Expression and analysis of the NS2 protein of influenza A virus.

Influenza NS2 protein was expressed in Saccharomyces cerevisiae using a copper-inducible promoter. The protein produced had a molecular weight of 13 kDa, was reactive with anti-NS2 antiserum and was localised to the yeast cell nucleus. Two-hybrid analysis identified a direct protein-protein interaction between NS2 and the M2 protein of the virus, involving the C-terminal 163 residues of M1.

Evaluation of complete genome sequences and sequences of individual gene products for the classification of hepatitis C viruses.

Comparisons of genome and polyprotein sequences of hepatitis C virus (HCV) isolates world-wide has led to the identification of nine major genotypes and many subtypes. This classification is based on either complete genome/polyprotein sequences or sequence data from the 5' noncoding region, core, E1, NS3 or NS5B genes. The relative merit of different gene segments as taxonomic markers and the validity of the resulting assignments is not clear at this stage.

Changes in the NS gene of neurovirulent strains of influenza affect splicing.

The nonstructural (NS) gene has been identified as an accessory factor in determining neurovirulence for influenza A virus. The nucleotide sequence of the NS gene of the neurovirulent variant A/NWS/33 was determined and compared with its parent, A/WS/33. Alterations in the mRNA structure of the gene were observed that serve to mask the 3' splice site.

Structure-based drug design.

There are now many successful examples of the design of new ligands based on knowledge of target protein structures. In most cases those ligands are unsuitable as drugs because of problems of toxicity, stability or bioavailability. The past twelve months have also seen the description of the structures of many proteins which are either known to be targets for existing drugs or have clear potential to be utilized in therapy.

A common structural motif incorporating a cystine knot and a triple-stranded beta-sheet in toxic and inhibitory polypeptides.

A common structural motif consisting of a cystine knot and a small triple-stranded beta-sheet has been defined from comparison of the 3-dimensional structures of the polypeptides omega-conotoxin GVIA (Conus geographus), kalata BI (Oldenlandia affinis DC), and CMTI-I (Curcurbita maxima). These 3 polypeptides have diverse biological activities and negligible amino acid sequence identity, but each contains 3 disulfide bonds that give rise to a cystine knot. This knot consists of a ring formed by the first 2 bonds (1-4 and 2-5) and the intervening polypeptide backbone, through which the third disulfide (3-6) passes.

Influenza virus neuraminidase: structure, antibodies, and inhibitors.

The determination of the 3-dimensional structure of the influenza virus neuraminidase in 1983 has served as a platform for understanding interactions between antibodies and protein antigens, for investigating antigenic variation in influenza viruses, and for devising new inhibitors of the enzyme. That work is reviewed here, together with more recent developments that have resulted in one of the inhibitors entering clinical trials as an anti-influenza virus drug.

Fusogenic activity of amino-terminal region of HIV type 1 Nef protein.

We have studied two isoforms of Nef, Nef-27 and Nef-25, which were produced in E. coli. Nef-25 lacked the first 18 N-terminal residues of Nef-27 and both were nonmyristylated.

Homology modelling and 1H NMR studies of human leukaemia inhibitory factor.

Human leukaemia inhibitory factor (LIF) is a glycoprotein with a diverse range of activities on many cell types. A molecular model of LIF has been constructed based mainly on the structure of the related cytokine granulocyte colony-stimulating factor, and refined using simulated annealing and molecular dynamics in water. The model was stable during molecular dynamics refinement and is consistent with known stereochemical data on proteins.

The structure of a complex between the NC10 antibody and influenza virus neuraminidase and comparison with the overlapping binding site of the NC41 antibody.

Background: While it is well known that different antibodies can be produced against a particular antigen, and even against a particular site on an antigen, up until now there have been no structural studies of cross-reacting antibodies of this type. One antibody-antigen complex whose structure is known is that of the influenza virus antigen, neuraminidase, in complex with the NC41 antibody. Another anti-neuraminidase antibody, NC10, binds to an overlapping site on the antigen.

Multiple conformations of the sea anemone polypeptide anthopleurin-A in solution.

Anthopleurin-A (AP-A) is a member of a family of sea anemone-derived polypeptides that interact with sodium channels in a voltage-dependent manner, producing a positive inotropic effect on the mammalian heart. There has been considerable interest in this molecule as a lead compound for the development of novel therapeutic agents. Earlier attempts to define the 3-dimensional structure of AP-A were complicated by the fact that it was found to exist in 2 conformations in solution.

Heterologous gene expression and protein secretion from Candida glabrata.

We have examined heterologous protein secretion from Candida glabrata with the aid of a stable C. glabrata vector and a secretion reporter cassette comprising the Saccharomyces cerevisiae PGK-gene promoter and a Kluveromyces Iactis secretion signal to drive secretion of Escherichia coli beta-lactamase. Abundant secretion of beta-lactamase from C.

Structure of phaseolin at 2.2 A resolution. Implications for a common vicilin/legumin structure and the genetic engineering of seed storage proteins.

The refinement to 2.2 A resolution of the three-dimensional structure of the seed storage protein phaseolin from the French bean (Phaseolus vulgaris) via an alternative crystal form is described. The refined structure reveals details of the molecule hitherto unobserved and in particular we identify the structural role of conserved residues within the broader 7 S (vicilin) family of seed storage proteins.

The three-dimensional structure of N-acetylneuraminate lyase from Escherichia coli.

Background: N-acetylneuraminate lyase catalyzes the cleavage of N-acetylneuraminic acid (sialic acid) to form pyruvate and N-acetyl-D-mannosamine. The enzyme plays an important role in the regulation of sialic acid metabolism in bacteria. The reverse reaction can be exploited for the synthesis of sialic acid and some of its derivatives.

High-level secretion of correctly processed beta-lactamase from Saccharomyces cerevisiae using a high-copy-number secretion vector.

We have sought to obtain a convenient system for the high-level production of secreted proteins in yeast. With the aid of a secretion reporter cassette we examined the secretion of beta-lactamase (Bla) as a model protein and found the highest expression in Saccharomyces cerevisiae using a high-copy-number plasmid. We further developed the high-copy-number plasmid introducing a secretion cassette that has a convenient cloning site coinciding with the sequence encoding the KEX2 cleavage site.

Vectors for Cu(2+)-inducible production of glutathione S-transferase-fusion proteins for single-step purification from yeast.

We describe six new yeast episomal vectors which encode glutathione S-transferase (GST) affinity tags. These allow for the production of GST-fusion proteins in Saccharomyces cerevisiae under the control of the CUP1 promoter. Affinity chromatography with glutathione-Sepharose permits convenient purification of the fusion protein from a yeast lysate.

Three-dimensional structures of two plant beta-glucan endohydrolases with distinct substrate specificities.

The three-dimensional structures of (1-->3)-beta-glucanase (EC 3.2.1.

Large-scale production and characterization of recombinant human immunodeficiency virus type 1 Nef.

Sequences encoding the 27K and 25K nef gene products (Nef 27 and Nef 25) were amplified by PCR from a human immunodeficiency virus type 1 infectious clone and subcloned directly into Escherichia coli, yeast and baculovirus expression vectors. The yeast- and baculovirus-derived Nef had native N termini but the expression levels were low. The expression levels of the E.

Expression and characterisation of the influenza A virus non-structural protein NS1 in yeast.

The influenza A virus non-structural protein NS1 was produced using a copper-inducible expression system in the yeast Saccharomyces cerevisiae. The protein produced had a molecular weight of 26 kDa by SDS-PAGE and was reactive with anti-NS1 antisera. The recombinant NS1 protein was targetted to the nucleolus/nuclear envelope fraction of the yeast cell nucleus, showing that its localisation signals remain functional in yeast.

NMR studies of a murine-human chimera of leukaemia inhibitory factor (LIF). Comparison with human LIF.

Leukaemia inhibitory factor (LIF) is a polyfunctional cytokine active on many cell types. We present here 1H NMR studies on the solution properties and stability of MH35, a chimera of murine and human LIF which can be expressed at high levels in Escherichia coli, thus enabling efficient labelling of the protein with the stable isotopes 13C and 15N. MH35 has 85% sequence identity with human LIF and similar activity in biological assays.

Shape complementarity at protein/protein interfaces.

A new statistic Sc, which has a number of advantages over other measures of packing, is used to examine the shape complementarity of protein/protein interfaces selected from the Brookhaven Protein Data Bank. It is shown using Sc that antibody/antigen interfaces as a whole exhibit poorer shape complementarity than is observed in other systems involving protein/protein interactions. This result can be understood in terms of the fundamentally different evolutionary history of particular antibody/antigen associations compared to other systems considered, and in terms of the differing chemical natures of the interfaces.

Three-dimensional structure in solution of the calcium channel blocker omega-conotoxin.

The 27 amino acid residue polypeptide omega-conotoxin GVIA, from venom of the cone shell Conus geographus, blocks neuronal voltage-activated calcium channels at picomolar concentrations. The three-dimensional structure in aqueous solution of synthetic omega-conotoxin has been determined from two-dimensional 1H n.m.

Sequence and structure alignment of paramyxovirus hemagglutinin-neuraminidase with influenza virus neuraminidase.

A model is proposed for the three-dimensional structure of the paramyxovirus hemagglutinin-neuraminidase (HN) protein. The model is broadly similar to the structure of the influenza virus neuraminidase and is based on the identification of invariant amino acids among HN sequences which have counterparts in the enzyme-active center of influenza virus neuraminidase. The influenza virus enzyme-active site is constructed from strain-invariant functional and framework residues, but in this model of HN, it is primarily the functional residues, i.

Expression of HIV-1 nef in yeast: the 27 kDa Nef protein is myristylated and fractionates with the nucleus.

The nef gene of human immunodeficiency virus type 1 (HIV-1) has been expressed in the yeast Saccharomyces cerevisiae to produce native Nef proteins. The proteins of M(r) 27 kDa and 25 kDa, produced by translation from the first and second start codons of the nef gene react with human HIV-1 antisera. Under low-level steady-state expression conditions, Nef27 undergoes myristylation and is targeted to the nuclear fraction while Nef25 is not myristylated and not nuclear localized.