Human glandular kallikrein-1 (hK2)

Details Human glandular kallikrein-1 (hK2) Human glandular kallikrein-1 (hK2)

1998-06-08

2001-10-16

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

C435S007400, C435S007920, C435S007930, C435S069100, C435S252300, C435S320100, C435S219000, C435S006120

Reexamination Certificate

active

06303361

ABSTRACT:

The present invention relates to novel proteins. In particular, it relates to a novel form of human glandular kallikrein-1 (hK2) encoded by a newly-identified hK2 gene and use of recombinant DNA techniques to obtain proteins encoded by the hK2 genes, including the mature protein encoded by the previously recognised hK2 gene, Arg
226
-hK2, as an active protein.
The human glandular kallikrein gene family is composed of genes encoding three different proteins: prostate specific antigen (PSA), glandular kallikrein-1 (hK2) and pancreatic/renal kallikrein (KLK1). These genes are located on chromosome 19 and the PSA and hK2 genes are aligned at a distance of 12 kb(1). The similarity of the coding region of the human KLK1 gene to the coding regions of the human PSA and K2 genes is 74% and 75% respectively. The coding regions of the hPSA and hK2 genes are 85% homologous and the promoter regions of the same genes are 91% homologous. The KLK1 gene encodes the true kallikrein. KLK1 has a kininogenase activity and it is expressed in kidney, pancreas and salivary gland (2). By in situ hybridization, the hPSA and hK2 genes have been shown to be expressed only in prostatic epithelial cells. Despite the similarity of the hK2 and hPSA genes, the expression level of the hK2 gene at mRNA level is only about 10-30% ot that of the hPSA gene in the prostate (3). When tested with LNCaP cells, clear up-regulation of the mRNA levels for both hK2 and hPSA was observed in the presence of androgens (4).
Recently, further results have indicated that expression of hK2 and hPSA is not in fact prostate specific as previously believed. By Southern blot analysis with gene-specific oligonucleotide probes after RT-PCR, expression of all three previously identified human kallikrein genes has been detected in the human endometrium (5). hPSA has also been detected in milk of lactating women by immunoassay (6). It has additionally been found that 30-40% of breast tumours as well as steroid hormone stimulated normal breast tissue samples contain hPSA (7).
An hK2 gene has been isolated from a human genomic fetal liver DNA library and sequenced (8a). The nucleotide sequence (SEQ ID NO:6) of the five coding exons of this hK2 gene was found to encode a 261 amino acid preproprotein (SEQ ID NO:7) with a signal peptide of 17 amino acids (SEQ ID NO:14) and an activation peptide of 7 amino acids (SEQ ID NO:13) like PSA. hK2 has been found to possess the typical catalytic triad (His41-Asp96-Serl89) of serine proteases. The presence of an aspartate residue at amino acid position 183 in hK2 fits with a trypsin-like substrate activity for this protein. In PSA, there is a serine residue at the same position which accounts for PSA in contrast exhibiting chymotrypsin-like activity (8).
The function of PSA is the cleavage of semenogelin clots (9), but the function of hK2 is unknown. The concentration of PSA in serum is increased in cancer and hyperplasia of the prostate gland and PSA is widely used as a marker for the detection and monitoring of prostate cancer (10). Because of the high homology between the hPSA and hK2 genes, an hK2 protein has, however, remained to be purified. Also, the purity of PSA is not always unambiguous. This leads to problems with hPSA assays that are based on polyclonal or monoclonal antibodies raised against hPSA.
An hK2 protein has now been obtained free of hPSA contamination by cloning hK2 cDNA from a human prostate cancer tissue cDNA library in an expression vector and expressing the cDNA in appropriate host cells. More specifically, an hK2 protein has been produced by providing a baculovirus expression vector encoding a prepro-hK2 protein in insect cells (see Examples 4 and 5). While only use of a baculovirus expression vector system for production of an hK2 protein is described herein, in particular a recombinant Autographa California Nuclear Polyhedrosis virus (AcNPV) containing a coding sequence for prepro-hK2 under the control of the polyhedrin promoter, other expression vector systems commonly employed for production of human proteins may be employed. If, however, an expression vector encoding a prepro-hK2 protein is used, for example, to transform bacterial cells, e.g.
E. coli
cells, it will be appreciated that unlike in the case of the above baculovirus expression vector system, the pre-pro-sequence will not be removed by post-translational processing to give directly the corresponding mature protein, e.g. active mature Arg
226
-hK2. By cloning an hK2 cDNA (SEQ ID NO: 8) from a human prostate cDNA library, an hK2 cDNA has now been identified which has one base difference from the coding sequence for hK2 previously reported. This difference at base position 790 (C to T) of SEQ ID NO: 8 equates with an amino acid change at amino acid position 226 of SEQ ID NO:9 from Arg to Trp. That this observed base difference was not an artifact of the cDNA cloning and sequencing procedure but reflects the existence of a previously unidentified hK2 gene was confirmed by using PCR to amplify the hK2 gene from genomic DNA of a number of human prostate and leucocyte samples (see Example 2).
In one aspect, the present invention thus provides a protein which is Trp
226
-hK2 (SEQ ID NO:9) having a sequence identical to Arg
226
-hK2 (SEQ ID NO:8) apart from change of Arg
226
to Trp or which is Trp
226
-hK2 having an N- and/or C-terminal extension and which retains the ability to bind Trp
226
-hK2 antibodies or hPSA antibodies, with the proviso that where said protein is a naturally-occurring protein it is substantially free of other proteins with which it is ordinarily associated.
Such proteins include, in addition to Trp
226
-hK2 in substantially pure form, for example, proTrp
226
-hK2 or prepro-Trp
226
-hK2 in substantially pure form wherein Trp
226
-hK2 is joined at the N-terminus to the pro- or prepro-sequences (SEQ ID NO:13 and SEQ ID NO:11, respectively) previously identified for Arg
226
-hK2. A further novel protein forming an embodiment of the present invention is Trp
226
-hK2 having the N-terminal dipeptide extension SerArg. It has been found that this derivative of Trp
226
-hK2, rather than Trp
226
-hK2 per se, is obtained by expression of a cDNA encoding prepro-Trp
226
-hK2 in insect cells. It is detectable using a conventional hPSA immunoassay as commercially available for use in clinical studies thus demonstrating cross-reactivity with known anti-hPSA antibodies, but is inactive (see Example 3).
The present invention additionally extends to fragments of Trp
226
-hK2 proteins as hereinbefore described which retain antigenicity and the Trp
226
amino acid residue. All such fragments and any protein having the Trp
226
-hK2 sequence are included within the term Trp
226
-hK2 protein used hereinafter.
In further aspects, the present invention provides an isolated DNA or recombinant DNA encoding a Trp
226
-hK2 protein of the invention. Preferably, such a DNA may include the natural coding sequence for Trp
226
-hK2, more preferably the complete natural coding sequence for prepro-Trp
226
-hK2(SEQ ID NO:8). Such a recombinant DNA may be in the form of a vector, e.g. a plasmid or viral vector. Desirably, such a vector may be an expression vector which when present in host cells is capable of directing production of a Trp
226
-hK2 protein of the invention in said cells or the culture medium. As hereinbefore indicated, particularly preferred, for example, is a baculovirus expression vector which when present in insect cells is capable of directing production of a Trp
226
-hK2 protein of the invention in the culture medium or in said cells.
In additional aspects, the invention provides host cells containing a vector of the invention as hereinbefore described and a method of preparing a Trp
226
-hK2 protein of the invention which comprises culturing host cells of the invention containing an expression vector under conditions whereby said protein is produced in the host cells or the culture medium and isolating said protein from said cells or medium. For example, where said cells are insect cells containing a baculov

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