Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
2001-01-22
2002-10-15
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C435S069100, C435S252300, C435S320100, C536S023100
Reexamination Certificate
active
06465622
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a novel protein and a DNA coding for the same. More particularly, it relates to a novel protein having inhibitory activity on the protease activity of hepatocyte growth factor activating factor (HGF activator) (hereinafter this protein is sometimes referred to also as “HAI-I”), a gene coding for the protein, an expression vector containing the gene, a transformant as transformed with the expression vector, and a method of producing HAI-I using the transformant.
BACKGROUND OF THE INVENTION
It is already reported that thrombin activates the precursor of HGF activator (JP-A-5-103670, JP-A-6-141859, JP-A-6-153946 and JP-A-6-153966 (the term “JP-A” as used herein means an “unexamined published Japanese patent application”); factor having activity to convert the single chain form of hepatocyte growth factor (HGF) to its double chain form) in the manner of positive activity control. However, human tissues-derived protease inhibitor capable of inhibiting, as a negative control factor, the physiological activity of HGF activator has not been known. Therefore, how HGF activator is controlled in human tissues remains unknown. Such a negative control factor might also influence indirectly on the activity of hepatocyte growth factor (HGF) on which HGF activator acts. Thus, for the analysis of the mechanism of action of HGF in vivo, as well, it has been demanded that such a human tissues-derived protease inhibitor be isolated and identified.
By using such a protease inhibitor and an antibody to the protease inhibitor, it would become possible to know the in vivo physiological activity of HGF activator, analyze the mechanism of action thereof or analyze the mechanism of control of HGF activation, from a standpoint different from those of the prior art.
Furthermore, for investigating the detailed in vivo function of HAI-I or the effect of HAI-I in hepatic disorder, for instance, HAI-I is required in large quantities. At present, however, there is only one method available for preparing HAI-I, which method comprises using, as a starting material, the culture supernatant obtained with a human cancer cell line such as MKN45 or A549 cells and purifying therefrom HAI-I occurring therein in trace amounts. This method is not always the best one from the labor, time and cost viewpoint. It encounters great difficulties in stably isolating the minor amount of HAI-I alone. Therefore, it has been desired that an expression system be constructed so that HAI-I can be obtained stably and in large quantities.
SUMMARY OF THE INVENTION
The present inventors have conducted screening of various cultured cell lines using, as an indicator, the inhibitory activity on the protease activity of hepatocyte growth factor activator and have found that a substance having the activity occurs in the culture supernatant of certain human cancer cell lines (MKN45 cells, A549 cells and like epithelial tumor cell lines). To reveal the nature of its inhibitory activity, they further attempted to purify the substance from the MKN45 cell culture supernatant using various column chromatography techniques. As a result, they have found a novel protein with a molecular weight of about 40,000 daltons as determined by SDS (sodium dodecyl sulfate)-polyacrylamide gel electrophoresis (PAGE) and they also have obtained an amino-terminal amino acid sequence of this protein by analyzing the protein on a protein sequencer. Further, they determined partial amino acid sequences by decomposing the protein using proteolytic enzymes, isolating the resultant peptides and subjecting each peptide to the same amino acid sequence analysis as mentioned above. Furthermore, they estimated DNA base sequences based on the partial amino acid sequences and conducted screening of a cDNA library using oligonucleotide probes prepared based on the sequences. As a result, they have succeeded in cloning a gene coding for the protein and have now completed the present invention.
Furthermore, as a result of various investigations to produce the protein stably and in large quantities using the recombinant DNA technique and, the present inventors have constructed a novel expression vector coding for the protein and have enabled expression of the protein. Thus, by constructing a plasmid for protein expression by inserting a DNA fragment coding for part or the whole of the amino acid sequence of the protein into a plasmid vector such as the expression vector pME18S for use in animal cells or an expression vector for use in yeasts,
Escherichia coli
and the like, at a site downstream from the promoter thereof and using the thus-obtained recombinant plasmid to transform host cells, they have now completed the present invention in another aspect.
The present invention thus relates to a protein having the following physico-chemical properties:
(1) a molecular weight of about 40,000 daltons as determined by SDS-polyacrylamide gel electrophoresis;
(2) inhibitory activity on the protease activity of hepatocyte growth factor activator; and
(3) one of the amino acid sequences depicted in the sequence listing under SEQ ID NO:1 through 7 or an amino acid sequence substantially equivalent thereto; proteins respectively having the amino acid sequences depicted in the sequence listing under SEQ ID NO:1 through 7 or amino acid sequences substantially equivalent thereto and having inhibitory activity on the protease activity of hepatocyte growth factor activator; a protein having the amino acid sequence depicted in the sequence listing under SEQ ID NO:18 or an amino acid sequence substantially equivalent thereto; and a protein having, as its amino acid sequence, that segment of the amino acid sequence depicted in the sequence listing under SEQ ID NO:18 which starts with the 36th amino acid (glycine) residue and ends with the 513th amino acid (leucine) residue, or an amino acid sequence substantially equivalent thereto; DNAs and genes coding for the proteins defined above; expression vectors respectively containing the DNA or genes; transformants obtained by transformation of host cells with the expression vectors; as well as a method of producing proteins having inhibitory activity on the protease activity of hepatocyte growth factor activator which comprises cultivating the transformants.
The base sequence shown in the sequence listing under SEQ ID NO:8 contains only one strand, with the other complementary base sequence being omitted. Starting with this gene and using the recombinant DNA technology, it is possible to cause expression of, for example, the protein having the amino acid sequence shown in the sequence listing under SEQ ID NO:18. On that occasion, the protein translated from mRNA coding for the protein contains a signal sequence. After extracellular excretion, however, the signal sequence has been cleaved off and the protein obtained has an amino acid sequence comprising the 36th amino acid (glycine) residue and the subsequent amino acid residues of the amino acid sequence shown in the sequence listing under SEQ ID NO:18. Signal sequences of other proteins may also be used as the signal sequence. For signal sequence-free mature protein expression in host cells, a gene having that portion of the base sequence shown in the sequence listing under SEQ ID NO:8 which comprises the 106th nucleotide (guanine) residue and the subsequent nucleotide residues may be used as the gene coding for the relevant protein and joined to the ATG codon of a vector. The present invention further includes, within the scope thereof, modifications of the proteins or DNAs mentioned above as derived therefrom by deletion, substitution and/or addition of one or more amino acid or nucleotide residues within limits not harmful to the inhibitory activity on the protease activity of HGF activator, namely those proteins or DNAs that respectively have “substantially equivalent amino acid sequences” or “substantially equivalent base sequences”.
REFERENCES:
patent: 5106833 (1992-04-01), Broze
patent: 6171790 (2001-06-01), Hillman et al.
patent: WO98/259
Kawaguchi Toshiya
Kitamura Naomi
Miyazawa Keiji
Shimomura Takeshi
Mitsubishi Chemical Corporation
Moore William W.
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