ASAP: analysis of peptide composition.

SUMMARY: ASAP is a web tool designed to search for specific dipeptides, tripeptides and tetrapeptides in a protein sequence database. The server allows for: (a) identification of frequent and infrequent peptides and the creation of peptide probability tables for a given database of sequences (GenerNet program), (b) determination of the compatibility of an amino-acid sequence to the given peptide probability tables (ClonErrNet program); and (c) comparison of different protein databases based on peptide composition (CompNet program). ASAP server can be useful in protein engineering and/or protein classification studies.

Expression in Escherichia coli and purification of soluble forms of the F protein of bovine respiratory syncytial virus.

Six fragments of the F gene from bovine respiratory syncytial virus (BRSV) were engineered into the pMAL-c2 Escherichia coli expression vector and expressed as C-terminal maltose-binding protein (MBP) fusion products. The resulting polypeptides were partially soluble and single-step purified by affinity chromatography. These fusion proteins were recognized in Western blots by several MAbs directed against human respiratory syncytial virus F protein. In addition, rabbit polyclonal antisera raised against two purified MBP-derived proteins reacted with the BRSV-F protein.

Glycoprotein E of bovine herpesvirus type 1 is involved in virus transmission by direct cell-to-cell spread.

In order to identify the role of the bovine herpesvirus type 1 (BHV-1) glycoprotein E (gE) in the viral infection cycle, we have constructed a BHV-1 gE deletion mutant strain (BHV-1 gE-). This strain was assayed in vitro by comparing its growth kinetics with the wild type strain used as a host of the deletion. Our results indicate that those conditions which prevent the infection by direct adsorption to the cells (presence of a semi-solid medium or presence of neutralizing antibodies in the medium) selectively inhibit the growth of the gE- strain, suggesting that gE plays a central role in the BHV-1 spread by direct cell-to-cell transmission, a major mechanism of the BHV-1 in vivo virulence.

Properties of a novel glucose-enhanced beta-glucosidase purified from Streptomyces sp. (ATCC 11238).

An inducible intracellular beta-glucosidase (EC 3.2.1.21) from Streptomyces sp. QM-B814 (ATCC 11238) has been purified and characterized. The purified polypeptide is monomeric with a relative molecular mass of 62 kDa by SDS-PAGE and 42 kDa by size-exclusion chromatography; its isoelectric point is 4.2. The difference in the molecular mass values can be attributed to the glycosylated nature of the protein. The purified enzyme has a pH optimum of 6.0-6.5. The temperature optimum for activity is 50 degrees C; at this temperature the enzyme is stable for 1 h. The enzyme hydrolyzes mainly aryl-beta-glucosides but also presents significant activity against beta-linked disaccharides and maltose. The enzyme displays an unusual kinetic behavior and biphasic Lineweaver-Burk and Eadie-Hofstee plots for p-nitrophenyl-beta-D-glucoside and cellobiose were obtained. The enzyme presents beta-glycosyltransferase activity and an exoglycosidase-type action on cellodextrins. It is inhibited by delta-gluconolactone (Ki 0.44 mM) but, remarkably, glucose in the range 25-200 mM enhances the rate of p-nitrophenyl-beta-D-glucoside hydrolysis.

Induction and preliminary characterization of intracellular beta-glucosidases from a cellulolytic Streptomyces strain.

The cellulolytic actinomycete Streptomyces sp. QM-B814 possess an intracellular beta-glucosidase system which is induced by cellobiose and carboxymethylcellulose. Maximal beta-glucosidase activity was attained 8-10 h after inducer addition to exponential phase growing cultures. The induction is depressed in the presence of glucose. The system is composed of two electrophoretically different beta-glucosidases forms showing relative molecular masses of about 60 and 35 kDa, and pI values in the range 4.2-4.5. Both beta-glucosidases are synthesized de novo. The enzymes share substrate preference and are both inhibited by delta-gluconolactone and p-chloromercuribenzoate. The induction pattern and glucose inhibition are similar for both enzymes.

Mapping, cloning and sequencing of a glycoprotein-encoding gene from bovine herpesvirus type 1 homologous to the gE gene from HSV-1.

In order to map and identify the glycoprotein-encoding gene from bovine herpesvirus type 1 (BHV-1), homologous to the gE glycoprotein from herpes simplex virus type 1 (HSV-1), a region of the unique short sequence from the BHV-1 genome has been sequenced. The sequenced region contains an ORF coding for a polypeptide of 575 amino acids (aa). The aa sequence presents substantial similarity to that of the glycoprotein gE from HSV-1 and to homologous proteins of related viruses such as pseudorabies virus, equine herpesvirus type 1 and varicella zoster virus. The aa sequence presents additional characteristics compatible with the structure of a viral glycoprotein: signal peptide, putative glycosylation sites and a long C-terminal transmembrane alpha-helix.

A beta-glucosidase gene (bgl3) from Streptomyces sp. strain QM-B814. Molecular cloning, nucleotide sequence, purification and characterization of the encoded enzyme, a new member of family 1 glycosyl hydrolases.

A beta-glucosidase gene (bgl3) from Streptomyces sp. QM-B814 (American Type Culture Collection 11238) has been cloned by functional complementation of a beta-glucosidase-negative mutant of Streptomyces lividans. An open-reading frame of 1440 nucleotides encoding a polypeptide of 479 amino acids was found by sequencing. The encoded protein (Bgl3) shows extensive similarity (over 45% identity) with beta-glycosidases from family-1 glycosyl hydrolases. The cloned enzyme, purified following ammonium sulphate precipitation and two chromatographic steps, is monomeric with molecular mass 52.6 kDa, as determined by mass spectrometry, and an isoelectric point of pI 4.4. The enzyme appears to be a beta-glucosidase with broad substrate specificity, is active on cellooligomers, and performs transglycosylation reactions. The estimated apparent Km values for p-nitrophenyl-beta-D-glucopyranoside and cellobiose are 0.27 mM and 7.9 mM, respectively. The Ki values for glucose and delta-gluconolactone, using p-nitrophenyl-beta-D-glucopyranoside as a substrate, are 65 mM and 0.08 mM, respectively. The purified enzyme has a pH optimum of pH 6.5 and the temperature optimum for activity is 50 degrees C.