Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
2001-02-28
2003-11-25
Horlick, Kenneth R. (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S006120, C435S007100, C435S069700, C435S071100, C435S071200, C435S320100, C530S412000
Reexamination Certificate
active
06653068
ABSTRACT:
BACKGROUND
The present invention relates to the generation of specific binding partners binding to (poly)peptides encoded by genomic DNA fragments or ESTs. The (poly)peptides are expressed as part of fusion proteins which are forming inclusion bodies on expression in host cells. The inclusion bodies are used to generate binding partners which bind specifically to said (poly)peptides. The specific binding partners, in particular immunoglobulins or fragments thereof, are useful for analysis and functional characterisation of proteins encoded by nucleic acid sequences comprising the corresponding genomic DNA fragments or ESTs.
The invention further relates to nucleic acid molecules, vectors and host cells to be used in the methods of the present invention.
The invention further relates to the use of fusion proteins comprising the first N-terminal domain of the geneIII protein of filamentous phage as fusion partner for the expression of a (poly)peptide/protein fused to said fusion partner, and to methods for the expression of (poly)peptide/proteins.
Since several years, massive efforts are being undertaken to sequence the human genome, and to identify and characterise structure and function of the proteins encoded therein. Finally, this will lead to novel targets for prevention, diagnosis and therapy of diseases (Collins & Galas, 1993; Adams et al., 1995). Currently, two different approaches are being pursued for identifying and characterising the genes distributed along the human genome. In one approach, large fragments of genomic DNA are isolated, cloned, and sequenced. Potential open reading frames in these genomic sequences are identified using bioinformatics software. However, this approach entails sequencing large stretches of human DNA which do not encode proteins in order to find the protein encoding sequences scattered throughout the genome. In addition to requiring extensive sequencing, the bioinformatics software may mischaracterize the genomic sequences obtained. Thus, the software may produce false positives in which non-coding DNA is mischaracterised as coding DNA or false negatives in which coding DNA is mislabelled as non-coding DNA.
In an alternative approach, complementary DNAs (cDNAs) are synthesised from isolated messenger RNAs (mRNAs) which encode human proteins. Using this approach, sequencing is only performed on DNA which is derived from protein coding sequences of the genome. Often, only short stretches of the cDNAs are sequenced to obtain sequences called expressed sequence tags (ESTs) (WO93/00353). In principle, the ESTs may then be used to isolate or purify extended cDNAs which include sequences adjacent to the EST sequences. These extended cDNAs may contain portions or the full coding sequence of the gene from which the EST was derived.
By analysing the genomic DNA or fragments thereof, ESTs, extended cDNAs, and/or the (poly)peptides/proteins encoded thereby, in certain cases, where homology, structural motifs etc. can be identified, it may be possible to assign a function to the (poly)peptide/protein which can be tested or verified in vitro or in vivo. However, the various EST-sequencing efforts have led to enormous numbers of ESTs, and to the problem how best to structure that information and how to identify interesting sequences. Hence, there is still a need for developing and using research tools directed against the (poly)peptide/protein of interest to analyse their localisation on cell and tissue types, their up- or down-regulation in certain disease or development stages or their role in activating or blocking certain interactions or signalling routes. One approach is to use antibodies or fragments thereof as such research tools. In WO93/00353 it was suggested to express the ESTs and to generate antibodies by immunising animals with the corresponding (poly)peptides. In a similar approach, DNA constructs comprising EST sequences have been injected into animals to generate an immune response against the (poly)peptide expressed in vivo (Sykes & Johnston, 1999). However, these approaches are not amenable to a high-throughput generation of antibodies. Alternatively, antibodies are generated against sets of overlapping peptides covering the EST sequence (Persic et al., 1999). In combination with screening recombinant antibody libraries, this approach can in principle be developed to generate antibody fragments as research tools with high throughput. However, it is often difficult to obtain anti-peptide antibodies with sufficiently high affinities.
Thus the technical problem underlying the present invention is to provide a generally applicable method for the generation of specific binding partners binding to (poly)peptides encoded by genomic DNA fragments or by ESTs, especially of antibodies or antibody fragments, for analysis aid functional characterisation of proteins corresponding to genomic DNA or ESTs. The solution to the above technical problem is achieved by providing the embodiments characterised in the claims. The technical approach of the present invention, to provide (poly)peptides encoded by genomic DNA fragments or ESTs for the generation of specific binding partners, such as antibodies or antibody-derived products, by expressing the (poly)peptides as fusions with (poly)peptide/protein fusion partners which lead to the formation of inclusion bodies on expression in host cells, such as
E. coli,
and to generate specific binding partners against the inclusion bodies and fusion proteins, obtainable therefrom, is neither provided nor suggested by the prior art.
A further problem related to the present invention was to devise a method for the expression of (poly)peptide/proteins which are not easily expressed in free form, e.g. since they are toxic to the host cell. The solution to that technical problem is also achieved by providing the embodiments characterised in the claims. The technical approach of the present invention, express the (poly)peptide/proteins as fusion proteins comprising the first N-terminal domain of the geneIII protein of filamentous phage leading to the formation of inclusion bodies, is neither provided nor suggested by the prior art.
SUMMARY
Thus, the present invention relates to a method for generating a specific binding partner to a (poly)peptide which is encoded by a nucleic acid sequence comprised in a genomic DNA fragment or an expressed sequence tag (EST) comprising:
a) expressing a nucleic acid molecule encoding a fusion protein in a host cell under conditions that allow the formation of inclusion bodies comprising said fusion protein, wherein said fusion protein comprises
aa) a (poly)peptide/protein fusion partner which is deposited in inclusion bodies when expressed in said host cell under said conditions and
ab) said (poly)peptide;
b) isolating said inclusion bodies; and
c) generating a specific binding partner that binds specifically to said (poly)peptide.
DETAILED DESCRIPTION
In the context of the present invention, a “specific binding partner” is a molecule which is able to specifically bind to a (poly)peptide of interest. Such a specific binding partner may be a peptide, a constrained peptide, an immunoglobulin or fragment thereof, or a cognate binding partner of a naturally occurring protein, e.g. a ligand to a receptor which comprises the (poly)peptide of interest. Such cognate ligand may be obtainable by screening a cDNA expression library for binding to the fusion protein of the present invention. The specific binding partner may also be a non-proteinaceous specific binding partner such as a small molecule, e.g. obtainable by screening of a combinatorial library of small molecules. A specific binding partner may further be modified to enable the detection of an interaction of a specific binding partner and the corresponding (poly)peptide. Such modification may be a detection and/or purification tag (Hochuli et al., 1988; Lindner et al., 1992; Hopp et al., 1988; Prickett et al., 1989; Knappik & Plückthun, 1994), or an enzyme (Blake et al., 1984) or a reporter molecule fused or coupled to the specific bi
Frisch Christian
Höss Adolf
Kretzschmar Titus
Von Rüden Thomas
Heller Ehrman White and McAuliffe
Morphosys AG
Strzelecka Teresa
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