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Last Updated: March 29, 2024

Claims for Patent: 6,500,647


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Summary for Patent: 6,500,647
Title: Recombinant expression vector of human parathyroid hormone
Abstract:The present invention relates to a recombinant expression vector which is prepared by inserting a human parathyroid hormone gene containing a urokinase-specific cleavage site into an L-arabinose inducible vector containing a phosphoribulokinase gene fragment of Rhodabacter sphaeroides or its mutated gene as a fusion partner, or its mutate gene as a fusion partner, a recombinant microorganism transformed with the said expression vector, and a process for preparing human parathyroid hormone on a large scale by cultivating the said microorganism in a medium containing L-arabinose. In accordance with the invention, a recombinant human PTH having the same activity of the native human PTH can be prepared in a high yield through the precise control of induction by a manufacturing process which comprises a step of inducing expression of fusion protein in the microorganism transformed with the recombinant expression vector by L-arabinose.
Inventor(s): Jung; Eun-Kyung (Seoul, KR), Park; Doo-Hong (Seoul, KR), Chung; Soo-Il (Sungnam-si, KR)
Assignee: Mogam Biotechnology Research Institute (Kyonggi-Do, KR)
Application Number:09/463,282
Patent Claims:1. A recombinant expression vector which is prepared by inserting a human parathyroid hormone gene comprising a urokinase-specific cleavage site into an L-arabinose inducible vector comprising, as a fusion partner to said human parathyroid hormone gene, a phosphoribulokinase gene fragment of Rhodobacter sphaeroides or a gene fragment encoding a mutated phosphoribulokinase, said mutated phosphoribulokinase comprising mutations in which the 30.sup.th, 58.sup.th and 94.sup.th arginine residues are substituted with valine residues and the 59.sup.th and 96.sup.th arginine residues are substituted with glycine residues.

2. The recombinant expression vector of claim 1, wherein the L-arabinose inducible vector containing a phosphoribulokinase (PRK) gene is pPRK whose gene map is shown in FIG. 4.

3. The recombinant expression vector of claim 1, wherein the phosphoribulokinase gene fragment is a DNA fragment coding for 113 to 153 amino acids from amino-terminus of phosphoribulokinase.

4. The recombinant expression vector of claim 1, wherein the urokinase-specific cleavage site is a DNA sequence deducible from the following amino acid sequence:

5. The recombinant expression vector of claim 1, wherein the urokinase-specific cleavage site is a DNA sequence deducible from an amino acid sequence of -Thr-Gly-Arg.

6. The recombinant expression vector of claim 1, wherein the human parathyroid hormone gene has the following DNA sequence (SEQ ID NO:26): TCT GTT TCG GAA ATC CAG CTT ATG CAT AAC CTG GGT AAA 39 CAT CTG AAC TCG ATG GAA CGT GTT GAA TGG CTG CGT AAA 78 AAA CTG CAG GAT GTT CAT AAC TTC GTT GCG CTG GGG GCT 117 CCA CTG GCG CCG CGC GAG GCG GGT TCG CAG CGC CCA CGT 156 AAA AAG GAA GAT AAC GTT CTG GTA GAG TCG CAT GAA AAG 195 TCT CTT GGC GAG GCT GAT AAA GCA GAC GTA GAC GTT TTG 234 ACT AAA GCA AAA TCT CAA TAA TGA 258.

7. A recombinant expression vector p153PTH which contains: a DNA fragment coding for 153 amino acids from amino-terminus of phosphoribulokinase of Rhodobacter sphaeroides; a DNA fragment coding for a urokinase-specific cleavage site of -Thr-Gly-Arg; and, a human parathyroid hormone gene.

8. The recombinant expression vector which contained the following genes in a serial manner: a DNA fragment coding for 153 amino acids from the amino-terminus of phosphoribulokinase of Rhodobacter sphaeroides, in which the arginine residues located at the 30.sup.th, 31.sup.th, 58.sup.th, 59.sup.th, 94.sup.th and 96.sup.th positions are substituted with different amino acids; a DNA fragment coding for a urokinase-specific cleavage site of X-Gly-Arg (wherein, X is Pro, Thr, Ile, Phe or Leu); and, a human parathyroid horomone gene.

9. A recombinant expression vector pm153PTH which contains the following genes in a serial manner: a DNA fragment coding for 153 amino acids from amino-terminus of phosphoribulokinase of Rhodobacter sphaeroides whose 30th, 58th, and 94th arginine residues are site-specifically mutated to be substituted with valines and 59th and 96th arginine residues with glycines; a DNA fragment coding for a urokinase-specific cleavage site of -Thr-Gly-Arg; and, a human parathyroid hormone gene.

10. Escherichia coli MC1061:p153PTH (KCCM-10101) transformed with the recombinant expression vector p153PTH of claim 7.

11. Escherichia coli MC1061:pm153PTH (KCCM-10102) transformed with the recombinant expression vector pm153PTH of claim 9.

12. A process for preparing a human parathyroid hormone which comprises the steps of: cultivating Escherichia coli MC1061:p153PTH (KCCM-10101); inducing the expression of a human parathyroid hormone by L-arabinose; and, recovering the human parathyroid hormone.

13. A process for preparing a human parathyroid hormone which comprises the steps of: cultivating Escherichia coli MC1061:pm153PTH (KCCM-10102); inducing the expression of a human parathyroid hormone by L-arabinose; and, recovering the human parathyroid hormone.

14. A process for preparing a human parathyroid hormone which comprises the steps of: cultivating a recombinant microorganism transformed with the recombinant expression vector of claim 1; inducing the expression of a human parathyroid hormone by L-arabinose; treating the expressed fusion protein of phosphoribulokinase and a human parathyroid hormone with urokinase to recover the human parathyroid hormone.

Details for Patent 6,500,647

Applicant Tradename Biologic Ingredient Dosage Form BLA Approval Date Patent No. Expiredate
Microbix Biosystems Inc. KINLYTIC urokinase For Injection 021846 01/16/1978 ⤷  Try a Trial 2017-07-25
Nps Pharmaceuticals, Inc. NATPARA parathyroid hormone For Injection 125511 01/23/2015 ⤷  Try a Trial 2017-07-25
>Applicant >Tradename >Biologic Ingredient >Dosage Form >BLA >Approval Date >Patent No. >Expiredate

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