Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2000-01-14
2003-03-04
Mertz, Prema (Department: 1646)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S071100, C435S071200, C435S252300, C435S252400, C435S320100, C435S325000, C435S254200, C435S348000, C435S471000, C536S023500, C530S350000
Reexamination Certificate
active
06528284
ABSTRACT:
The present invention relates to the isolation, purification and characterization of proteins mediating switch recombination. The present invention further relates to the microbial production via recombinant DNA technology of recombination protein SRTA-70, a member of the proteins mediating switch recombination. The present invention further relates to the use of these proteins as therapeutically active agents in immune response modulation, specifically, in augmentation and suppression of the immune response.
Higher eukaryotes produce immunoglobulins (Ig) of diffent classes, which are defined by the constant region (C) of the heavy (H) chain. Upon stimulation by antigen expression of the early IgM class changes to that of another H chain class. This switch from one H chain class to another, named simply “class switching”, occurs via DNA recombination. Switch recombination imprecisely joins two so-called switch (S) regions, which lie upstream of the H chain genes and contain highly repetitive sequences (for reviews see Esser and Radbruch, Annu. Rev. Immunol. 8, 717-735 [1990] and Harriman et al., Annu. Rev. Immunol. 11, 361-384 [1993]). The recombination mechanism for most class switching events can be described by the loop-excision model (Jäck et al., Proc. Natl. Acad. Sci. USA 85, 1581-1585 [1988]). The biochemistry of the class switch recombination process, however, remains largely unknown.
In order to study the mechanism of class switch recombination an assay that measures DNA-transfer activity was devised which makes use of two S (S&mgr; and S&ggr;2b) regions, cloned into two different, largely non-homologous vectors (FIG.
1
). Using this assay three proteins in the S-Region Transfer Activity (SRTA) were identified: B23 (nucleophosmin), poly (ADP) ribose polymerase (PARP) and a novel 70-KDa protein SRTA-70.
Thus, in a first aspect of this invention, there are provided SRTA-70 proteins, specifically recombinantly produced SRTA-70 protein. The term “recombinantly produced SRTA-70 protein” refers to the protein of SEQ ID No. 1 or any protein or polypeptide having an amino acid sequence which is substantially homologous to the amino acid sequence SEQ ID No. 1 and further having the biological activities of the protein of SEQ ID No. 1.
As used hereinbefore the term “substantially homologous” means that a particular subject sequence, for example, a mutant sequence, varies from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which does not result in an adverse functional dissimilarity between the reference and subject sequences. For purposes of the present invention, sequences having greater than 95 percent homology, equivalent biological activity and equivalent expression characteristics are considered substantially homologous. For purposes of determining homology, truncation of the sequence should be disregarded. Sequences having lesser degrees of homology, comparable bioactivity, and equivalent expression characteristics, e.g., fragments of the amino acid sequence SEQ ID No: 1 are considered substantial equivalents.
As used herein the term recombinantly produced SRTA-70 protein includes proteins modified deliberately, as for example, by addition of specific sequences that preferably bind to an affinity carrier material. Examples of such sequences are sequences containing at least two adjacent histidine residues (see in this respect European Patent No. 282 042). Such sequences bind selectively to nitrilotriacetic acid nickel chelate resins (Hochuli and Döbeli, Biol. Chem. Hoope-Seyler 368, 748 [1987]; European Patent No. 253 303). SRTA-70 proteins which contain such a specific sequence can, therefore, be separated selectively from the remaining polypeptides. The specific sequence can be linked either to the C-terminus or the N-terminus of the SRTA-70 protein.
There are further provided isolated DNA sequences encoding SRTA-70 proteins or fragments thereof. Specifically, the DNA sequences of this invention are defined to include the nucleotide sequence SEQ ID No: 2 or a fragment thereof or any DNA sequence which is substantially homologous to the nucleotide sequence SEQ ID No: 2 or a fragment thereof.
As used hereinbefore the term “substantially homologous”, means that a particular subject sequence, for example, a mutant sequence, varies from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which does not result in an adverse functional dissimilarity between the reference and subject sequences. For purposes of the present invention, DNA sequences having greater than 95 percent homology, encoding equivalent biological properties, and showing equivalent expression characteristics are considered substantially homologous. For purposes of determining homology, truncation of the DNA sequence should be disregarded. Sequences having lesser degrees of homology, encoding comparable bioactivity, and showing equivalent expression characteristics, e.g., fragments of the nucleotide sequence SEQ ID No: 2 are considered substantial equivalents. Generally, homologous DNA sequences can be identified by cross-hybridization under standard hybridization conditions of moderate stringency.
There are also provided vectors and expression vectors containing the DNA sequences of the present invention, hosts containing such vectors for the production of SRTA-70 proteins, and processes for the production of such DNA sequences, recombinant vectors and host cells.
Methods for the expression, isolation and purification of the SRTA-70 proteins are also provided.
The following steps outline the methods for recombinantly expressing the SRTA-70 proteins.
1) Cloning of DNA Sequences Encoding SRTA-70 Proteins
DNA sequences encoding SRTA-70 proteins can be cloned using a variety of techniques. Using the methods described in this application cDNAs encoding SRTA-70 proteins or fragments thereof can be produced. These cDNAs can be isolated and amplified by PCR technique using oligodeoxynucleotide DNA primers by conventional techniques.
The cDNA (SEQ ID No: 2) encoding the amino acid sequence SEQ ID No:1 is obtained using the DNA primers described in the examples. By using conventional technique, this cDNA has been isolated from a mouse spleen cDNA library.
The cDNA may be obtained not only from cDNA libraries, but by other conventional techniques, e.g., by cloning genomic DNA, or fragments thereof, purified from the desired cells. These procedures are described by Sambrook et al., in “DNA Cloning: A Practical Approach”, Vol. I and II, D. N. Glover, ed., 1985, MRL Press, Ltd., Oxford, U. K.; Benton and Davis, Science 196, 180-182 [1977]; Grunstein and Hogness, Proc. Nat. Acad. Sci. 72, 3961-3965 [1975]; and Maniatis et al., in “Molecular Cloning-A Laboratory Manual”, Cold Spring Harbor Laboratory [1989].
To obtain the cDNA encoding the SRTA-70 proteins cDNA libraries are screened by conventional DNA hybridization techniques by the methods of Benton and Davis, supra, or Grunstein and Hogness, supra, using radioactive SRTA-70 gene fragments. Clones which hybridize to the radioactive gene fragments are analyzed, e.g., by restriction endonuclease cleavage or agarose gel electrophoresis. After isolating several positive clones the positive insert of one clone is subcloned, e.g., into phagemids, and sequenced by conventional techniques.
Clones derived from genomic DNA may contain regulatory and intron DNA regions in addition to coding regions; clones derived from cDNA will not contain intron sequences. In the molecular cloning of the gene from genomic DNA, DNA fragments are generated, some of which will encode the desired gene. The DNA may be cleaved at specific sites using various restriction enzymes. Alternatively, one may use DNAse in the presence of manganese to fragment the DNA, or the DNA can be physically sheared, as for example, by sonication. The linear DNA fragments can then be separated according to size by standard techniques, including but not limited to,
Jessberger Rolf
Wabl Matthias
Borden Paula A.
Bozicevic Field & Francis LLP
F. Hoffman La Roche, Ltd.
Mertz Prema
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