We have constructed a series of random N-terminal deletions of the large subunit (R1) of the herpes simplex virus type 1 ribonucleotide reductase. Deletions extended throughout the R1 gene open reading frame and, in total, 31 different truncated polypeptides were expressed in Escherichia coli using the T7 expression system. N-Terminal truncations were analyzed for their interaction with the small subunit (R2) of ribonucleotide reductase using a sensitive enzyme-linked immunosorbent assay (ELISA) method and for their ability to complement R2 in ribonucleotide reductase assays. Truncated proteins were also tested for homodimerization using gel-filtration chromatography. The results identified a region of R1 between amino acids 349 and 373 which was essential for subunit interaction. Proteins lacking up to 348 amino-terminal residues associated with R2 and complemented R2 in ribonucleotide reductase assays. Proteins commencing at amino acid 373 and beyond did not interact with R2 and were inactive in enzyme assays. Using a plasmid which expressed an N-terminal deleted protein commencing at amino acid 247, we constructed two defined C-terminal deletions to give proteins comprising amino acids 247-434 and 247-996 of R1. Neither of these truncated proteins bound R2 and we concluded that a second region between amino acids 996 and 1137 (the C-terminus) is required for interaction with R2. Gel-filtration studies indicated that deletion of the first 420 amino acids from R1 did not affect dimerization. However, deletions of 457 amino acids and larger gave proteins which existed as monomers.(ABSTRACT TRUNCATED AT 250 WORDS)

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Glutamate Carboxypeptidase II (also known as Prostate Specific Membrane Antigen-PSMA) is an important marker in the diagnosis of prostate cancer, however, relatively little is known about its biochemical and structure-function characteristics. We have expressed mutant forms of PSMA and have started to address the roles of three putative domains of PSMA in its cellular localization and peptidase activity. Three mutants, a full-length recombinant PSMA (rPSMA-FL), one expressing only the proposed extracellular domain of PSMA (rPSMA-ECD) and one form omitting the proposed transmembrane domain (rPSMA-deltaTMD) have been produced in human cells via a mammalian expression vector system. We show that rPSMA-FL is associated with the cell surface membrane; so too is rPSMA-deltaTMD even though it lacks the proposed transmembrane domain, whereas rPSMA-ECD has a cytosolic localization. Only rPSMA-FL retains functional hydrolytic activity and is similarly glycosylated to PSMA found in the cultured prostate cancer cell line LNCaP.

This transaction reports on the on-going attempt to isolate anti-peptide scFv antibodies from an scFv phage-display library capable of detecting the native protein from which the peptide sequence was derived.The display of antibody fragments on the surface of filamentous bacteriophage by fusion to the minor coat protein (plll) and selection of phage with antigen, has provided a powerful method of generating antibodies of pre-defined specificity without the need for immunization (for review seeHaving successhlly generated antibodies against human cathepsin S utilizing homology-based molecular modelling of cathepsin S, epitope prediction and multiple antigenic peptide synthesis followed by polyclonal antibody production in rabbits [2], it was wondered whether a linear version of the peptides would enable the isolation of scFv antibodies from a phage display library capable of cross-reacting with native cathepsin S (Table 1) Conc. of antigen Washing (Pglml) protocol 50 1 OxMPBS, 1 OxPBS 50 20xMPBS, 20xPBS 25 I O W B S , IOxPBS 25 20xMF'BS, 20xPBS 10 20xMPBS, 20xPBSFollowing each stage of biopanning a polyclonal phage mixture of the eluted phage prepared by precipitation using polyethylene glycol [5] was utilized in a dot-blotting experiment to determine binding to antigen. Essentially, antigen (100-300 ng, in water) was dotted onto nitrocellulose strips and allowed to dry. Following blocking of non-specific binding sites on the strips using 3% MPBS the blot was challenged with the polyclonal phage solution (in PBS) overnight with shaking at room temperature. Following extensive washing with PBS, a monoclonal anti-MI3 horseradish peroxidase conjugated antibody (Pierce) was used to detect bound phage (according to manufacturer's protocols).Atter five rounds of panning, polyclonal phage solution was seen to detect antigen (100 ng) well above background staining (by visual inspection).Polyclonal phage isolated from the fifth biopanning were reinfected into Ikherichin coli TGI cells and plated onto large bioassay dishes using appropriate selective media. Ninety individual colonies were selected at random from these plates and propagated to produce monoclonal phage solutions. Each of these phage solutions were utilized in a dot-blotting experiment essentially as above with the difference that a 96well dot-blot apparatus was used (Biorad, according to manufacturer's instructions) and I00 ng antigen was used. From the ninety clones, at least 23 were seen to recognize the antigen (by visual inspection) above background levels.Fourteen positive and three negative clones were chosen for characterization utilizing DNA sequencing of the CDR3 region.Analysis of the DNA sequences and assessment as to whether any of the clones isolated will cross-react with native cathepsin S and/or cathepsins B and L is in progress.This approach if successful will allow the generation of antibodies to proteins of biological interest, but of limited supply, if the primary sequence of the protein is known and possible antigenic regions can be predic...

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