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A modified recombinant protein derived from the viral capsid of the human immunodeficiency virus (HIV) was coupled to a copolymer of maleic anhydride and methyl vinyl ether (MAMVE). The conjugated copolymer was spotted versus free protein in 96-well microplates. The conjugated copolymer largely improved detection sensitivity of anti-P24 antibodies in infected human sera. Results obtained with immuno-linked sorbent array (ILISA) were correlated with those generated with a standard enzyme-linked immunosorbent assay (ELISA). Serum dilution dynamic range was varied from 1/5000 to 1/150, and inter-well coefficient of variation was below 8%. Copyright © 2004 Society of Chemical Industry

Pyrrolidone carboxyl peptidase (EC 3.4.11.8) (Pcp) is an enzyme that catalyzes the removal of the N-terminal pyroglutamyl group from some poptides or proteins. Its value in protein chemistry and bacterial diagnosis makes this enzyme an interesting subject of study. The present paper reports for the first time the cloning and characterization of a pyrrolidone carboxyl peptidase gene ( pcp). This gene is present in a single copy in the genome of Bacillus subtilisas indicated by Southern blot hybridization analysis. The pcptranscripts were analyzed in Escherichia coliby Northern blot hybridization and S1 nuclease mapping. The deduced amino acid sequence predicts a protein of 215 amino acids with a calculated molecular weight of 23,777 Da. The pcpgene has been over-expressed in E. coli, allowing the identification and partial characterization of Pcp protein.

Pyrrolidone carboxyl peptidase (EC 3.4.11.8) (Pcp), an enzyme which selectively removes pyrrolidone carboxylic acid (PCA) from some PCA-peptides and -proteins, was demonstrated in bacteria and in plant, animal and human tissues. In this paper we describe the purification to homogeneity of the enzyme of Streptococcus pyogenes, over-expressed in Escherichia coli. This was achieved, for the first time in one step, by hydrophobic interaction chromatography. Analysis under non-denaturing conditions revealed a molecular mass of 85 kDa and in the presence of sodium dodecyl sulfate gave a molecular mass of 23.5 kDa. Investigations on enzymatic properties showed that the Pcp over-expressed in E. colidisclosed properties similar to those found for the enzyme extracted from S. pyogenesor for some other Pcps studied previously. Thus the over-expressed enzyme should serve as a suitable source for N-terminal unblocking prior to some PCA protein sequencing.

Pyrrolidone carboxyl peptidase (EC 3.4.11.8) (Pcp), an enzyme which selectively removes pyrrolidone carboxylic acid (PCA) from some PCA‐peptides and ‐proteins, was demonstrated in bacteria and in plant, animal and human tissues. In this paper we describe the purification to homogeneity of the enzyme of Streptococcus pyogenes, over‐expressed in Escherichia coli. This was achieved, for the first time in one step, by hydrophobic interaction chromatography. Analysis under non‐denaturing conditions revealed a molecular mass of 85 kDa and in the presence of sodium dodecyl sulfate gave a molecular mass of 23.5 kDa. Investigations on enzymatic properties showed that the Pcp over‐expressed in E. colidisclosed properties similar to those found for the enzyme extracted from S. pyogenesor for some other Pcps studied previously. Thus the over‐expressed enzyme should serve as a suitable source for N‐terminal unblocking prior to some PCA protein sequencing.

Pyrrolidone carboxyl peptidase (EC 3.4.11.8) (Pcp) is an enzyme that catalyzes the removal of the N‐terminal pyroglutamyl group from some poptides or proteins. Its value in protein chemistry and bacterial diagnosis makes this enzyme an interesting subject of study. The present paper reports for the first time the cloning and characterization of a pyrrolidone carboxyl peptidase gene (pcp). This gene is present in a single copy in the genome of Bacillus subtilisas indicated by Southern blot hybridization analysis. The pcptranscripts were analyzed in Escherichia coliby Northern blot hybridization and S1 nuclease mapping. The deduced amino acid sequence predicts a protein of 215 amino acids with a calculated molecular weight of 23,777 Da. The pcpgene has been over‐expressed in E. coli, allowing the identification and partial characterization of Pcp protein.

The occurrence of β-glucuronidase activity, a main characteristic of Escherichia coli and the presence of the uid chromosomal region of E. coli, coding for this enzyme, were tested on representative members of enteric bacteria. DNA hybridization techniques using uid probes and ampplification experiments of uidA gene by the polymerase chain reaction (PCR) confirmed the specificity of uid genes fro E. coli and Shigella spp. (i.e., S. boydii, S. dysenteriae, S. flexneri and S. sonnei), independent of the β-glucuronidase phenotype of bacterial strains. This specificity seemed to be conserved when studies were extended to a wide range of bacteria. It was not possible to distinguish E. coli from Shigella spp. The detection sensitivity using double stranded DNA radiolabeled probes was 3 × 104 bacteria and could be brought down to 8 bacteria by PCR. Thus, the uid genes appeared to be ideal candidates for DNA probes technology to detect E. coli-Shigella species.