scholarly journals Characterization of antithrombins produced by active site mutagenesis of human alpha 1-antitrypsin expressed in yeast

Blood ◽  
1989 ◽  
Vol 73 (2) ◽  
pp. 490-496 ◽  
Author(s):  
PM George ◽  
P Pemberton ◽  
IC Bathurst ◽  
RW Carrell ◽  
HL Gibson ◽  
...  
Keyword(s):  
Active Site ◽  
Recombinant Dna ◽  
Thrombin Inhibition ◽  
Congenital Deficiency ◽  
At Iii ◽  
Alpha 1 Antitrypsin ◽  
Recombinant Dna Techniques ◽  
Site Mutagenesis

Abstract Both congenital and acquired antithrombin-III (AT-III) deficiencies are amenable to replacement therapy. We describe two antithrombins produced by recombinant DNA techniques from human alpha 1-antitrypsin (alpha 1AT) cDNA in yeast. Alteration of the alpha 1AT active site, replacing methionine 358 with arginine, results in a thrombin inhibition rate similar to that of heparin-activated AT-III. Alteration of two further residues, to give a five-residue sequence identical to AT-III, does not increase this rate further. Neither antithrombin is activated by heparin; both are unglycosylated and have shorter in vivo half-lives (t1/2) than human alpha 1AT. These antithrombins should be suitable for therapeutic replacement of AT-III in cases of congenital deficiency and in conditions associated with acquired AT-III deficiency, such as disseminated intravascular coagulation.

scholarly journals Characterization of antithrombins produced by active site mutagenesis of human alpha 1-antitrypsin expressed in yeast

Blood ◽  
1989 ◽  
Vol 73 (2) ◽  
pp. 490-496
Author(s):  
PM George ◽  
P Pemberton ◽  
IC Bathurst ◽  
RW Carrell ◽  
HL Gibson ◽  
...  
Keyword(s):  
Active Site ◽  
Recombinant Dna ◽  
Thrombin Inhibition ◽  
Congenital Deficiency ◽  
At Iii ◽  
Alpha 1 Antitrypsin ◽  
Recombinant Dna Techniques ◽  
Site Mutagenesis

Both congenital and acquired antithrombin-III (AT-III) deficiencies are amenable to replacement therapy. We describe two antithrombins produced by recombinant DNA techniques from human alpha 1-antitrypsin (alpha 1AT) cDNA in yeast. Alteration of the alpha 1AT active site, replacing methionine 358 with arginine, results in a thrombin inhibition rate similar to that of heparin-activated AT-III. Alteration of two further residues, to give a five-residue sequence identical to AT-III, does not increase this rate further. Neither antithrombin is activated by heparin; both are unglycosylated and have shorter in vivo half-lives (t1/2) than human alpha 1AT. These antithrombins should be suitable for therapeutic replacement of AT-III in cases of congenital deficiency and in conditions associated with acquired AT-III deficiency, such as disseminated intravascular coagulation.

scholarly journals Characterization of an associated microfibril protein through recombinant DNA techniques.

1992 ◽  
Vol 267 (14) ◽  
pp. 10087-10095
Author(s):  
S.K. Horrigan ◽  
C.B. Rich ◽  
B.W. Streeten ◽  
Z.Y. Li ◽  
J.A. Foster
Keyword(s):  
Recombinant Dna ◽  
Recombinant Dna Techniques

scholarly journals Influenza virus hemagglutinin-specific cytotoxic T cell response induced by polypeptide produced in Escherichia coli.

1985 ◽  
Vol 162 (2) ◽  
pp. 663-674 ◽  
Author(s):  
A Yamada ◽  
M R Ziese ◽  
J F Young ◽  
Y K Yamada ◽  
F A Ennis
Keyword(s):  
Influenza Virus ◽  
Recombinant Dna ◽  
Cytotoxic T Lymphocyte ◽  
Nonstructural Protein ◽  
T Cell Response ◽  
Cytotoxic T Cell ◽  
Protein Immunization ◽  
Recombinant Dna Techniques

We have tested the abilities of various polypeptides of A/PR/8/34 (H1N1) virus, constructed by recombinant DNA techniques, to induce influenza virus-specific secondary cytotoxic T lymphocyte (CTL) responses. A hybrid protein (c13 protein), consisting of the first 81 amino acids of viral nonstructural protein (NS1) and the HA2 subunit of viral hemagglutinin (HA), induced H-2-restricted, influenza virus subtype-specific secondary CTL in vitro, although other peptides did not. Using a recombinant virus, the viral determinant responsible for recognition was mapped to the HA2 portion of c13 protein. Immunization of mice with c13 protein induced the generation of memory CTL in vivo. The CTL precursor frequencies of A/PR/8/34 virus- and c13 protein-immune mice were estimated as one in 8,047 and 50,312, respectively. These results indicate that c13 protein primed recipient mice, even though the level of precursor frequency was below that observed in virus-immune mice.

Isolation and Partial Characterization of Equine Antithrombin III.

1979 ◽  
Author(s):  
D.W. Estry ◽  
T.G. Bell ◽  
G.H. Tishkoff ◽  
J.C. Mattson ◽  
S.C. Estry
Keyword(s):  
Molecular Weight ◽  
Antithrombin Iii ◽  
Stable Complex ◽  
Sds Page ◽  
At Iii ◽  
Human Thrombin ◽  
Horse Plasma ◽  
Stoichiometric Complex

A protein analogous to human antithrombin III was isolated from fresh horse plasma. The procedure for purification was a modification of Thaler and Schmer’s two-step isolation procedure. The horse protein was homogeneous on 7.5% SDS-PAGE gels and had a molecular weight of 62,000 to 64,000 daltons in both reducing and non-reducing systems (human; 62,300). Rabbit anti-human antithrombin III was used to demonstrate a line of partial identity by Immunoelectrophoresis between the horse and human protein. The horse protein rapidly neutralizes human thrombin (34,000 daltons) and the reaction appears to be greatly potentiated by heparin. In order to establish the formation of 1:1 covalent stoichiometric complex between horse AT III and thrombin (IIa), time studies were run in the presence and absence of heparin. AT III (62,000) at 15 seconds, 2, 5, 10 and 60 minutes formed a stable complex with thrombin (32,000) having a molecular weight of 86,000 daltons. Additional bands developing with time are due to the autolytic capabilities of the uncomplexed IIa. The major autolytic band had a molecular weight of 70,000 daltons. Addition of heparin potentiated the interaction although it did not change the stoichio-metry of the complexes formed. The data accumulated to date demonstrates the similarities between the human and horse protein and the possibilities of using the horse as a model system for the evaluation of AT III replacement therapy in vivo.

scholarly journals Semisynthetic human [[3H2]Phe1]proinsulin

1987 ◽  
Vol 247 (3) ◽  
pp. 785-788 ◽  
Author(s):  
R M L Jones ◽  
K Rose ◽  
R E Offord
Keyword(s):  
Time Course ◽  
Recombinant Dna ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Specific Radioactivity ◽  
Reversed Phase ◽  
British Library ◽  
Recombinant Dna Techniques ◽  
Phenylalanine Residue

Biosynthetic human proinsulin (obtained by recombinant DNA techniques) was used as the starting material for the preparation, by semisynthetic methods, of [3H]proinsulin with the label at the N-terminal phenylalanine residue. The labelled proinsulin was characterized by its retention time on reversed-phase h.p.l.c., by polyacrylamide-gel electrophoresis, by the time course of its enzymic conversion into insulin and by chromatographic analysis after extensive proteolytic degradation. The specific radioactivity of the product was 5 Ci/mmol. Experimental details of the preparation of human [[3H]Phe1]proinsulin, the isolation of this product by isocratic h.p.l.c. and gel filtration, and further characterization of protein intermediates have been deposited as supplement SUP 50138 (12 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on prepayment [see Biochem. J. (1987) 241, 5].

scholarly journals Hereditary Deficiency of Antithrombin III.

1977 ◽  
Author(s):  
E. Marciniak
Keyword(s):  
Antithrombin Iii ◽  
Binding Capacity ◽  
Antigenic Determinants ◽  
Moderate Increase ◽  
Congenital Deficiency ◽  
At Iii ◽  
Inhibitor Level ◽  
New Perspective ◽  
Thrombotic Tendency

The recognition of biological importance of antithrombin III (AT III) stems from the description of a familial thrombotic tendency associated with reduced levels of this protein in blood. A limited number of such families has been reported in the literature. All of the affected members are heterozygous for the abnormal gene. In many, the quantity of AT III as evaluated by antigenic determinants correlates with the capacity of plasma to inactivate coagulation enzymes. Both the existence of genetic polymorphism and the prevalence of the trait are still open to speculation. Although low level of AT III is an important risk factor for the development of venous thromboembolism, it does not preclude longevity. Liver is the major source of AT III synthesis but little is otherwise known of its metabolism. Subjects with hereditary deficiency treated withCoumadin often respond with a moderate increase in both AT III antigen and binding capacity. One can assume that this reflects a diminished rate of AT III utilization in vivo since the requirement for inhibitor apparently decreases with the reduction in procoagulants. More puzzling, however, is the fact that continuous intravenous treatment with heparin markedly diminishes the quantity of circulating AT III in man. This decrease is without proportion to the starting AT III level and a patient with congenital deficiency is at risk of attaining alarmingly low inhibitor level during therapy with heparin. Although the mechamism remains conjectural, the fact that heparin markedly lowers AT III in blood gives some new perspective to AT III metabolism.

scholarly journals Preparation and characterization of bovine growth hormones produced in recombinant Escherichia coli

1987 ◽  
Vol 243 (3) ◽  
pp. 829-839 ◽  
Author(s):  
P T Wingfield ◽  
P Graber ◽  
G Buell ◽  
K Rose ◽  
M G Simona ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Recombinant Dna ◽  
Polyacrylamide Gel Electrophoresis ◽  
Full Length ◽  
Sds Page ◽  
Rat Tibia ◽  
Bovine Sequence ◽  
Recombinant Dna Techniques

Two analogues of bovine growth hormone (BGH) have been produced in Escherichia coli by recombinant DNA techniques. In analogue Delta-1, the N-terminal alanine residue of the full-length bovine sequence is replaced by methionine. In analogue Delta-9, which is expressed at much higher levels than is Delta-1, the full-length bovine sequence is truncated at the N-terminus by eight residues and there is a serine-for-glycine substitution in the first position of the truncated protein. Both analogues, which were characterized by isoelectric focusing (i.e.f.), polyacrylamide-gel electrophoresis in the presence of SDS (SDS/PAGE), amino acid analysis and N-terminal amino acid sequence determination using combined g.l.c.-m.s., are compared with BGH isolated from pituitaries. In contrast with pituitary-derived BGH, the recombinant-derived proteins are homogeneous on SDS/PAGE and on i.e.f. In a radioimmunoassay, a radioreceptor assay and a bioassay in vivo (rat tibia), Delta-9 BGH showed very similar characteristics to the pituitary-derived hormone. Similar results have also been obtained with the Delta-1 analogue.

Isolation and Partial Characterization of Equine Antithrombin III

1979 ◽  
Author(s):  
D. W. Estry ◽  
T. G. Bell ◽  
G. H. Tishkoff ◽  
J. C. Mattson ◽  
S. C. Estry
Keyword(s):  
Molecular Weight ◽  
Antithrombin Iii ◽  
Stable Complex ◽  
Sds Page ◽  
At Iii ◽  
Human Thrombin ◽  
Horse Plasma ◽  
Stoichiometric Complex

A protein analogous to human antithrombin III was isolated from fresh horse plasma. The procedure for purification was a modification of Thaler and Schmer’s two-step isolation procedure. The horse protein was homogeneous on 7.5% SDS-PAGE gels and had a molecular weight of 62,000 to 64,000 daltons in both reducing and non-reducing systems (human; 62,300). Rabbit anti-human antithrombin III was used to demonstrate a line of partial identity by immunoelectrophoresis between the horse and human protein. The horse protein rapidly neutralizes human thrombin (34,000 daltons) and the reaction appears to be greatly potentiated by heparin. in order to establish the formation of 1:1 covalent stoichiometric complex between horse AT III and thrombin (IIa), time studies were run in the presence and absence of heparin. AT III (62,000) at 15 seconds, 2, 5, 10 and 60 minutes formed a stable complex with thrombin (32,000) having a molecular weight of 86,000 daltons. Additional bands developing with time are due to the autolytic capabilities of the uncomplexed IIa. The major autolytic band had a molecular weight of 70,000 daltons. Addition of heparin potentiated the interaction although it did not change the stoichiometry of the complexes formed. The data accumulated to date demonstrates the similarities between the human and horse protein and the possibilities of using the horse as a model system for the evaluation of AT III replacement therapy in vivo.

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