Mutant proteins of human DNA topoisomerase I

Details Mutant proteins of human DNA topoisomerase I Mutant proteins of human DNA topoisomerase I

1996-11-26

1998-12-15

LeGuyader, John L.

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving antigen-antibody binding, specific binding protein...

435183, 435233, G01N 3353, C12N 990

Patent

active

058495037

DESCRIPTION:

BRIEF SUMMARY
BACKGROUND OF THE INVENTION

1. Field of the Invention
This invention relates to novel mutant proteins of human DNA topoisomerase I. These mutant proteins are preferably produced by genetic engineering and useful for a diagnosis of autoimmune diseases and for analysis of topological interconversion of genes.
2. Description of the Prior Art
The autoantibodies against various nuclear antigens are detected in the sera of patients with autoimmune diseases, and it is possible to diagnose autoimmune diseases by detecting these autoantibodies.
It is known that, among these autoantibodies, the autoantibodies against a nuclear antigen, Scl-70 having a molecular weight of 70 kD, are detected in sera of many patients with an autoimmune disease named diffuse scleroderma. In recent years it has been demonstrated that Scl-70 is a 70 kD-molecular-weight protein of the C-terminal fragment of DNA topoisomerase I (J. H. Sero et al., 1986, Science, Vol. 231, pp. 737-740). DNA topoisomerases catalyze the breaking and rejoining of DNA strands in a way that allows the strands to pass through one another, thus altering the topology of DNA. There exist two kinds of DNA topoisomerases, Type I and Type II. Type I cleaves a single chain DNA while Type II cleaves a double-chain DNA.
The topological interconversion is brought about by these DNA topoisomerases I and II, which are necessary for the transcription from DNA to RNA and for replication of DNA. These DNA topoisomerases I and II are also essential proteins for cell proliferation and functional maintenance such as protein synthesis. In particular, Scl-70, namely, 70 kD-molecular-weight protein of the C-terminal fragment of human DNA topoisomerase I, is useful for a diagnosis of autoimmune diseases such as diffuse scleroderma. However, it is not commercially feasible to extract and purify human DNA topoisomerase I from natural materials due to scarce content of the protein in organisms. It is preferably by genetic engineering that the enzyme is practically produced.
The cDNA of human DNA topoisomerase I was cloned by P. D'Arpa in 1988 (Proc. Natl. Acad. Sci., USA, Vol. 85, pp. 2543-2547). However, production of a sufficient amount of the targeted recombinant protein was unsuccessful. A trial was made to express human DNA topoisomerase I by integrating it into a conventional expression vector in E. coli according to conventional genetic engineering methods. A large amount of the enzyme production in E. coli caused inhibition of the transcription of the E. coli DNA itself and caused inhibition of DNA replication. Thus, growth of the E. coli was inhibited, and death of the E. coli was observed.


DETAILED DESCRIPTION OF THE INVENTION

The present invention provides for mutant proteins of human DNA topoisomerase I which are preferably produced by genetic engineering. It is estimated that the active site of human DNA topoisomerase I is tyrosine (Tyr) at amino acid 723 (Proc. Natl. Acad. Sci., 1989, Vol. 86, pp. 3559-356). Moreover, a mutant protein lacking 95 amino acids at terminal fragments and 723-phenylalanine (Phe) whose active also inhibited the growth of E. coli, and failed to produce a sufficient amount of the mutant protein. Then, upon producing a mutant protein of human DNA topoisomerase I (FF mutant hereinafter, shown in SEQ ID NO.:1 of the Sequence Listing) by substituting Tyr at the 592nd amino acid with Phe, FF mutant reacted sufficiently with anti-Scl-70, autoantibodies in the sera of autoimmune disease patients. In addition the production of FF mutuant in E. coli was found very high, which led to the completion of the invention.
Further, it was found that the mutant protein of human DNA topoisomerase I which consists of Ala at the 96th amino acid to Phe at the 765th amino acid, Tyr at the 723rd (i.e., the amino acid of the wild type protein) and substituting Tyr at the 592nd amino acid with Phe (FY mutant hereinafter) was recovered in a large quantity at 4 to 5 hours after induction of transformed E. coli by reagents. FY mutant as well as FF mutant has antigenicity an

REFERENCES:
patent: 5070192 (1991-12-01), Earnshaw et al.
patent: 5541291 (1996-07-01), Keene et al.
J.H. Shero et al., Science, 231:737-740 (1986).
P. D'Arpa et al., Proc. Natl. Acad. Sci., USA 85(8):2543-2547. (1988).
R.M. Lynn, et al., Proc. Natl. Acad. Sci., USA 86 (10):3559-3563. (1989).
Benton, W.D., and Davis, R.W., Science, 196(4286):180-182. (1977).
Sanger, Proc. Natl. Acad. Sci., USA, 74:(12):5463 (1977).

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