Browsing by Author "Jacobs, P."
Now showing 1 - 9 of 9
Results Per Page
Sort Options
- Cloning and expression in Escherichia coli of full-length complementary DNA coding for human α1-antitrypsinPublication . Bollen, A.; Herzog, A.; Cravador, A.; Hérion, P.; Chuchana, P.; Vander Straten, A.; Loriau, R.; Jacobs, P.; Van Elsen, A.A cDNA library prepared from human liver was screened for α₁-antitrypsin, a major constituent of plasma which functions as inhibitor of proteolytic enzyms. The library was screened using a 12-base-long synthetic oligodeoxyribonucleotide corresponding to a known DNA fragment of human α₁-antitrypsin and by hybrid-selection of α₁-antitrypsin mRNA.
- High-level production of fully active human alpha 1-antitrypsin in Escherichia coli.Publication . Johansen, H.; Sutiphong, J.; Sathe, G.; Jacobs, P.; Cravador, A.; Bollen, A.; Rosenberg, M; Shatzman, A.The human alpha-1-antitrypsin (A1AT) gene expressed in Escherichia coli as a full-length, non-fusion gene product accumulates to a relatively low level approaching less than or equal to 0.1% of total cellular protein. In contrast, deletion of the first 5, 10 or 15 codons leads to production of truncated A1AT derivatives at levels between 10 and 30% of total cellular protein. The protein with the largest truncation was insoluble and inactive following solubilization by chaotropic agents. In contrast, the two derivatives with the smaller truncations were found to be soluble, and exhibit identical specific activities in both trypsin and elastase inhibition assays to authentic human A1AT. The expression of the full-length A1AT was also optimized by making silent third position mutations within its first 15 codons. These mutations were chosen to optimize codon usage and minimize the possibility of RNA secondary structure formation in this region. Via this approach, expression of full-length, authentic, fully active A1AT was increased at least 20-fold to 2% of total cellular protein. Optimal expression was obtained using as few as three silent mutations in the first five codons, confirming the importance of this 5'-terminal region as had been defined by our deletion mutants. Both the full-length derivatives as well as the small N-terminal deletion derivative can be readily purified from bacterial extracts in fully active form suitable for the examination of their potential therapeutic application.
- Molecular cloning, sequencing, and expression in Escherichia coli of human preprourokinase cDNAPublication . Jacobs, P.; Cravador, A.; Loriau, R.; Brockly, F.; Colau, B.; Chuchana, P.; Van Elsen, A.; Herzog, A.; Bollen, A.A cDNA library derived from human carcinoma cells was used to isolate a clone, pULB1000, coding for the preproenzyme form of human urokinase. This clone carries the full-length sequence coding for the signal peptide and for the A chain (157 amino acids) and B chain (253 amino acids) of urokinase in tandem. The sequence of the cDNA predicts the presence of a single lysine residue between the last amino acid of the mature A polypeptide (Phe-157) and the first amino acid of the mature B polypeptide (Ile-1). The amino acid sequence deduced from the cDNA sequence fits the published amino acid sequence data with three exceptions, the reported cysteine residue at position 131 in the A chain is a tryptophan, and glycine 366 and alanine 410 in the B chain are, respectively, a cysteine and a valine in our clone. A large Bgl I fragment (1482 bp), derived from the clone pULB1000 coding for most of the signal peptide and for the A and B chains, has been subcloned into the expression vector pCQV2. Heat induction of E. coli cells carrying the recombinant plasmid leads to the production of urokinase-like polypeptides having the expected molecular weights and being specifically recognized by antibodies raised against natural human urokinase.
- Mutant and chimeric recobinant plasminogen activatorsproduction in eukaryotic cellsand preliminary characterizationPublication . Pierard, L.; Jacobs, P.; Gheysen, D.; Hoylaerts, M.; Andre, B.; Topisirovic, L.; Cravador, A.; Deforesta, F.; Herzog, A.; Collen, D.; Dewilde, M.; Bollen, A.Mutant urokinase-type plasminogen activator (u-PA) genes and hybrid genes between tissue-type plasminogen activator (t-PA) and u-PA have been designed to direct the synthesis of new plasminogen activators and to investigate the structure-function relationship in these molecules. The following classes of constructs were made starting from cDNA encoding human t-PA or u-PA: 1) u-PA mutants in which the Arg156 and Lys158 were substituted with threonine, thus preventing cleavage by thrombin and plasmin; 2) hybrid molecules in which the NH2-terminal regions of t-PA (amino acid residues 1-67, 1-262, or 1-313) were fused with the COOH-terminal region of u-PA (amino acids 136-411, 139-411, or 195-411, respectively); and 3) a hybrid molecule in which the second kringle of t-PA (amino acids 173-262) was inserted between amino acids 130 and 139 of u-PA. In all cases but one, the recombinant proteins, produced by transfected eukaryotic cells, were efficiently secreted in the culture medium. The translation products have been tested for their ability to activate plasminogen after in situ binding to an insolubilized monoclonal antibody directed against urokinase. All recombinant enzymes were shown to be active, except those in which Lys158 of u-PA was substituted with threonine. Recombination of structural regions derived from t-PA, such as the finger, the kringle 2, or most of the A-chain sequences, with the protease part or the complete u-PA molecule did not impair the catalytic activity of the hybrid polypeptides. This observation supports the hypothesis that structural domains in t-PA and u-PA fold independently from one to another.
- Mutant and chimeric recombinant plasminogen activatorsPublication . Pierard, L.; Jacobs, P.; Gheysen, D.; Hoylaerts, M.; Cravador, A.; Herzog, A.; Collen, D.; Bollen, A.Mutant urokinase-type plasminogen activator (u-PA) genes and hybrid genes between tissue-type plasminogen activator (t-PA) and u-Pa have been designed to direct the synthesis of new plasminogen activators.
- 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.
- Total DNA synthesis and cloning in Escherichia coli of a gene coding for the human growth hormone releasing factorPublication . Cravador, A.; Jacobs, P.; Van Elsen, A.; Lacroix, C.; Colau, B.; Van Alphen, P.; Herzog, A.; Bollen, A.A DNA containing a sequence coding for the human growth hormone releasing factor (hGRF) has been obtained by enzymatic assembly of chemically synthesized DNa fragments. The synthetic gene consists of a 140 base-pair fragment containing initiation and termination signals for translation and appropriate protruding ends for cloning into a newly constructed plasmid vector (pULB1219). Eleven oligodeoxyribonucleotides, from 14 to 31 bases in length, sharing pairwise stretches of complementary regions of at least 13 bases were prepared by phosphotriester solid-phase synthesis. The DNA sequence was designed to take into account the optimal use of E. coli codons. Oligomers were annealed in one step and assembled by ligation. The DNA fragment of the expected size (140 bp) was recovered and cloned into the pULB1219 vector. The expected sequence was confirmed by DNA sequencing. © 1985 Masson, Paris.