Claims for Patent: 5,100,788

Summary for Patent: 5,100,788

Title:	Method of producing and isolating IGG-binding protein a fusion peptides and a vector therefor
Abstract:	A method of producing and selectively isolating a desired protein or polypeptide or derivative thereof by constructing a recombinant vector comprising a DNA sequence coding for said desired protein or polypeptide operatively linked to a DNA sequence coding for protein A or an active polypeptide fragment thereof or any other macromolecule capable of binding to the constant regions of immunoglobulins, such that said DNA sequences together code for an IgG-binding fusion product between said desired protein or polypeptide and said protein A, active polypeptide fragment thereof or macromolecule; transforming a compatible host with said recombinant vector such that the combined DNA sequences coding for said fusion protein or polypeptide can be expressed by the host, and culturing the transformed host in a suitable growth medium to produce said fusion protein or polypeptide; selectively isolating said fusion protein or polypeptide by adsorption to an IgG-supporting carrier material; and optionally desorbing said fusion protein or polypeptide from said IgG-supporting carrier, said fusion protein or polypeptide coded for by said combined DNA-sequence optionally comprising a unique cleavage site between said protein A part and said desired protein or polypeptide part, said desired protein or polypeptide part then being cleaved off from the rest of the fusion protein or polpeptide either while the latter is adsorbed to the IgG-supporting carrier or after desorption thereof from the carrier. Also a hybrid vector for use herein, a method and an expression vector for its preparation and a host organism transformed by said hybrid vector are disclosed.
Inventor(s):	Lofdahl; Sven (Uppsala, SE), Uhlen; Mathias (Uppsala, SE), Lindberg; Martin (Uppsala, SE), Sjoquist; John (Uppsala, SE)
Assignee:	Pharmacia LKB Biotechnology AB (Uppsala, SE)
Application Number:	07/196,846
Patent Claims:	1. A method of producing and selectively isolating a desired protein or polypeptide as a fusion product, characterized by the steps of constructing a recombinant vector comprising a DNA sequence coding for said desired protein or polypeptide in correct reading frame with a DNA sequence coding for an IgG-binding protein A or polypeptide fragment thereof, capable of binding to the constant region of IgG, such that said DNA sequences together code for an IgG-binding fusion product transforming a compatible host with said recombinant vector, culturing the transformed host in a suitable growth medium to produce said fusion product, selectively isolating said fusion product by adsorption to an IgG-supporting carrier material, and optionally desorbing said fusion protein or polypeptide fusion product from the IgG-supporting carrier. 2. A method according to claim 1, characterized in that the fusion protein or polypeptide comprises a unique cleavage site between the desired protein or polypeptide and said IgG-binding protein A or polypeptide fragment thereof, said cleavage site not being present in the desired protein or polypeptide and may or may not be present in the IgG-binding protein A or polypeptide fragment thereof, and that said desired protein or polypeptide is cleaved off from the rest of the fusion product either while the latter is adsorbed to the IgG-supporting carrier or after desorption. 3. A method according to claim 2, characterized in that said unique cleavage site is an amino acid sequence susceptible to cleavage by a cleaving agent selecting from the group consisting of proteases, hydroxylamine, cyanogen bromide and formic acid. 4. A method according to claim 1, characterized in that said recombinant vector is constructed by providing an expression vector comprising a functional DNA sequence coding for said IgG-binding protein A or polypeptide fragment thereof, and a multilinker sequence located before any stop codon of said sequence; and inserting a DNA sequence coding for the desired protein or polypeptide into an appropriate restriction site of said multilinker sequence, and optionally inserting a DNA sequence coding for a unique cleavage site between the DNA sequence coding for the desired protein A or polypeptide fragment thereof, and the DNA sequence coding for the IgG-binding protein or polypeptide, said cleavage site coding sequence may or may not being provided in the expression vector or in the junction end of the desired protein or polypeptide coding DNA sequence before the insertion thereof into the expression vector. 5. A method according to claim 1, characterized in that said desorption of the fusion protein or polypeptide from the IgG-supporting carrier is effected by subjecting the carrier to low pH conditions, high salt concentrations, chaotrophic ions or to competitive protein elution with an excess of soluble protein A or IgG or fragments thereof. 6. A recombinant vector, comprising a DNA sequence coding for an IgG-binding protein A or polypeptide fragment thereof, capable of binding to the constant region of IgG, in correct reading frame with a DNA sequence coding for a desired protein or polypeptide such that the combined sequences together code for a fusion product between said IgG-binding protein A or polypeptide fragment thereof and said desired protein or polypeptide, wherein said fusion product has IgG-binding activity. 7. A recombinant vector according to claim 6 characterized in that the DNA sequence encoding protein A or polypeptide fragment thereof extends from the 5'-end of the combined DNA sequence for coding said fusion protein or polypeptide, said protein A coding sequence may or may not comprising the promoter and signal sequence of the structural protein A coding gene. 8. A recombinant vector according to claim 6, characterized in that a junction between said combined DNA sequences comprises a DNA sequence coding for a unique cleavage site, which is not present in said desired protein or polypeptide and may or may not be present in said IgG-binding protein A or polypeptide fragment thereof, and which may be cleaved by a cleaving agent. 9. A recombinant vector according to claim 8, characterized in that said cleavage site is an amino acid sequence susceptible to cleavage by a cleaving agent selected from the group consisting of proteases, cyanogen bromide, hydroxylamine, and formic acid. 10. A recombinant vector according to claim 8, characterized in that it is a plasmid. 11. A recombinant vector according to claim 6, characterized in that it is a plasmid. 12. A recombinant vector according to claim 7, characterized in that it is a plasmid. 13. A host organism transformed by a recombinant vector of any one of claims 6, 7-8, 9 or 10-12, characterized in that it is a strain of Escherichia, Bacillus or Staphylococcus. 14. A method of producing and selectively isolating a desired protein or polypeptide, characterized by the steps of constructing a recombinant vector comprising DNA sequences, linked in correct reading frame, coding for said desired protein or polypeptide, a unique cleavage site and an IgG-binding protein A or polypeptide fragment thereof, capable of binding to the constant region of IgG, such that said DNA sequence together code for an IgG-binding fusion product, comprising a unique cleavage site between the desired protein or polypeptide and the IgG binding protein A or polypeptide fragment thereof, transforming a compatible host with said recombinant vector, culturing the transformed host in a suitable growth medium to produce said fusion product, selectively isolating said fusion product by adsorption to an IgG-supporting carrier material, and cleaving off the desired protein or polypeptide from the rest of the fusion product either while the latter is adsorbed to the IgG-supporting carrier or after desorption. 15. A method according to claim 1, wherein the desired protein or polypeptide is subsequently further purified by gel filtration or ion exchange techniques. 16. A method according to claim 1, wherein the desired protein or polypeptide to be isolated as a fusion product with the IgG-binding protein A or polypeptide fragment thereof is selected from the group consisting of transferases, hydrolases, lyases, isomerases, ligases, insulin, ACTH, somatostatin, prolactin, placental, lactogen, melanocyte stimulating hormone, thyrotropin, parathyroid hormone, calcitonin, enkephalin, angiotensin, fibrinogen, fibronectin, prothrombin, thromboplastin, globulin, heparin, oxytocin, albumins, actin, myosin, hemoglobin, ferritin, cytochrome, myoglobin, lactoglobulin, histones, avidin, thyroglobulin, interferon, transcortial kinins and peptide antigens for use in making vaccines. 17. A recombinant vector according to claim 6, wherein the desired protein or polypeptide is selected from the group consisting of transferases, hydrolases, lyases, isomerases, ligases, insuling, ACTH, somatostatin, prolactin, placental lactogen, melanocyte stimulating hormone, thyrotropin, parathyroid hormone, calcitonin, enkephalin, angiotensin, fibrinogen, fibronectin, prothrombin, thromboplastin, globulin, heparin, oxytocin, albumins, actin, myosin, hemoglobin, ferritin, cytochrome, myoglobin, lactoglobulin, histones, avidin, thyroglobulin, interferon, transcortial kinins and peptide antigens for use in making vaccines. 18. A method according to claim 14, characterized in that said unique cleavage site is an amino acid sequence susceptible to cleavage by a cleaving agent selecting from the group consisting of proteases, hydroxylamine, cyanogen bromide and formic acid. 19. A method according to claim 1, wherein the desired protein or polypeptide to be isolated as a fusion product with the IgG-binding protein A or polypeptide fragment thereof is selected from the group consisting of enzymes, hormones, serum proteins, coagulation factors, complement factors, and plasma proteins. 20. A recombinant vector according to claim 6 wherein the desired protein or polypeptide to be isolated as a fusion product with the IgG-binding protein A or polypeptide fragment thereof is selected from the group consisting of enzymes, hormones, serum proteins, coagulation factors, complement factors, and plasma proteins.

Details for Patent 5,100,788

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Nps Pharmaceuticals, Inc.	NATPARA	parathyroid hormone	For Injection	125511	01/23/2015		2009-03-31