RECOMBINANT HUMAN PROSTATE SPECIFIC ANTIGEN AND USES THEREOF

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.

Description: Recombinant human prostate specific antigen and uses thereof The present invention relates to the production and puπfi- cation of human prostate specific antigen (hPSA) by recom- binant-DNA-technology, and the active and inactive protein forms obtained, as well as uses thereof. Human prostate specific antigen (hPSA) is a 30-34 kDa single chain glycoprotein synthesized in the epithelial cells of the prostate gland and secreted into seminal fluid occurring at levels of 0.5-2 mg/ml. 1"3 hPSA is a potent proteolytic enzyme belonging to the group of extra¬ cellular serine proteases and has some features common with chymotrypsin-like enzymes. 4,5 According to the primary structure of hPSA it belongs to the human kallikrein family of proteases. 5,6 The two other members of this family are the human pancreatic/renal kallikrein and the human glandu¬ lar kallikrein-1 (hGK-1) . The hGK-1 is most closely related to hPSA having « 80% sequence identity and is also expres¬ sed in human prostate tissue. 6-10 Since Papsidero and colleagues' 1 demonstrated that hPSA can be regularly detected in sera of prostatic cancer patients various commercial hPSA assays were developed using mono¬ clonal or polyclonal antibodies, such as radioimmunoassay (RIA) , enzyme-linked immunosorbent assay (ELISA) , sandwich immunoassays and fluorescent immunoassays. 12 The clinical use of serum hPSA measurements have become the most central in the diagnosis and follow-up of treatment of prostatic cancer. 13,14 Because serum hPSA concentrations are also increased in patients with benign prostatic hyperplasia and in inflammatory conditions affecting the prostate, hPSA measurements alone are not sufficient screening tool for undiagnostised prostatic cancer. However, by combining serum hPSA concentrations to other methods as digital rectal examination and transrectal ultrasound the early diagnosing of prostatic cancer is improved. 15"17 The recent findings have shown that hPSA can perhaps no longer be considered an absolutely prostate tissue-specific marker. Using immunohistochemical methods hPSA could be detected in female periurethral glands, apocrine sweat glands, apocrine breast cancers, salivary gland neoplasma and male urethra. 18 hPSA is also found m very low concen¬ trations in human breast milk. 19 In the serum the proteolytic activity of hPSA is inhibited by the formation of irreversible complexes with serum protease inhibitors such as αι-antιchymotrypsin and α 2 -macroglobulin. 20~23 However, uncomplexed forms of hPSA also exist in bloodstream. The free form of hPSA and the form complexed with (Xi-antichymotrypsin are detected im¬ munologically whereas α 2 -macroglobulιn encapsulates the reactive hPSA epitopes in such a way that complexes are not quantifiable immunologically by conventional two-site immunometric assays. 24 Because of the complexity of the hPSA molecule and anti-hPSA antibodies, there is currently no internationally standardized method for the quantification of serum hPSA. Also it will be important to have consensus on what form of hPSA clinical assays should measure, the free, α 2 -macroglobulιn bound, αi-antichymotrypsin bound or combinations of these forms of hPSA. But before any standardization of hPSA assay can be done a common standard is urgently required. In the present work hPSA was produced as a recombinant protein in insect cells. The active form of hPSA was sepa- rated from the inactive form, and these were purified and characterized. Both of the protein forms were found to be suitable for use as a standard protein m hPSA assays, and also as an antigen in the preparation of very specific antibodies against hPSA. In the present application we describe for the first t me the development of an efficient expression system for hPSA by recombinant baculovirus-mfected Sf9 insect cells. The recombinant hPSA was secreted into the culture medium both in an active and an inactive form which were able to be separated in the last purification step with cation exchange chromatography. In the active form the signal and activation peptides were properly cleaved, as confirmed by N-terminal peptide sequencing. The mature hPSA consists of a single chain polypeptide of 237 ammo acids with a molecular weight (M r ) of 26079 for the peptide moiety of the molecule and contains a single N-lmked carbohydrate side cham attached to asparagme- 45. 6 ' 34 It has been reported that when analyzing hPSA puri¬ fied from seminal fluid by ion spray mass spectrometry (ISMS) the average M r of 28430 was detected indicating that the protein contams a carbohydrate residue of M r 2351. 35 Analysis of the active recombinant hPSA by ISMS showed a M r of 26500 which indicates almost total absence of N-termmal sugars. Degiycosylation studies also confirmed the finding that the recombinant hPSA was not glycosylated. After N-glycosidase F or O-glycosidase treatment no detectable reduction in the molecular weight of 31 kDa of recombinant hPSA could be seen m SDS-PAGE analysis whereas the molecu¬ lar weight of commercial hPSA reduced after N-glycosidase F treatment from 34 to 31 kDa. Despite the differences in the glycosylation of commercial and recombinant hPSAs they both were recognized in the fluoroimmunoassay by two mouse mono¬ clonal antibodies. In the native PAGE and isoelectric focusing our recombi¬ nant hPSA was shown to be more homogeneous than the commer¬ cial one. The commercial hPSA had five different forms in silver-stained native PAGE whereas recombinant hPSA had only one. In the isoelectric focusing the commercial hPSA had several isoforms with isoelectric points in the range pH 6.0 to 7.0 whereas the recombinant hPSA had one major pl of 7.7 and smaller one of 7.4. The specific activities of recombinant and commercial hPSAs were similar and compara- ble to the previously measured values when synthetic pepti¬ de substrate was used. 20 The active form of the recombinant hPSA forms stable comp¬ lexes with αi-antichymotrypsm and α.-macroglobulm the similar manner as the commercial hPSA. oti-antichymotrypsm forms complexes of approximately 80 kDa and inactivates both hPSAs by binding to the active site of the protem. 20 It is known that α 2 -macroglobulιn encapsulates the anti¬ genic epitopes of hPSA in such a way that the protein escapes immunodetection in native conditions. Reducing SDS-PAGE of α 2 -macroglobulin-protemase complexes produces α 2 -macroglobulin-fragments which have a molecular weight of approximately 85 kDa. 36 When recombinant hPSA (31 kDa) or commercial hPSA (34 kDa) were covalently bound to these fragments of α 2 -macroglobulin weak bands of approximately 110 kDa could be detected in immunoblots. Using small synthetic peptide substrates the chromogenic activities of the recombinant and commercial hPSAs increased immediately after α 2 -macroglobulm addition. The inactive form of recombinant hPSA has two additional ammo acid residues in its N-termmus. The N-termmal ammo acid sequence thereof is Ser-Arg-Ile-Val-Gly-Gly-Trp-Glu- Cys-Glu-Lys-His-Ser. This protein does not form complexes with αι-antιchymotrypsm or α 2 -macroglobulm, and it has no activity on either on MeO-Suc-Arg-Pro-Tyr-pNA » HCl or H-D- Pro-Phe-Arg-pNA-2HCl substrates. Having the recombinant hPSA we can avoid the contamination of hPSA with very similar hGK-1 protein. As these two protems are expected to be identical size and also share significant similarity m hydrophilic areas, they may be predicted to manifest extensive immunological cross- reactivity. We have also produced in insect cells the recombinant hGK-1 which is also recognized the hPSA fluoroimmunoassay. Due to that we conclude that it is very important to use recombinant hPSA in the standardization of PSA-assay either as a standard protein or as an antigen in the preparation of proper antibodies aga st hPSA. Antibodies can either be monoclonal or polyclonal antibo- dies which are raised agamst either the active or the inactive form of the recombinant hPSA of the invention. The antibodies can be produced by various processes known m the art, depending on the type of the antibody desired. To obtam polyclonal antibodies, a vertebrate, e.g. a rabbit is hyperimmunized with the antigen, blood is collected after repeated immunizations and the gamma globulin is isolated. Suitable processes for preparing of polyclonal antibodies are disclosed e.g. Harlow and Lane, Eds., Antibodies, a laboratory manual, 1988 Cold Spring Harbor Press. To obtain monoclonal antibodies a small animal, typically a mouse or a rat, is immunized with the antigen, the spleen or popliteal lymph node is removed, and the lymphocytes are combined with myeloma cells in the presence of a suitable fusion promoter. The hybrid cells so ob¬ tained, called hybridomas, are screened to isolate indi¬ vidual clones each of which secretes one unique antibody to the antigen. A general method for obtaining monoclonal antibodies was described by Kohler and Milstein in Nature 256:495-497 (1975) . Brief description of the drawings Figure 1: Silver-stained native (A.) and reduced SDS (B.) polyacrylamide gel electrophoresis of the commercial hPSA purified from seminal fluid (Line 1) and the recombinant hPSA (active form) (Line 2) Figure 2: Silver-stained isoelectric focusing of the recom¬ binant hPSA (active form) (Line 1) and commercial hPSA (Line 2) Figure 3: Interaction of recombinant and commercial hPSAs with αi-antichymotrypsin Formation of complexes with pure recombinant hPSA (active form) (A.) and with commercial hPSA (B.) was followed by Western blotting of SDS-PAGE. All samples were reduced with 5% 2-mercaptoethanol before analysis. Lines 2-5 show the time course of the interaction, incubation times were: (2) 0.1 min, (3) 1 min, (4) 15 min, (5) 60 mm. Line A.l is nonco plexed recombinant hPSA and Line B.l noncomplexed commercial hPSA. The chromogenic activity of recombinant hPSA (■) and commercial hPSA (D) was followed during comp¬ lex formation in part C. Figure 4: Interactions of recombinant and commercial hPSAs with α 2 -macroglobulm Formation of complexes with pure recombinant hPSA (active form) (A.) and with commercial hPSA (B.) was followed by Western blotting of SDS-PAGE. All samples were reduced with 5% 2-mercaptoethanol before analysis. Lines 2-5 show the time course of the interaction, incubation times were: (2) 0.1 mm, (3) 1 mm, (4) 15 mm, (5) 60 mm. Line A.l is noncomplexed recombinant hPSA and Line B.l noncomplexed commercial hPSA. The chromogenic activity of recombinant hPSA (■) and commercial hPSA (D) was followed durmg comp- lex formation in part C. Figure 5: Quantitative recovery of purified recombinant hPSA ana commercial hPSA measured by time-resolved fluo- roimmunoassay for hPSA Pure recombinant hPSA (active form) (•) and commercial hPSA (O) were diluted with human serum contammg buffer to concentrations 1, 5, 10, 100, 500 μg/L. These concentra¬ tions were assayed in duplicate by fluoroimmunoassay kit (Wallac) for hPSA. - 100% recovery. Figure 6: The last purification step of recombinant hPSA with cation exchange chromatography. Elution with linear sodium chloride gradient. Active rec-hPSA: peak 2. Inactive rec-hPSA: peak 5. Peaks 3 and 4 contain the mixture of both forms. Experimental Construction of the recombinant plasmid transfer vector The hPSA-cDNA (1464 bp) was cloned mto EcoRI site of the pVL1392 nonfusion transfer vector (Invitrogen) and introdu¬ ced with homologous recombination into the genome of Auto- grafa californica nuclear polyhedrosis virus (AcNPV) . Generation of the recombinant bacul ovirus and protein production Recombinant virus contammg the hPSA DNA fragment, was used to infect Spodoptera frugiperda (Sf9) cells (ATCC CRL1711) . Recombinant protein was produced using exponen¬ tially growing Sf9 insect cells (2 x loVml) at 27°C infec¬ ted by hPSA-AcNPV at a multiplicity of infection 1 in 1000 ml spinner flasks (Bellco) containing 10% FCS complete TNM-FH insect medium (Sigma) with antibiotics. For harves- ting the cells were centrifuged and the supernatant was collected and stored for further purification of the recom¬ binant hPSA. The amount of hPSA secreted mto the culture medium was determined by time-resolved fluoroimmunoassay kit (DELFIA, Wallac) . Expression of hPSA in Sf9 cells The recombinant hPSA was overproduced m Sf9 cells. The protein was secreted mto the culture medium and the pro- duction reached a maximum at 4 days after infection being 3 - 4 mg/liter. Scalmg-up of Sf9 insect cell cultures was effected using the following series: T-flasks, spinner bottles, 2-liter bioreactor and 30-liter bioreactor. Purification of recombinant hPSA The harvested medium from the recombinant virus infection was concentrated with a Pellicon cassette system (cut-off, 10 kDa; Millipore) and dialyzed m 50 mM sodium acetate buffer (pH 6.8) . The concentrate was then loaded onto a fiuidized bed column (Streamline 50, 5 x 100 cm, 100 ml/mm) contammg strong cation-exchange matrix (600 ml, Streamline SP, Pharmacia) . After washing the recombinant hPSA was eluted from the column with linear salt gradient from 0 to 0.25 M NaCl. The fractions reacting with rabbit anti-hPSA serum (slot-blot) were concentrated (Amicon) for gel filtration chromatography with Sephacryl S-200 (2.6 x 80 cm, 0.7 ml/mm, Pharmacia) column eluted with 50 mM sodium phosphate buffer (pH 7.0) containing 0.15 M NaCl. The fractions with the highest hPSA content were pooled and dialyzed m 50 mM sodium phosphate buffer (pH 5.8) for cation-exchange chromatography with Mono-S column (0.5 x 5 cm, 0.5 ml/mm, Pharmacia) . The active (90-110 mM) and inactive (120-140 mM) pools of recombinant hPSA were eluted from the column with a linear salt gradient of NaCl. The Streamline 50 and the Sephacryl S-200 columns were connec¬ ted to BioPilot and the Mono-S column to FPLC automated chromatography systems (Pharmacia) . The purification procedure is illustrated in Scheme I Scheme I Purification of recombinant hPSA Sf9 CULTURE MEDIUM [2-4 mg rec-hPSA/liter; centrifugation Concentration of culture medium by ULTRAFILTRATION (MW cut-off 10000 Da) dilution CATION EXCHANGE CHROMATOGRAPHY with expanded bed adsorption column BioPilot (Streamline SP 50, 5x100 cm, 100 ml/min) 0.25M NaCl- elution concentration GEL FILTRATION CHROMATOGRAPHY Superdex 75 BioPilot (2,6 x 80 cm, 0,7 ml/min) CATION EXCHANGE CHROMATOGRAPHY MonoS HP FPLC 0,5 x 5 cm 0-0.25M NaCl- elution The active and inactive forms of recombinant hPSA were obtained in purity (Fig. 6) . Characterization of recombinant hPSA The recombinant hPSA was produced into culture medium in two forms, active and inactive, which were able to be separated in the last purification step with cation- exchange chromatography. The active form of hPSA was eluted with lower salt concentration than the inactive one. The specific activity of the pure protein was 73 nmol x mm "1 x mg "1 with the synthetic peptide MeO-Suc-Arg-Pro-Tyr-pNA-HCl as the substrate. The specific activity for commercial hPSA was 79 nmol x min "1 x g "1 . Purified active recombinant hPSA was present in silver-stained SDS-PAGE m one band of 26 kDa and the commercial hPSA purified from seminal fluid had one band of 34 kDa (Fig. IB) . In the silver-stamed native PAGE the active recombinant hPSA showed one band of 200 kDa whereas hPSA from seminal fluid had four bands between 70 and 150 kDa (Fig.lA) . All these forms of recombinant and commercial hPSAs were recognized in Western blottings of SDS-PAGE and native PAGE with the rabbit polyclonal antibo- dy raised against hPSA purified from seminal fluid . In the immunoblot of the native PAGE there can be seen one additional band in the sample of recombinant hPSA and three in commercial hPSA comparing to the silver-stamed native PAGE of the same samples (Fig.lA) . Silver-stamed isoelecric focusing (Fig.2) shows the commercial hPSA to exist in several isoforms with isoelectric points in the pH range from 6.0 to 7.0. Our active recombinant hPSA showed one major band in the pH 7.7 and the smaller one m the pH 7.4. Both forms were recognized the immunostaining of blotted IEF gel (data not shown) . Quanti tative recovery of recombinant hPSA by f l uoroimmuno¬ assay The protein concentration of purified recombinant hPSA was measured by the method of Lowry et al . with bovine gamma globulin (Bio-Rad) as the standard. 25 The recovery of recombinant hPSA was further measured by time-resolved fluoroimmunoassay (DELFIA PSA kit, Wallac) which recognized both free PSA and complexed with α--antichymotrypsin. 21,22 For the assay the recombinant hPSA antigen was diluted with "zero" standard of fluoroimmunoassay kit containing human serum in Tris-HCl buffered salt solution (pH 7.8) . The recombinant and commercial hPSAs were diluted with human serum containing buffer to yield concentrations of 1, 5, 10, 100 and 500 μg/L which were tested in duplicate for hPSA concentrations with fluoroimmunoassay. The recoveries of the recombinant and the commercial hPSAs were 119±3% (n=5) and 149+13% (n=5) , respectively when compared with the hPSA calibrator in the fluoroimmunoassay kit (Fig.5) . Separating of acti ve and inacti ve forms of recombinant hPSA produced The last purification step of recombinant hPSA was done with cation exchange chromatography. With linear sodium chloride gradient we could separate the active form of hPSA in peak 2 from the inactive form m peak 5. Peaks 3 and 4 contained the mixture of both forms. (Fig.6) . The condi- tions were as follows. Column: MonoS HP (0 0.5 x 5 cm) Buffer B: 50 mM Na-phosphate, pH 5.8 Buffer A: 50 mM Na-phosphate, pH 5.8 + IM NaCl Sample: Partially purified rec-hPSA Sample volume: 5 ml Flow rate: 0.5 ml/min. In silver stained native PAGE the active form of recom¬ binant hPSA had the molecular weight of 195 kDa and the inactive form 370 kDa (not shown) . Gel electrophoresis and immunoblotting The purity and characterization of recombinant hPSA were evaluated by SDS-PAGE and native PAGE. Both electrophoresis were carried out on a PhastSystem (Pharmacia) with PhastGel gradient media 10-15 for SDS-PAGE and 8-25 for native PAGE and were silver-stained. 26-30 In Western blotting, the proteins were transferred onto nitrocellulose membranes with PhastTransfer (Pharmacia) . 31 Rabbit polyclonal antibo¬ dy raised against hPSA purified from seminal fluid 32 was used together with a ProtoBlot AP system (Promega) to detect expressed protein. In all electrophoresis the recom¬ binant hPSA was compared to the commercial hPSA purified from seminal fluid (Calbiochem) . Isoelectric focusing was performed on a PhastSystem with PhastGel IEF media, pH range 3-9, and gels were silver-stained. Formation of complexes between PSA and proteinase inhibi ¬ tors Reaction mixtures were prepared in which 10 μg of recom- binant or commercial purified hPSAs were incubated with 40 μg of purified αi-antichymotrypsin (Calbiochem) or 200 μg of α 2 -macroglobulin (Calbiochem) . All reactions were per¬ formed at 37°C for different periods of time (0.1, 1, 15, 60 min) in 20 mM Tris buffer containing 150 mM NaCl (pH 7.4) . Reaction mixtures were analysed by activity measure¬ ments and Western blotting of SDS-PAGE. The active form of our recombinant hPSA formed stable complexes in reduced SDS-PAGE with α-.-antichymotrypsin and α 2 -macroglobulin as did commercial hPSA. After 1 min appro¬ ximately 80 kDa band can be seen in Western blots when both hPSAs were complexed with αi-antichymotrypsin (a twofold molar excess of αi-antichymotrypsin) (Fig.3A,B) . The loss of hPSA-mediated hydrolysis of MeO-Suc-Arg-Pro-Tyr-pNA-HCl was achieved immediately after complex formation begins (Fig.3C) . After 1 min 53% of recombinant hPSA and 35% of commercial hPSA activities were left. When adding α 2 -macroglobulin to the incubation mixtures containing commercial or our recombinant hPSAs (in a 1:1 molar ratio) the complex formation begins immediately and weak band of approximately 110 kDa can be seen in Western blots of SDS-PAGE (Fig.4A,B) . The addition of α 2 -macroglobulin increased the activities of both PSAs, threefold with recombinant hPSA and fourfold with the commercial hPSA (Fig.4C) . Measurements of PSA acti vi ty PSA-hydrolysis of MeO-Suc-Arg-Pro-Tyr-pNA*HCl (Chromogenix AB) at a final concentration of 1 mM was measured at 405 nm. The reactions were performed at 37°C and initiated by addition of the chromogenic substrate (50 μl) to 200 μl 50 mM Tris buffer (pH 7.8) with 100 mM NaCl containing hPSA (25 μg) . After one hour the reaction was stopped by adding 800 μl 0.6 M acetic acid and the reaction rate (nmol pNA formed per min) was calculated from the standard curve of p-nitroaniline (pNA) . Enzymatic degiycosylation of hPSA Ten micrograms of recombinant (13 μl) and commercial (3 μl) hPSAs were mixed with 0.5 μl of 10% SDS, the total volumes were adjusted to 15 μl with 0.1 M sodium phosphate containing 25 mM EDTA (pH 7.2), and the mixtures were boiled for 3 min. After the pretreatment, 5 μl of 10% (vol/vol) Triton X-100, 3 μl N-glycosidase F solution (0.6 unit) or 5 μl O-glycosidase solution (2.5 milliunits) were added to the protein solutions (both enzyme solutions were purchased from Boehringer Mannheim) . The final volume was adjusted to 30 μl with 0.1 M sodium phosphate buffer con¬ taining 25 mM EDTA (pH 7.2) . The mixtures were incubated at 37°C overnight and analyzed by SDS-PAGE under nonreducing conditions. O-Glycosidase and N-glycosidase F did not have any detect¬ able effects on the molecular mass of recombinant hPSA in SDS-PAGE whereas after degiycosylation of commercial hPSA with N-glycosidase F the reduction of 3 kDa m molecular weight can be seen. References 1. Sensabaugh GF. Isolation and characterization of a semen-specific protein from human seminal plasma: A poten¬ tial new marker for semen identification. J Forensic Sci 1978; 23:106-115. 2. Wang MC, Valenzuela LA, Murphy GP, Chu TM. Purification of human prostate specific antigen. Invest Urol 1979; 17: 159-163. 3. Wang MC, Papsidero LD, Kuπyama M, et al. Prostate antigen: A new potential marker for prostatic cancer. The Prostate 1981; 2: 89-96. 4. Hara M. Kimura H. Two prostate-specific antigens, γ-semmoprotein and β-microsemmoprotem. J Lab Cim Med 1989; 113: 541-548. 5. Watt KWK, Lee P-J, Timkulu TM, et al. Human prostate- specific antigen: Structural and functional similarity with serine proteases. Proc Natl Acad Sci USA 1986; 83:3166-3170. 6. Lundwall A, Lilja H. Molecular cloning of human prostate specific antigen cDNA. FEBS Let t 1987; 214: 317-322. 7. Henttu P, Vihko P. cDNA coding for the entire human prostate specific antigen shows high homologies to the human tissue kallikrein genes. Bi ochem Bi ophys Res Commun 1989; 160: 903-910. 8. Schedlich LJ, Bennets BH, Morris BJ. Primary structure of a human glandular kallikrein gene. DNA 1978; 6: 429-437. 9. Chapdelaine P, Paradis G, Tremblay RR, Dube J. High level of expression in the prostate of a human glandular kallikrein mRNA related to prostate-specific antigen. FEBS Lett 1988; 236: 205-208. 10. Henttu P, Lukkarinen 0, Vihko P. Expression of the gene coding for human prostate-specific antigen and related hGK-1 in benign and malignant tumors of the human prostate. Int J Cancer 1990; 45: 654-660. 11. Papsidero LD, Wang MC, Valenzuela LA, et al . A prostate antigen in sera of prostatic cancer patients. Cancer Res 1980; 40: 2428-2432. 12. Takayama TK, Vessella RL, Lange PH. A brief review of ultrasensitive prostate-specific antigen assays for the evaluation of patients after radical prostatectomy. World J Urol 1993; 11: 192-195. 13. Sta ey TA, Yang N, Hay AR, et al. Prostate-specific antigen as a serum marker for adenocarcinoma of the prosta¬ te. N Engl J Med 1987; 317: 909-916. 14. Kablin JN. Prostate specific antigen: Clinical use in the diagnosis and management of prostate cancer. Geria trics 1992; 47: 26-32. 15. Cooner WH, Mosley BR, Rutherford CL et al. Prostate cancer detection in a clinical urological practice by ultrasonography, digital rectal examination and prostate specific antigen. J Urol 1990; 143: 1146-1154. 16. Catalona WJ, Smith DS, Ratliff TL et al. Measurement of prostate-specific antigen in serum as a screening test for prostate cancer. N Engl J Med 1991; 324: 1156-1161. 17. Partin AW, Oesterling JE. The clinical usefulness of prostate specific antigen: Update 1994. J Urol 1994; 152: 1358-1368. 18. Graves HCB. Nonprostatic sources of prostate-specific antigen: a steroid hormone-dependent phenomen. Clin Chem 1995; 41: 7-9. 19. Yu H, Diamandis EP. Prostate-specific antigen in milk of lactating women. Clin Chem 1995; 41: 54-58. 20. Christensson A. Laurell C-B. Lilja H. Enzymatic activi- ty of prostate-specific antigen and its reactions with extracellular serine protease inhibitors. E J Biochem 1990; 104: 755-763. 21. Stenman UH, Leinonen J, Alfthan H, et al. A complex between prostate-specific antigen and ai-antichymotrypsin is the major form of prostate-specific antigen in serum of patients with prostatic cancer: assay of the complex impro¬ ves clinical sensitivity for cancer. Cancer Res 1991; 51: 222-226. 22. Lilja H, Christensson A, Dahlen U, et al. Prostate- specific antigen in human serum occurs predominantly in complex with α- . -antichymotrypsin. Clin Chem 1991; 37: 1618-1625. 23. Christensson A, Bjork T, Nilsson 0, et al. Serum pros¬ tate specific antigen complexed to αi-antichymotrypsin as an indicator of prostate cancer. J Urol 1993; 150: 100-105. 24. Zhou AM, Tewari PC, Bluestein BI, et al . Multiple forms of prostate-specific antigen in serum: differences in im unorecognition by monoclonal and polyclonal assays. Clin Chem 1993; 39: 2483-2491. 25. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193: 265-275. 26. Jovin TM. Multiphasic zone electrophoresis. I. Steady- state moving boundary systems formed by different electro¬ lyte combinations. Biochem 1973; 12: 871-890. 27. Wyckoff M, Rodbard D, Crambach A. Polyacrylamide gel electrophoresis in sodium dodecyl sulphate containing buffers using multiphasic buffer systems: Properties of the stack, valid Rf measurements and optimized procedure. Anal Biochem 1977; 78: 459482. 28. Davis BJ. Disc Electrophoresis II: Methods and Applica- tion to human serum proteins. Ann N Y Acad Sci 1964; 121: 404-427. 29. Andrews AT. Molecular weight measurement and the use of gel concentration gradients. In Electrophoresis. Theory, Techniques and Biochemical and Clinical Applications, Peacoce AR, Harrington WF, eds. Clarendon Press, Oxford, 1981: 63-80. 30. Heukeshoven J, Dernick R. Simplified method for silver staining of proteins in polyacrylamide gels and the mechanism of silver staining. El ectrophoresis 1985; 6: 103-112. 31. Prieur B, Russo-Marie F. An automated Western blot analysis using the Phast-System. Anal Biochem 1988; 172: 338-343. 32. Vihko P, Kurkela R, Ramberg J, et al . Time-resolved immunofluorometric assay of human prostate-specific anti- gen. Clin Chem 1990; 36: 92-95. 33. Schaller J, Akiyama K, Tsuda R, et al. Isolation, characterization and amino-acid sequence of γ-semino- protein, a glycoprotein from human seminal plasma. Eur J Biochem 1987; 170: 111-120. 34. Belanger As van Halbeek H, Graves HCB, et al. 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Claims: Claims 1. Recombinant human prostate specific antigen (hPSA) which is characterized in that a) it is obtainable by recombmant-DNA-technology using baculovirus expression system in infected insect cells, b) it has a molecular weight of about 26.5 kDa as determined by ion spray mass spectrometry, c) it has a major isoelectric point of 7.7, d) it has a specific activity of 73 nmol/mm/mg with the synthetic peptide MeO-Suc-Arg-Pro-Tyr-pNAΗCl as a substrate, e) it has no activity on the substrate H.-D.-Pro- Phe-Arg-pNA • 2HC1, f) it forms complexes with ι-antιchymotrypsm and α 2 -macroglobulm. 2. Recombinant human prostate specific antigen (hPSA) which is characterized in that a) it is obtainable by recombmant-DNA-technology using baculovirus expression system in infected insect cells, b) it has the followmg N-termmal ammo acid sequence: Ser-Arg-Ile-Val-Gly-Gly-Trp-Glu-Cys-Glu-Lys- His-Ser, c) it does not form complexes with α-.- antichymotrypsm or α 2 -macroglobulιn, d) it has no activity on the substrates MeO-Suc-Arg- Pro-Tyr-pNA » HCl and H-D-Pro-Phe-Arg-pNA • 2HC1. 3. Process for the production of the recombinant hPSA of claim 1 or 2, comprising a) incorporating 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. 4. Process according to claim 3 wherein the purification of step e) is effected by dialyzing the culture medium, loading the dialysate onto a fiuidized 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. 5. In a method for immunometric assay of human prostate specific antigen (hPSA) in human body fluids, an improve- ment comprising use of the hPSA according to claim 1 or claim 2 as a standard protein. 6. The improvement according to claim 5 wherein the body fluid is serum. 7. Use of the hPSA protein according to claim 1 or claim 2 as a standard protein in immunometric hPSA assays. 8. An antibody directed against the hPSA according to claim 1 or 2.

Inventors: VIHKO PIRKKO, VIHKO, PIRKKO
Applicants: BOEHRINGER MANNHEIM GMBH, VIHKO PIRKKO, VIHKO, PIRKKO
Application year: 1996
Main classification: C12N15
IPC codes: C07K16, C12N9, C12N15, C12P21, C12R1, G01N33