Abstract: Methods are disclosed for producing hybrid phospholipid-binding proteins from eukaryotic cells. DNA constructs comprising a transcriptional promoter, at least one signal sequence and a hybrid phospholipid-binding protein coding sequence comprising at least one lipocortin lipid-binding domain joined to a gla-domainless, vitamin K-dependent protein and a transcriptional terminator are also disclosed.

Abstract: Promoters associated with expression of specific enzymes in the glycolytic pathway are used for expression of alien DNA, particularly yeast promoters known to provide high enzyme levels of enzymes in the glycolytic pathway are employed for expressing a mammalian protein, such as .alpha..sub.1 -antitrypsin. The promoters include promoters involved in expression of pyruvate kinase, triose phosphate isomerase, phosphoglucose isomerase, phosphoglycerate mutase, hexokinase 1, hexokinase 2, glucokinase, phosphofructokinase, and aldolase, as well as the glycolytic regulation gene. Particularly, the glycolytic regulation gene can be used in conjunction with promoters in the glycolytic pathway for regulated production of desired proteins.

Abstract: Methods for purifying factor XIII from a biological fluid are provided. The methods comprise precipitation of factor XIII by adjusting the pH of the biological fluid to 5.5 to 6.5 and recovering the precipitated factor XIII.

Abstract: Zymogens of proteins having fibrinolytic activity are disclosed. The proteins are cleavable by thrombin, the cleavage resulting in the stimulation of fibrinolytic activity. Suitable proteins which may be modified in accordance with the present invention include tissue plasminogen activator, urokinase, and plasminogen variants. The modified molecules are substantially clot-specific, in view of the large amounts of thrombin associated with clots in vivo.

Abstract: Methods for purifying factor VIII:C, von Willebrand factor (vWF) or complexes thereof from heterogeneous biological fluids are disclosed. The methods utilize a binding peptide, specific to either factor VIII:C or vWF, bound to an insoluble matrix. Peptides suitable for use within the methods are also disclosed.

Abstract: Methods for expressing a variety of biologically active PDGF analogs in eucaryotic cells are disclosed. The methods generally comprise introducing into a eucaryotic host cell a DNA construct capable of directing the expression and secretion of biologically active PDGF analogs in eucaryotic cells. The DNA construct contains a transcriptional promoter followed downstream by a suitable DNA sequence. The DNA sequence may encode a protein substantially homologous to the A-chain or the B-chain of PDGF, or a portion thereof, or an A-B heterodimer. In addition, a portion of the DNA sequence may encode at least a portion of the A-chain, while another portion encodes at least a portion of the B-chain of PDGF. Eucaryotic cells transformed with these DNA constructs are also disclosed. Methods of promoting the growth of mammalian cells, comprising incubating the cells with a biologically active PDGF analog expressed by a eucaryotic host cell transformed with such a DNA construct, are also disclosed.

Abstract: Methods for producing secreted receptor analogs and biologically active peptide dimers are disclosed. The methods for producing secreted receptor analogs and biologically active peptide dimers utilize a DNA sequence encoding a receptor analog or a peptide requiring dimerization for biological activity joined to a dimerizing protein. The receptor analog includes a ligand-binding domain. Polypeptides comprising essentially the extracellular domain of a human PDGF receptor fused to dimerizing proteins, the portion being capable of binding human PDGF or an isoform thereof, are also disclosed. The polypeptides may be used within methods for determining the presence of and for purifying human PDGF or isoforms thereof.

Abstract: Purified cytotoxic proteins for use within therapeutic compositions are disclosed. The proteins inhibit protein synthesis in vitro, exhibit abortifacient activity in mice, and contain proline residues with equivalent positions at residue 43 and residue 46 in ricin A-chain. A method for preparing such a cytotoxic protein from the tissue of Trichosanthes kirilowii is also disclosed. The proteins may also be used within a method for inhibiting protein synthesis in selected cells.

Abstract: Tissue plasminogen activator analogs exhibiting greater specificity for fibrin than native t-PA are disclosed. The analogs include the K1 domain of native t-PA replaced with another kringle domain mediating the binding of the analog to fibrin. The kringle contains six cysteine residues. The t-PA analogs may further include a variety of substitutions and modifications. Pharmaceutical compositions containing one or more of the t-PA analogs along with a physiologically acceptable carrier or diluent are also disclosed.

Abstract: Methods for producing a heterologous protein or polypeptide are disclosed. A preferred method utilizes a fungal cell carrying a defect in a gene whose product is required for the addition of outer chain oligosaccharide moieties to glycoproteins, the cell transformed with a first DNA construct comprising a regulated promoter followed downstream by a DNA sequence which complements the defect, and a second DNA construct comprising a second promoter followed downstream by a DNA sequence encoding a secretion signal and a DNA sequence encoding a heterologous protein or polypeptide. A yeast cell having a Mnn9.sup.- phenotype and capable of producing colonies of normal morphologies in the absence of osmotic stabilization is also disclosed.

Abstract: Proteins having substantially the same biological activity as PDGF are provided. In one aspect, a protein homodimer having two polypeptide chains is disclosed, each of the chains being a mosaic of amino acid sequences substantially identical to portions of the A- and B-chains of PDGF, the protein being chemotactic or mitogenic for fibroblasts. Therapeutic compositions comprising such proteins in combination with a physiologically acceptable carrier or diluent are also provided. Such therapeutic compositions may be used within methods for enhancing the wound-healing process in warm-blooded animals.

Abstract: Monoclonal antibodies (MAbs) capable of binding to native PDGF, and MAbs capable of specifically binding to the PDGF-AA, PDGF-BB and PDGF-AB isoforms are disclosed. The subject MAbs may be used in the detection or purification of native PDGF or selected PGDF isoforms. In addition, the MAbs may be labeled with an imaging agent and used for in vivo diagnostic purposes, or combined with a pharmaceutically acceptable carrier or diluent for use within wound healing compositions.

Abstract: Methods for cloning cDNA and producing mRNA from the cloned cDNA are disclosed. The cDNA is cloned in one orientation through the use of a vector containing a directional cloning site and through the use of primer-adapter sequences complementary to portions of the directional cloning site. The vector may also contain a promoter, one or more terminators and a polyadenylation signal to facilitate transcription of the cloned cDNA and translation of the mRNA.

Abstract: Biologically active PDGF analogs expressed in eucaryotic cells are disclosed. The analogs are produced by yeast strains transformed with an extrachromosomal element composed of a strong transcriptional promoter directing the expression of a gene which encodes a protein having substantially the same biological activity as PDGF. Suitable genes include the v-sis gene or a derivative of the v-sis gene of simian sarcoma virus or portions thereof, or the human cDNA gene for PDGF or portions thereof. In particular, DNA sequences encoding polypeptides substantially homologous to the B chain of PDGF are preferred. A secretory signal sequence may be provided upstream of the gene, enabling secretion of the gene product from the host cell. Mitogenic activity is one of the biological activites possessed by these PDGF analogs, making them useful in promoting the growth of mammalian cells.

Abstract: Methods for producing heterologous proteins in a host organism whereby the proteins are processed through the secretory pathway of the host are provided. Secretion is achieved by transforming a host organism with a DNA construct comprising a transcriptional promoter operably linked to DNA sequences encoding a signal peptide, at least a portion of the BAR1 C-terminal domain capable of directing the export of heterologous proteins and a heterologous protein or polypeptide. DNA constructs and transformants are also provided wherein the DNA sequence encoding at least a portion of the C-terminal domain of BAR1 capable of directing the export of heterologous proteins further comprises a DNA sequence encoding a proteolytic cleavage site operably linked to the DNA sequence encoding a heterologous protein.

Abstract: Purified cytotoxic proteins for use within therapeutic compositions are disclosed. The proteins inhibit protein synthesis in vitro, exhibit abortifacient activity in mice, and contain proline residues with equivalent positions at residue 43 and residue 46 in ricin A-chain. A method for preparing such a cytotoxic protein from the tissue of Trichosanthes kirilowii is also disclosed. The proteins may also be used within a method for inhibiting protein synthesis in selected cells.

Abstract: Genomic and cDNA sequences coding for a protein having substantially the same biological activity as human protein C and recombinant transfer vectors comprising these sequences are disclosed.Methods are disclosed for producing a protein which has substantially the same biological activity as human protein C. The protein, which may be in the form of activated protein C, is produced by mammalian host cells transfected with a plasmid capable of integration in mammalian host cell DNA. The plasmid includes a promoter followed downstream by a nucleotide sequence which encodes a protein having substantially the same structure and/or activity as human protein C, the nucleotide sequence being followed downstream by a polyadenylation signal.

Abstract: Proteins having therapeutic potential as both anticoagulants and as anti-inflammatory agents are disclosed. The present invention also discloses the use of lipocortins in reducing blood coagulation in warm-blooded animals.

Abstract: A method for expressing higher eucaryotic genes in Aspergillus through the use of a recombinant plasmid capable of integration into the chromosomal DNA of Aspergillus is disclosed. It is preferred to utilize a transcriptional promoter within a DNA construct contained in the plasmid that is of a DNA sequence encoding an ADH enzyme or a TPI enzyme. Promoters capable of directing the expression of a heterologous gene in Aspergillus, as well as other filamentous fungal genera are also disclosed.

Abstract: Methods are provided for producing .alpha.-1-antitrypsin in host cells and for selecting transformed cells comprising the step of transforming the host cell with a DNA molecule comprising a gene which complements a deficiency in the host cell. The host cell is a strain having a deficiency in a function necessary for normal cell growth. The gene in the DNA molecule, such as a plasmid, which complements the deficiency serves as a selectable marker whereby the growth conditions for selection may comprise a conventional complex medium.