Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Patent
1997-12-29
2000-10-31
Burke, Julie
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
536 235, 435348, 435 691, 435 701, 435 703, 530416, 530417, C07K 1400
Patent
active
061404683
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to the production and purification of human prostate specific antigen (hPSA) by recombinant-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..sup.1-3 hPSA is a potent proteolytic enzyme belonging to the group of extracellular serine proteases and has some features common with chymotrypsin-like enzymes..sup.4,5 According to the primary structure of hPSA it belongs to the human kallikrein family of proteases..sup.5,6 The two other members of this family are the human pancreatic/renal kallikrein and the human glandular kallikrein-1 (hGK-1). The hGK-1 is most closely related to hPSA having .apprxeq.80% sequence identity and is also expressed in human prostate tissue..sup.6-10
Since Papsidero and colleagues.sup.11 demonstrated that hPSA can be regularly detected in sera of prostatic cancer patients various commercial hPSA assays were developed using monoclonal or polyclonal antibodies, such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), sandwich immunoassays and fluorescent immunoassays..sup.12 The clinical use of serum hPSA measurements have become the most central in the diagnosis and follow-up of treatment or prostatic cancer..sup.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..sup.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..sup.18 hPSA is also found in very low concentrations in human breast milk..sup.19
In the serum the proteolytic activity of hPSA is inhibited by the formation of irreversible complexes with serum protease inhibitors such as .alpha..sub.1 -antichymotrypsin and .alpha..sub.2 -macroglobulin..sup.20-23 However, uncomplexed forms of hPSA also exist in bloodstream. The free form of hPSA and the form complexed with .alpha..sub.1 -antichymotrypsin are detected immunologically whereas .alpha..sub.2 -macroglobulin encapsulates the reactive hPSA epitopes in such a way that complexes are not quantifiable immunologically by conventional two-site immunometric assays..sup.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, .alpha..sub.2 -macroglobulin bound, .alpha..sub.1 -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 separated 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 in 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 time the development of an efficient expression system for hPSA by recombinant baculovirus-infected 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
REFERENCES:
Ausubel et al. Current Protocols in Molecular Biology. Unit 16.8 "Expression of proteins in insect cells using baculoviral vectors" pp. 16.8.1-16.11.7, 1990.
Bei et al. "Generation, purification, and characterization of a recombinant source of human prostate-specific antigen" Journal of Clinical Laboratory Analysis. Vol. 9, pp. 261-268, Jan. 1, 1995.
Henttu et al. "cDNA coding for the entire human prostate specific antigen shows high homologies to the human tissue kallikrein genes" Biochem. and Biophys. Res. Commun. vol. 160. No. 2. pp. 903-910, Apr. 28, 1989.
Lundwall et al. "Molecular cloning of human prostate specific antigen cDNA" Febs Letters. vol. 214. No. 2, pp. 317-322, Apr. 1987.
Medin et al. "Efficient, low-cost protein factories: Expresssion of human adenosine deaminase in baculovirus-infected insect larvae" PNAS. vol. 87. pp. 2760-2764, Apr. 1990.
Boehringer Mannheim GmbH
Burke Julie
Helms Larry R
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