Browsing by Author "Guillaume, J. P."
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- Porcine D-amino acid oxidase: determination of the mRNA nucleotide sequence by the characterization of genomic and cDNA clonesPublication . Jacobs, P.; Brockly, F.; Massaer, M.; Loriau, R.; Guillaume, J. P.; Ciccarelli, E.; Heinderyckx, M.; Cravador, A.; Biemans, R.; Van Elsen, A.; Herzog, A.; Bollen, A.Oligodeoxynucleotide probes derived from the published amino acid (aa) sequence for D-amino acid oxidase (DAO) [Ronchi et al. J. Biol. Chem. 259 (1982) 8824-8834] were used to screen cDNA libraries made from porcine kidney cortex and liver. Whereas no clones were obtained from kidney mRNAs, 20 independent ones were isolated from the liver library. Surprisingly, all of them carried only partial cDNAs for DAO starting around aa 100 in the coding sequence and extending for up to 250 bp in the 3'-noncoding sequence. One of these clones, pULB9103, was used to screen a porcine genomic library and allowed the isolation of DAO gene clone phULB001. Four exons encoding aa 1-151 were identified and sequenced, as well as the relevant exon-intron junctions. The mRNA sequence coding for DAO has been reconstituted from the genomic and cDNA sequences; its analysis by computer did not reveal and significant secondary structure, or particular feature, which could explain the failure to obtain full-length cDNAs.
- Porcine D-amino acid oxidase: production of the biologically active enzyme in Escherichia coliPublication . Ciccarelli, E.; Massaer, M.; Guillaume, J. P.; Herzog, A.; Loriau, R.; Cravador, A.; Jacobs, P.; Bollen, A.DNA molecules coding either for mature porcine D-amino acid oxidase or for truncated forms of the enzyme have been obtained by stepwise addition of synthetic oligonucleotides to a partial cDNA. Under the control of the λ P(L) thermoregulatable promoter, these DNAs were respectively expressed in Escherichia coli as 36, 28 and 25 kilodalton polypeptides, specifically recognised by antibodies raised against the natural enzyme. None of the truncated proteins were biologically active whereas the mature recombinant species was able to hydrolyze D-alanine in vitro as efficiently as the natural product.
- Production of human recombinant proapolipoprotein A-I in Escherichia coli: purification and biochemical characterizationPublication . Moguilevsky, N.; Roobol, C.; Loriau, R.; Guillaume, J. P.; Jacobs, P.; Cravador, A.; Herzog, A.; Brouwers, L.; Scarso, A.; Gilles, P.; Homquist, L.; Carlson, L. A.; Bollen, A.A human liver cDNA library was used to isolate a clone coding for apolipoprotein A-I (Apo A-I). The clone carries the sequence for the prepeptide (18 amino acids), the propeptide (6 amino acids), and the mature protein (243 amino acids). A coding cassette for the proapo A-I molecule was reconstructed by fusing synthetic sequences, chosen to optimize expression and specifying the amino-terminal methionine and amino acids -6 to +14, to a large fragment of the cDNA coding for amino acids 15-243. The module was expressed in pOTS-Nco, an Escherichia coli expression vector carrying the regulatable X P^ promoter, leading to the production of proapolipoprotein A-I at up to 10% of total soluble proteins. The recombinant polypeptide was purified and characterized in terms of apparent molecular mass, isoelectric point, and by both chemical and enzymatic peptide mapping. In addition, it was assayed in vitro for the stimulation of the enzyme lecithin: cholesterol acyltransferase. The data show for the first time that proapo A-I can be produced efficiently in E. coli as a stable and undegraded protein having physical and functional properties indistinguishable from those of the natural product.