to anticancer therapies. However, the clinical utility of TNF has been limited by generalized toxicity and hypotension. Recently, studies have begun to dissect the individual proinflammatory and immunologic responses that result from TNF binding to its two cellular receptors, p55 and p75, in an attempt to develop TNF receptor agonists with reduced systemic toxicity. To evaluate a p75 receptor selective TNF mutant (p75TNF), TNF and p75TNF were administered to healthy anesthetized baboons. Intravenous infusion of the p75TNF produced none of the hemodynamic changes seen after the infusion of TNF. Infusion of p75TNF also failed to induce the plasma appearance of interleukins 6 and 8. However, p75TNF enhanced in vitro baboon thymocyte proliferation to concanavalin A, and infusion of p75TNF resulted in increased soluble p55 and p75 receptor plasma concentrations. Local skin necrosis and tissue neutrophil infiltration were seen after subcutaneous injections of TNF and p55TNF. Subcutaneous injection of p75TNF did not result in skin necrosis but did result in a modest dermal infiltration of lymphocytes and macrophages. The findings suggest that p75TNF may stimulate T cell proliferation without the systemic and local toxicity seen with TNF.

The three-dimensional high resolution structure of human interferon alpha-2a determined by heteronuclear NMR spectroscopy in solution.

The solution structure of recombinant human interferon alpha-2a (Roferon-A) has been determined by multidimensional heteronuclear NMR spectroscopy. The calculations using simulated annealing produced a family of 24 convergent structures which satisfy the experimental restraints comprising 1541 NOE-derived inter-proton distances, 187 dihedral restraints, 66 pairs of hydrogen bond restraints, and six upper and lower limits for two disulfide bridges. The fractional labeling of methyl groups allowed their direct and unambiguous stereospecific assignment which proved to be essential for obtaining a high resolution of the structures. A best fit superposition of residues 10 to 47, 50 to 101 and 111 to 157 gives an rms deviation of 0.62 A for the backbone heavy atoms and 1.39 A for all heavy atoms of these segments. The dominant feature of the structure is a cluster of five alpha-helices, four of which are arranged to form a left-handed helix bundle with an up-up-down-down topology and two over-hand connections. The interpretation of heteronuclear 15N-¿1H¿ NOE data shows the co-existence of flexible regions within an otherwise rigid framework of the protein. Four stretches of pronounced flexibility can be located: Cys1-Ser8, Gly44-Ala50, Ile100-Lys112, and Ser160-Glu165. Among the structurally related four-helical bundle cytokines, the structure of IFN alpha-2a is most similar to that of human interferon alpha-2b and murine interferon-beta. From this structural information and mutagenesis data, areas on the surface of the protein are identified which seem to be important in receptor interactions.

A single amino acid substitution in Staphylococcus aureus dihydrofolate reductase determines trimethoprim resistance.

A single amino acid substitution, Phe98 to Tyr98, in dihydrofolate reductase (DHFR) is the molecular origin of trimethoprim (TMP) resistance in Staphylococcus aureus. This active site amino acid substitution was found in all S. aureus TMP-resistant clinical isolates tested. In order to explore the structural role of Tyr98 in TMP-resistance the ternary complexes of the chromosomal S. aureus DHFR (SaDHFR) with methotrexate (MTX) and TMP in the presence of nicotinamide adenine dinucleotide phosphate (NADPH) as well as that of mutant Phe98Tyr DHFR SaDHFR(F98Y) ternary folate-NADPH complex have been determined by X-ray crystallography. Critical evidence concerning the resistance mechanism has also been provided by NMR spectral analyses of 15N-labelled TMP in the ternary complexes of both wild-type and mutant enzyme. These studies show that the mutation results in loss of a hydrogen bond between the 4-amino group of TMP and the carbonyl oxygen of Leu5. This mechanism of resistance is predominant in both transferable plasmid-encoded and non-transferable chromosomally encoded resistance. Knowledge of the resistance mechanism at a molecular level could help in the design of antibacterials active against multi-resistant Staphylococcus aureus (MRSA), one of todays most serious problems in clinical infectology.

A biotechnological method provides access to aggregation competent monomeric Alzheimer's 1-42 residue amyloid peptide.

Senile plaques, a neuropathological hallmark of Alzheimer's disease, consist primarily of insoluble aggregates of beta-amyloid peptide (A beta). A 42-residue peptide (A beta 1-42) appears to be the predominant form. In contrast to A beta 1-40, A beta 1-42 is characterized by its extreme tendency to aggregate into fibers or precipitate. A tailored biotechnological method prevents aggregation of A beta 1-42 monomers during its production. The method is based on a protein tail fused to the amino terminus of A beta. This tail leads to a high expression in E. coli, and a histidine affinity tag facilitates purification. Selective cleavage of the fusion tail is performed with cyanogen bromide by immobilizing the fusion protein on a reversed phase chromatography column. Cleavage then occurs only at the methionine positioned at the designed site but not at the methionine contained in the membrane anchor sequence of A beta. Furthermore, immobilization prevents aggregation of cleaved A beta. Elution from the HPLC column and all succeeding purification steps are optimized to preserve A beta 1-42 as a monomer. Solutions of monomeric A beta 1-42 spontaneously aggregate into fibers within hours. This permits the investigation of the transition of monomers into fibers and the correlation of physico-chemical properties with biological activities. Mutations of A beta 1-42 at position 35 influence the aggregation properties. Wild-type A beta 1-42 with methionine at position 35 has similar properties as A beta with a methionine sulfoxide residue. The fiber formation tendency, however, is reduced when position 35 is occupied by a glutamine, serine, leucine, or a glutamic acid residue.

Non-GMP plasmid production for transient transfection in bioreactors.

We describe a generic plasmid purification process for producing DNA for larger-scale transient transfection. Data on plasmid quality with regard to residual protein, endotoxin content and presence of different plasmid forms is given. The effects of contaminants and plasmid forms on expression levels of TNFRp55 and SEAP are discussed. Transient transfection of serum-free suspension grown mammalian cells represents a suitable approach to provide research quantities of proteins (50-100 mg) within1-2 weeks.

NMR studies in aqueous solution fail to identify significant conformational differences between the monomeric forms of two Alzheimer peptides with widely different plaque-competence, A beta(1-40)(ox) and A beta(1-42)(ox).

NMR studies of amyloid beta-peptides (A beta) in aqueous solution provide a novel way in which to characterize the apparent Alzheimer's disease-related conformational polymorphism of A beta. In the aqueous medium, neither of the polypeptides A beta(1-40)(ox) or A beta(1-42)(ox) (both of which contain a methionine sulfoxide at position 35) is folded into a globular structure, but they both deviate from random coil behavior by local conformational preferences of several short segments along the amino-acid sequence. Differences between the solution structures of A beta(1-40)(ox) and A beta(1-42)(ox) are indicated only by decreased flexibility of the region from about residue 32 to the C-terminus in A beta(1-42)(ox) when compared to A beta(1-40)(ox). The lack of the observation of more extensive conformational differences between the two molecules is intriguing, considering that A beta(1-42)(ox) in aqueous solution has much higher plaque-competence than A beta(1-40)(ox).

Both homodimeric isoforms of PDGF (AA and BB) have mitogenic and chemotactic activity and stimulate phosphoinositol turnover.

Platelet-derived growth factor (PDGF) occurs as three dimeric isoforms, AA, BB, and AB, which were previously shown to bind to two receptors with different isoform-specificity, the A/B-type (binds all three isoforms) and the B-type (binds only PDGF-BB). Results from competition binding experiments with Swiss 3T3 cells suggest the existence of a third receptor type, which recognizes PDGF-AB and PDGF-BB. Furthermore, Swiss 3T3 cells and human dermal fibroblasts express different relative and absolute levels of these receptor types. In particular, Swiss 3T3 cells express 90,000 PDGF-AA binding sites (A/B-receptors) per cell, whereas human fibroblasts express only 20,000 A/B-receptors per cell. All three PDGF isoforms were tested in either cell type for their effect on DNA synthesis. PDGF-BB and PDGF-AA were also tested in Swiss 3T3 cells for their effect on inositol phospholipid metabolism and chemotaxis. Each isoform promoted all three processes dose-dependently, but there were differences in the maximum cellular responses elicited. These responses reflect the capacity of the cells to bind the individual isoforms. These results demonstrate that the previous distinctions in responsiveness to the different PDGF isoforms are primarily a consequence of the differences in the levels of surface expression of the various isoform-specific receptor types, rather than of the differences in the intrinsic activity of these isoforms. Furthermore, these results suggest that all types of PDGF receptors are capable of responding to their respective ligands by mediating phosphoinositide breakdown, chemotactic responses, and DNA synthesis. Whether they exhibit other functional differences remains to be seen.

HMQC and HSQC experiments with water flip-back optimized for large proteins.

An HMQC experiment is proposed, dubbed FHMQC, where water flip-back is achieved by a single water-selective pulse preceding the basic HMQC pulse sequence. The scheme is demonstrated with a 15N,1H-HMQC spectrum of uniformly 15N/2H-labelled S. aureus DNA gyrase B with a molecular weight of 45 kDa for the unlabelled protein. The sensitivity of the experiment is improved compared to that of an FHSQC spectrum. It is further shown that the original FHSQC experiment can be shortened by the use of bipolar gradients. Relaxation times of different 15N magnetizations and coherences were measured. The new FHMQC scheme is implemented in 3D NOESY-15N-HMQC and 3D 15N-HMQC-NOESY-15N-HMQC pulse sequences which are demonstrated with a 24 kDa fragment of uniformly 15N/13C/2H-labelled S. aureus DNA gyrase B.

Two-dimensional map of the proteome of Haemophilus influenzae.

We have constructed a two-dimensional database of the proteome of Haemophilus influenzae, a bacterium of medical interest of which the complete genome, comprising about 1742 open reading frames, has been sequenced. The soluble protein fraction of the microorganism was analyzed by two-dimensional electrophoresis, using immobilized pH gradient strips of various pH regions, gels with different acrylamide concentrations and buffers with different trailing ions. In order to visualize low-copy-number gene products, we employed a series of protein extraction and sample application approaches and several chromatographic steps, including heparin chromatography, chromatofocusing and hydrophobic interaction chromatography. We have also analyzed the cell envelope-bound protein fraction using either immobilized pH gradient strips or a two-detergent system with a cationic detergent in the first and an anionic detergent in the second-dimensional separation. Different proteins (502) were identified by matrix-assisted laser desorption/ionization mass spectrometry and amino acid composition analysis. This is at present one of the largest two-dimensional proteome databases.

Purification and crystallization of the extracellular domain of human neutral endopeptidase (neprilysin) expressed in Pichia pastoris.

Neutral endopeptidase (NEP) is a mammalian zinc metalloprotease involved in the inactivation of a wide variety of regulatory peptides such as enkephalins and atrial natiuretic factor. The soluble extracellular domain of NEP (sNEP) was expressed in the methylotrophic yeast Pichia pastoris. The protein was purified to homogeneity and single crystals have been obtained. Enzymatic deglycosylation of the enzyme was essential for the production of crystals suitable for X-ray analysis for both the NEP-phosphoramidon binary complex and the apo enzyme.

Recombinant fusion proteins for the industrial production of disulfide bridge containing peptides: purification, oxidation without concatamer formation, and selective cleavage.

We report the biotechnical production of peptides of approximately 35-50 amino acids in length containing one intramolecular disulfide bridge, using a recombinant fusion tail approach. This method fills the technological gap when either (a) chemical synthesis fails due to known problematic peptide sequences or (b) if simple recombinant expression is unsuccessful due to degradation. The fusion tail described here serves several purposes: (i) it enables high expression levels in Escherichia coli to be achieved; (ii) it renders the fusion protein fairly soluble; (iii) it contains a histidine affinity tag for easy purification on Ni-chelate resins, which also serves as a catalyst for the oxygen-dependent formation of the disulfide bridge; and (iv) it suppresses the formation of concatamers during the oxidation process through steric hindrance. The purified fusion protein is then immobilized on a reversed phase column for two purposes: (i) chemical cleavage of the fusion tail by cyanogen bromide and (ii) subsequent purification of the peptide. A very hydrophilic fusion partner is required so that immobilization on the reversed phase column always occurs due to the peptide. Sensitive hydrophobic residues are thereby protected from the cleavage reagent while the cleaved hydrophilic fusion tail is easily separated from the hydrophobic peptide. The method is exemplified by eight peptides representing an immunodominant epitope of the human immunodeficiency virus, but may be useful for a significant variety of similar peptides.