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E Degryse

Publications and source records attributed to E Degryse.

33 records · Page 2Linked to original sources
PubMed1991

Stability of a host-vector system based on complementation of an essential gene in Escherichia coli.

Antibiotic selection is the most common selection system for plasmid-containing bacteria. This technique, nevertheless, can be a source of problems during the expression of heterologous genes in Escherichia coli. We have developed an alternative selection system based on the complementation of a chromosomal auxotrophic (dapD2) mutation by the corresponding wild type gene carried on a plasmid. We show that the system effectively selects for the presence of plasmid on solid and liquid medium. In addition, we have observed a loss of viability associated with high levels of gene expression and accumulation of a heterologous protein, but the selective power and improved intrinsic stability of the dap+ plasmid, compared to a beta-lactamase (bla) based vector, excludes overgrowth of the culture by plasmidless cells.

Cloning, Molecular↗
PubMed1990

Influence of the second and third codon on the expression of recombinant hirudin in E. coli.

The effect of all possible codons corresponding to the second and third amino acid (isoleucine and threonine) on the expression level of hirudin in E. coli has been analysed. These levels could not be correlated with changes in primary and secondary mRNA structure. A decrease in the rate of synthesis and of product accumulation follows the introduction for ile of the ATA codon which is of very low usage, and for thr of the ACC codon, which results in homology of the mRNA with the 3'-end of 16S rRNA. The results are discussed according to current concepts of protein expression in E. coli.

Base Sequence↗
PubMed1990

Conversion of recombinant hirudin to the natural form by in vitro tyrosine sulfation. Differential substrate specificities of leech and bovine tyrosylprotein sulfotransferases.

Hirudin, a tyrosine-sulfated protein secreted by the leech Hirudo medicinalis, is one of the most potent anticoagulants known. The hirudin cDNA has previously been cloned and has been expressed in yeast, but the resulting recombinant protein was found to be produced in the unsulfated form, which is known to have an at least 10 times lower affinity for thrombin than the naturally occurring tyrosine-sulfated hirudin. Here we describe the in vitro tyrosine sulfation of recombinant hirudin by leech and bovine tyrosylprotein sulfotransferase (TPST). With both enzymes, in vitro sulfation of recombinant hirudin occurred at the physiological site (Tyr-63) and rendered the protein biochemically and biologically indistinguishable from natural hirudin. However, leech TPST had an over 20-fold lower apparent Km value for recombinant hirudin than bovine TPST. Further differences in the catalytic properties of leech and bovine TPSTs were observed when synthetic peptides were tested as substrates. Moreover, a synthetic peptide corresponding to the 9 carboxyl-terminal residues of hirudin (which include Tyr-63) was sulfated by leech TPST with a similar apparent Km value as full length hirudin, indicating that structural determinants residing in the immediate vicinity of Tyr-63 are sufficient for sulfation to occur.

Adrenal Medulla↗
PubMed1990

Inhibition by recombinant hirudins of experimental venous thrombosis and disseminated intravascular coagulation induced by tissue factor in rats.

Antithrombotic potency of recombinant hirudins rHV2, rHV2-Lys47 and rHV2-Arg47 was studied in a model of experimental thrombosis induced by tissue factor in the rat. Venous thrombosis was induced by i.v. injection of 25 mg/kg tissue factor followed by stasis of the inferior vena cava. In this model natural recombinant hirudins, rHV2 and rHV2-Lys47 injected 5 min before thrombo-plastin totally inhibited thrombosis in the same micrograms range as heparin or natural hirudin extracted from leeches. However, the mutant variant rHV2-Arg47 gave a maximal 60% inhibition of thrombosis. Variants rHV2-Lys47 (30 micrograms/kg) and rHV2-Arg47 (157 micrograms/kg) injected 5 min before thromboplastin prevented by 90 to 100% the drop in platelet count observed during the disseminated intravascular coagulation induced by thromboplastin injection. Recombinant hirudins were less anticoagulant than heparin as measured by an APTT on rat plasma. After rat tail transection, rHV2-Lys47 caused a 2-fold smaller prolongation of the bleeding time than an equivalent antithrombotic dose of heparin. Plasmatic elimination of rHV2-Lys47 from rat plasma after i.v. injection had a fast distribution phase with a half-life of 3 min during which 90% of injected rHV2-Lys47 was lost and was followed by a slower elimination phase. Thus recombinant hirudin rHV2-Lys47 appears as a promising potent antithrombotic agent for the prevention of thrombin-dependent venous thrombosis and disseminated intravascular coagulation.

Animals↗
PubMed1990

Evidence for extended maintenance of the corpus luteum by uterine infusion of a recombinant trophoblast alpha-interferon (trophoblastin) in sheep.

Ovine trophoblastin (oTP) is a natural interferon of the class-II interferon-alpha subfamily. Recombinant ovine trophoblastin (r.oTP), produced by genetic engineering, was purified by anion-exchange HPLC. The product exhibited a high degree of homogeneity (greater than 98%), and similar immunological cross reaction and antiviral activity to natural oTP. Antiluteolytic activity of r.oTP was established by intrauterine injection in two groups of cyclic recipient ewes. Control group A included 10 ewes which received sterile BSA in saline twice daily for 8 days (from day 10-12 of oestrous cycle). Experimental group B included 17 ewes which received 80 micrograms (4 ewes), 170 micrograms (8 ewes) or 340 micrograms (5 ewes) r.oTP daily for 8 days. Maintenance of functional corpora lutea for 1 month or more was observed in 4 out of 5 ewes which received high doses of r.oTP. These results indicate that oTP alone extends luteal secretory activity.

Animals↗
PubMed1989

Production and evaluation of recombinant hirudin.

Hirudin, a 65 amino acid polypeptide form the medicinal leech, is an extremely efficient and specific thrombin inhibitor whose therapeutic potential has been demonstrated in a number of animal models. We have developed protocols for the production of recombinant hirudin by secretion from S. cerevisiae and carried out a full biologic evaluation of the purified product. These studies showed that natural and recombinant hirudin was similar in structure and in biologic function in vitro. Moreover, the recombinant protein displayed strong antithrombotic activity in several experimental thrombosis models in vivo, confirming the molecule's promise in the therapy of thrombotic disorders.

Amino Acid Sequence↗
PubMed1989

Point mutations modifying the thrombin inhibition kinetics and antithrombotic activity in vivo of recombinant hirudin.

The hirudin variant HV2 was modified by in vitro site-specific mutagenesis of HV2 cDNA to generate HV2(Asn-47----Lys), HV2(Asn-47----Arg) and HV2(Lys-35----Thr, Asn-47----Lys). Residues 35 and 47 are positioned respectively within the finger and prothrombin-like domains of hirudin, both of which have been suggested as thrombin binding sites. The modified polypeptides were synthesized in Saccharomyces cerevisiae using a secretion vector and purified from culture supernatants. By analysis of the human alpha-thrombin:hirudin inhibition reaction in steady-state conditions it was shown that the dissociation constants for HV2(Lys-47) and HV2(Arg-47) were 5- to 14-fold lower than for unmodified HV2, whereas mutation of Lys-35 did not significantly alter the inhibition kinetics. Furthermore, HV2(Lys-47), whose sequence is identical to a natural hirudin variant, displayed enhanced anti-thrombotic activity in vivo, having a 100-fold lower ED50 compared to HV2 in the rabbit Wessler venous thrombosis model. These results support a role for the prothrombin-like domain in thrombin binding and, moreover, demonstrate that in vivo antithrombotic efficiency correlates with the dissociation constant of the inhibition reaction.

Amino Acid Sequence↗
PubMed1988

Use of human surfactant low molecular weight apoproteins in the reconstitution of surfactant biologic activity.

Two low molecular weight (LMW) apoproteins were isolated from human pulmonary surfactant. SDS polyacrylamide gel analysis showed one protein (SP 18) to have an apparent molecular weight of 18,000 when unreduced and 9,000 D after reduction. The second protein (SP 9) migrated at approximately 9,000 D in the presence or absence of reducing agents. Both proteins contain a high number of hydrophobic amino acids. The NH2-terminal sequence of SP 18 was determined to be: NH2-phe-pro-ile-pro-leu-pro-tyr-. A cDNA clone isolated from a human adult lung cDNA library contained a long open reading frame encoding at an internal position the human SP 18 amino-terminal sequence. Mixtures of phospholipids (PL) and SP 9 and SP 18 were assessed for their capacity to reduce surface tensions on a pulsating bubble surfactometer. The addition of 1% apoprotein resulted in a reduction of surface tension after 15 s from 42.9 dyn/cm for PL alone to 16.7 and 6.3 dyn/cm for preparations containing SP 9 and SP 18, respectively. In vivo assessment of reconstituted surfactant activity was performed in fetal rabbits. Reconstituted surfactant consisting of PL + 0.5% SP 18 instilled intratracheally at delivery resulted in a marked increase in lung compliance, while the incorporation of 0.5% SP 9 yielded a moderate increase. These data show the ability to produce biologically active surfactant by the addition of isolated LMW apoproteins to defined PL.

Amino Acid Sequence↗
PubMed1986

Cloning and expression of a cDNA coding for the anticoagulant hirudin from the bloodsucking leech, Hirudo medicinalis.

Cloned cDNAs have been isolated that encode a variant of hirudin, a potent thrombin inhibitor that is secreted by the salivary glands of the medicinal leech, Hirudo medicinalis. This variant probably corresponds to a form that has been purified from leech heads but differs in amino acid sequence from the hirudin purified from whole leeches. There are at least three hirudin transcripts detectable in leech RNAs that are different in size, site of synthesis, inducibility by starvation, and relationship to hirudin activity. The new hirudin variant predicted by the cDNA and the heterodisperse transcription products suggest a hirudin protein family. The hirudin cDNA was expressed in Escherichia coli under the control of the bacteriophage lambda PL promoter. The recombinant product is biologically active, inhibiting the cleavage by thrombin of fibrinogen and a synthetic tripeptide substrate.

Amino Acid Sequence↗
PubMed1978

A comparative analysis of extreme thermophilic bacteria belonging to the genus Thermus.

Several extreme thermophilic Gram negative bacteria found in a thermally polluted river in Belgium have been compared with Thermus strains isolated from widely distant geographical areas. This analysis has become possible after the design of a new culture medium (162). All strains examined (including the isolate successively denominated Flavobacterium thermophilum and Thermus thermophilus) were found to be morphologically identical with strain YT-1 of Thermus aquaticus. The cells are immotile, rod-like, strictly aerobic, catalase and oxidase positive. They produce amylase, hydrolyze gelatin and are confirmed to be highly sensitive towards penicillin. The nutritional pattern of all strains has been analysed extensively, by testing a broad spectrum of possible substrates. The strains display a uniform response to the microbiological tests applied and most probably belong to the same species: Thermus aquaticus.

Aerobiosis↗
PubMedDate not supplied

Use of synthetic oligonucleotides in gene isolation and manipulation.

Great progress has occurred in the techniques of synthesis of DNA molecules of defined sequences in terms of speed, length of the obtained oligonucleotides, and automation of the processes. Corresponding progress also occurred in the ways of using synthetic DNA in molecular biology and recombinant DNA research. Screening of cloned DNA sequence banks with long, unique oligonucleotides, provided a new approach to isolate the genes for proteins which are present in very small quantity. This technique can present considerable advantages over the more classical use of mixtures of oligonucleotides, in reducing the number of potentially positive clones on a primary screen, and enabling cloning with a minimum of amino acid sequence data. Synthetic oligonucleotides also provide the basis of a set of techniques for site-directed mutagenesis of DNA sequences. This allows the possibility of engineering the structure of particular proteins, and the properties of new variants can be tested by expressing the protein in a heterologous host. An example of this approach is the production of variants of human alpha 1-antitrypsin. A variant where valine replaces the methionine at the active site is equally active as an antielastase, but no longer susceptible to oxidative inactivation. A second variant, where arginine replaces the methionine, now functions as an antithrombin, but no longer inhibits elastase. Total gene synthesis is now feasible for larger and larger genes, and some of the recent strategies of whole gene synthesis are presented.

Amino Acid Sequence↗