Bio-technology General Corporation

Savient Pharmaceuticals, Inc.

Tikva Vogel

Avigdor Levanon

Amos B Oppenheim

Marian Gorecki

Moshe M Werber

Rachel Guy

Amos Panet

Haim Aviv

Menachem Zeevi

Elisha P Zeelon

Jacob R Hartman

Hadassa Shaked

Hilla Locker-Giladi

Pinhas E Zeelon

This invention provides an imaging agent which comprises a polypeptide labeled with an imageable marker, such polypeptide having an amino acid sequence substantially present in the fibrin binding domain of naturally-occurring human fibronectin and being capable of binding to fibrin. The invention further provides a method wherein the imaging agent is used for imaging a fibrin-containing substance, i.e., a thrombus or atherosclerotic plaque. Further provided are plasmids for expression of polypeptides having an amino acid sequence substantially present in the fibrin binding domain of naturally-occurring human fibronectin and being capable of binding to fibrin, hosts containing these plasmids, methods of producing the polypeptides, methods of treatment using the polypeptides, and methods of recovering, refolding and reoxidizing the polypeptides. The invention also provides for purified polypeptides substantially free of other substances of human origin which have an amino acid sequence substantially present in the fibrin binding domain of naturally-occurring human fibronectin and which are capable of binding to fibrin.

Fibrin binding domain polypeptides and uses and methods of producing same

An improved vector upon introduction into a suitable bacterial host containing the thermolabile repressor C.sub.I renders the host cell capable, upon increasing the temperature of the host cell to a temperature at which the repressor is destroyed, of effecting expression of a desired gene inserted into the vector and production of polypeptide encoded by the gene. The vector is a double-stranded DNA molecule which includes in 5' to 3' order the following: a DNA sequence which contains the promoter and operator P.sub.L O.sub.L from lambda bacteriophage; the N utilization site for binding antiterminator N protein produced by the host cell; a DNA sequence which contains a ribosomal binding site for rendering the mRNA of the desired gene capable of binding to ribosomes within the host cell; an ATG initiation codon or a DNA sequence which is converted into an ATG initiation codon upon insertion of the desired gene into the vector; a restriction enzyme site for inserting the desired gene into the vector in phase with the ATG initiation codon; and additionally a DNA sequence which contains an origin of replication from a bacterial plasmid capable of autonomous replication in the host cell and a DNA sequence which contains a gene associated with a selectable or identifiable trait which is manifested when the vector is present in the host cell.

Expression vectors for enhanced production of polypeptides, plasmids

This invention provides an imaging agent which comprises a polypeptide labeled with an imageable marker, such polypeptide having an amino acid sequence substantially present in the fibrin binding domain of naturally-occurring human fibronectin and being capable of binding to fibrin. The invention further provides a method wherein the imaging agent is used for imaging a fibrin-containing substance, i.e., a thrombus-or atherosclerotic plaque. Further provided are plasmids for expression of polypeptides having an amino acid sequence substantially present in the fibrin binding domain of naturally-occurring human fibronectin and being capable of binding to fibrin, hosts containing these plasmids, methods of producing the polypeptides, methods of treatment using the polypeptides, and methods of recovering, refolding and reoxidizing the polypeptides. The invention also provides for purified polypeptides substantially free of other substances of human origin which have an amino acid sequence substantially present in the fibrin binding domain of naturally-occurring human fibronectin and which are capable of binding to fibrin.

An improved vector upon introduction into a suitable host containing the thermolabile repressor C.sub.I renders the host capable of effecting expression of a desired gene. The vector is a double-stranded DNA molecule which includes in 5' to 3' order the following: the promoter and operator P.sub.L O.sub.L from lambda bacteriophage; the N utilization site; a first restriction enzyme site permitting replacement of the ribosomal binding site which follows thereafter; a ribosomal binding site; an ATG initiation codon or DNA which is converted into an ATG initiation codon upon insertion of the desired gene into the vector; a second restriction enzyme site for inserting the gene in phase with the ATG codon; a T.sub.1 T.sub.2 rRNA transcription termination sequence; an origin of replication and a gene associated with a selectable or identifiable phenotypic trait manifested when the vector is present in the host. The distance between the 3' end of the P.sub.L O.sub.L promoter and operator sequence and the 5' end of the N utilization site is less than about 80 base pairs and the distance between the 3' end of the N utilization site and the 5' end of the ribosomal binding site is less than about 300 base pairs. Plasmids have been constructed from the vectors and used to produce bovine, chicken and porcine growth hormones, human apolipoprotein E and human superoxide dismutase.

Expression vectors containing .lambda.P.sub.L promoter and T.sub.1

This invention provides an imaging agent which comprises a polypeptide labeled with an imageable marker, such polypeptide having an amino acid sequence substantially present in the fibrin binding domain of naturally-occurring human fibronectin and being capable of binding to fibrin. The invention further provides a method wherein the imaging agent is used for imagifig a fibrin-containing substance, i.e., a thrombus or atherosclerotic plaque. Further provided are plasmids for expression of polypeptides having an amino acid sequence substantially present in the fibrin binding domain of naturally-occurring human fibronectin and being capable of binding to fibrin, hosts containing these plasmids, methods of producing the polypeptides, methods of treatment using the polypeptides, and methods of recovering, refolding and reoxidizing the polypeptides. The invention also provides for purified polypeptides substantially free of other substances of human origin which have an amino acid sequence substantially present in the fibrin binding domain of naturally-occurring human fibronectin and which are capable of binding to fibrin.

Plasmids for production of human growth hormone or polypeptide analog

Fibrin binding domain polypeptide and method of producing

A plasmid for the production of porcine growth hormone or an analog thereof which upon introduction into a suitable bacterial host containing the thermolabile repressor C.sub.I renders the host cell capable, upon increasing the temperature of the host cell to a temperature at which the repressor is destroyed, of effecting expression of DNA encoding porcine growth hormone and production of porcine growth hormone or analogs thereof. The plasmid is a double-stranded DNA molecule which includes in 5' to 3' the following: a DNA sequence which contains the promoter and operator PLOL from lambda bacteriophage; the N utilization site for binding antiterminator N protein produced by the host cell; a DNA sequence which contains a ribosomal binding site for rendering the mRNA of the gene encoding porcine growth hormone capable of binding to ribosomes within the host cell; an ATG initiation codon; a restriction enzyme site for inserting the gene encoding porcine growth hormone into the plasmid in phase with the ATG initiation codon; a gene encoding porcine growth hormone; and additionally a DNA sequence which contains an origin of replication from a bacterial plasmid capable of autonomous replication in the host cell and a DNA sequence which contains a gene associated with a selectable or identifiable trait which is manifested when the plasmid is present in the host cell.

Bacterial expression of porcine growth hormone

Plasmids for production of met-asp-gln bovine growth hormone, hosts

A method is provided for recovering a purified animal growth hormone or a polypeptide analog thereof having substantially the same amino acid sequence as, and the biological activity of, the corresponding naturally-occurring animal growth hormone from a bacterial cell in which the animal growth hormone or polypeptide analog has been produced by means of expression of a plasmid encoding the hormone or polypeptide analog which comprises: (a) disrupting the cell wall of the bacterial cell in a buffered neutral pH solution so as to produce a lysate containing precipitated hormone or polypeptide analog; (b) recovering the resulting precipitated hormone or polypeptide analog; (c) suspending the precipitated hormone or polypeptide analog so recovered in distilled water; (d) treating the resulting precipitate-containing suspension with a sodium hydroxide solution having an alkaline pH of about 11.8 so as to solubilize the precipitate and thus the hormone or polypeptide analog contained therein; (e) separating the solubilized hormone or polypeptide analog from other soluble components by gel filtration chromatography; and (f) subjecting the hormone or polypeptide analog thus separated to ion exchange chromatography to purify the hormone or analog and thereby recover purified hormone or polypeptide analog.

Method for recovering a purified animal growth hormone or polypeptide

Analogs of hGH having the activity of naturally occurring hGH and a similar amino acid sequence varying from the sequence of natural hGh by the addition of one or more amino acids, e.g. methione or methionine-leucine, to the N-terminus of natural hGH have been produced, recovered and purified. Such analogs may be incorporated into pharmaceutical compositions and administered to a subject to increase the level of hGH in the subject. Analogs of hGH which comprise the amino acid sequence of natural hGH from the N-terminus of which one or more amino acids have been deleted, e.g. Met.sup.14 hGh, have been produced, recovered and purified. Such analogs may be incorporated into pharmaceutical compositions and administered to a subject to lower the level of hGH in the subject. A plasmid has been constructed which directs the expression of an analog of hGH having the amino acid sequence methionine-leucine added to the N-terminus of natural hGH. This plasmid has been introduced into Escherichia coli, the resulting Escherichia coli grown and the analog recovered and purified.

Growing community of inventors

Rehovot, Israel

Tikva Vogel