WO1997002350B1 - Recombinant human prostate specific antigen and uses thereof - Google Patents
Recombinant human prostate specific antigen and uses thereofInfo
- Publication number
- WO1997002350B1 WO1997002350B1 PCT/EP1996/002779 EP9602779W WO9702350B1 WO 1997002350 B1 WO1997002350 B1 WO 1997002350B1 EP 9602779 W EP9602779 W EP 9602779W WO 9702350 B1 WO9702350 B1 WO 9702350B1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- hpsa
- recombinant
- column
- specific antigen
- arg
- Prior art date
Links
- 102000007579 human kallikrein-related peptidase 3 Human genes 0.000 title claims abstract 19
- 108010071652 human kallikrein-related peptidase 3 Proteins 0.000 title claims abstract 19
- 239000001963 growth media Substances 0.000 claims abstract 4
- 102000004169 proteins and genes Human genes 0.000 claims abstract 4
- 108090000623 proteins and genes Proteins 0.000 claims abstract 4
- 241000238631 Hexapoda Species 0.000 claims abstract 3
- 238000000034 method Methods 0.000 claims abstract 3
- 238000005277 cation exchange chromatography Methods 0.000 claims abstract 2
- 238000002523 gelfiltration Methods 0.000 claims abstract 2
- 238000004519 manufacturing process Methods 0.000 claims abstract 2
- 238000000746 purification Methods 0.000 claims abstract 2
- 239000000758 substrate Substances 0.000 claims abstract 2
- 241000701447 unidentified baculovirus Species 0.000 claims abstract 2
- 210000001124 Body Fluids Anatomy 0.000 claims 2
- 238000004166 bioassay Methods 0.000 claims 2
- 239000010839 body fluid Substances 0.000 claims 2
- 150000003839 salts Chemical class 0.000 claims 2
- 239000011780 sodium chloride Substances 0.000 claims 2
- 229920002676 Complementary DNA Polymers 0.000 claims 1
- 125000001429 N-terminal alpha-amino-acid group Chemical group 0.000 claims 1
- 210000002966 Serum Anatomy 0.000 claims 1
- 241000256251 Spodoptera frugiperda Species 0.000 claims 1
- 238000005341 cation exchange Methods 0.000 claims 1
- 238000004113 cell culture Methods 0.000 claims 1
- 239000003541 chymotrypsin inhibitor Substances 0.000 claims 1
- 239000002299 complementary DNA Substances 0.000 claims 1
- 239000012141 concentrate Substances 0.000 claims 1
- 229920003013 deoxyribonucleic acid Polymers 0.000 claims 1
- 238000003306 harvesting Methods 0.000 claims 1
- 239000011159 matrix material Substances 0.000 claims 1
- 239000002609 media Substances 0.000 claims 1
- 102000007312 Recombinant Proteins Human genes 0.000 abstract 2
- 108010033725 Recombinant Proteins Proteins 0.000 abstract 2
- 102000001399 Kallikreins Human genes 0.000 abstract 1
- 108060005987 Kallikreins Proteins 0.000 abstract 1
- 125000000539 amino acid group Chemical group 0.000 abstract 1
Abstract
The present invention relates to the production and purification of human prostate specific antigen (hPSA) by recombinant-DNA-technology. hPSA was produced in an active and an inactive form by the baculovirus expression vector system. Recombinant protein was secreted into the culture medium at a high yield by infected insect cells and purified to homogeneity by three-step procedure containing cation-exchange chromatography and gel filtration. The active protein obtained differs from the native protein in that it is unglycosylated and that it does not degrade the kallikrein-specific substrate H-D-Pro-Phe-Arg-pNA.HC1. The inactive form of hPSA obtained has two additional amino acid residues in its N-terminus, and it does not form complexes. Possible uses of the recombinant protein are also disclosed.
Claims
1. Recombinant human prostate specific antigen (hPSA) which is characterized in that
a) it is obtainable by recombinant-DNA-technology using baculovirus expression system in infected insect cells, b) it has the following N-terminal amino acid sequence: Ser-Arg-Ile-Val-Gly-Gly- Trp-Glu-Cys-Glu-Lys-His-Ser, c) it does not form complexes with αrantichymo trypsin or α2-macroglobulin, d) it has no activity on the substrates MeO-Suc-Arg-Pro-Tyr-pNAΗCl and H-D- Pro-Phe-Arg-pNA • 2HC1.
2. Process for the production ofthe recombinant hPSA of claim 1, comprising
a) incoφorating the cDNA encoding hPSA into a transfer vector, b) cotransfecting said vector with baculovirus-DNA into Spodoptera frugiperda cells, c) cultivating said cells in a suitable insect cell cultivation medium, d) harvesting the culture medium, and e) purifying the hPSA secreted into the culture medium.
3. Process according to claim 2 wherein the purification of step e) is effected by - dialyzing the culture medium, loading the dialysate onto a fluidized bed column having strong cation-exchange matrix, eluting the hPSA from the column by salt gradient, concentrating the hPSA fragments, - gel filtrating the concentrate obtained, dialyzing the hPSA fractions eluted from the gel filtration column, loading the dialyzate onto a cation-exchange chromatography column, and eluting the active and inactive pools of recombinant hPSA from the column with a linear salt gradient.
4. In a method for immunometric assay of human prostate specific antigen (hPSA) in human body fluids, an improvement comprising use ofthe hPSA according to claim 1 as a standard protein.
5. The improvement according to claim 4 wherein the body fluid is serum.
6. Use of the hPSA protein according to claim 1 as a standard protein in immunometric hPSA assays.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US08/973,719 US6140468A (en) | 1995-06-30 | 1996-06-26 | Recombinant human prostate specific antigen |
| EP96923930A EP0835317A1 (en) | 1995-06-30 | 1996-06-26 | Recombinant human prostate specific antigen and uses thereof |
| JP9504778A JPH11508449A (en) | 1995-06-30 | 1996-06-26 | Recombinant human prostate specific antigen and uses thereof |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FI953257A FI953257A (en) | 1995-06-30 | 1995-06-30 | Recombinant human prostate-specific antigen and its uses |
| FI953257 | 1995-06-30 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| WO1997002350A1 WO1997002350A1 (en) | 1997-01-23 |
| WO1997002350B1 true WO1997002350B1 (en) | 1997-02-13 |
Family
ID=8543716
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/EP1996/002779 WO1997002350A1 (en) | 1995-06-30 | 1996-06-26 | Recombinant human prostate specific antigen and uses thereof |
Country Status (5)
| Country | Link |
|---|---|
| US (1) | US6140468A (en) |
| EP (1) | EP0835317A1 (en) |
| JP (1) | JPH11508449A (en) |
| FI (1) | FI953257A (en) |
| WO (1) | WO1997002350A1 (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0725139A3 (en) * | 1995-02-03 | 1997-11-26 | Bayer Corporation | Recombinant prostate-specific antigen and its use as an immunoassay calibrator |
| US6548260B1 (en) | 1997-01-21 | 2003-04-15 | Bayer Corporation | Detection of PSA-α2-macroglobulin complex in a biological fluid |
| DE19916242C2 (en) * | 1999-04-10 | 2003-04-10 | Seratec Ges Fuer Biotechnologi | PSA-containing substance and its use |
| US20090118139A1 (en) * | 2000-11-07 | 2009-05-07 | Caliper Life Sciences, Inc. | Microfluidic method and system for enzyme inhibition activity screening |
| US7425700B2 (en) * | 2003-05-22 | 2008-09-16 | Stults John T | Systems and methods for discovery and analysis of markers |
| CN117233413A (en) | 2019-08-05 | 2023-12-15 | 禧尔公司 | Systems and methods for sample preparation, data generation, and protein crown analysis |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ZA941645B (en) * | 1994-03-09 | 1995-01-13 | South To South Co Operation In | Antibodies specifically reactive against human prostate specific antigen. |
| EP0725139A3 (en) * | 1995-02-03 | 1997-11-26 | Bayer Corporation | Recombinant prostate-specific antigen and its use as an immunoassay calibrator |
-
1995
- 1995-06-30 FI FI953257A patent/FI953257A/en unknown
-
1996
- 1996-06-26 JP JP9504778A patent/JPH11508449A/en active Pending
- 1996-06-26 EP EP96923930A patent/EP0835317A1/en not_active Withdrawn
- 1996-06-26 WO PCT/EP1996/002779 patent/WO1997002350A1/en not_active Application Discontinuation
- 1996-06-26 US US08/973,719 patent/US6140468A/en not_active Expired - Fee Related
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