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
2000-08-16
2002-08-27
Yucel, Remy (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S006120, C435S489000
Reexamination Certificate
active
06440700
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to a type of protein synthesis utilizing combinatorial translation of a single gene sequence interspersed with programmed ribosomal frameshifting sequences. Specifically this type of protein synthesis can be utilized to make numerous, varied proteins from a single gene. More particularly, libraries can be made utilizing this combinatorial protein synthesis and the libraries can then be used to screen for specific protein activities.
Unusual translational events, such as frameshifting, are known to play an important role in some diseases. For example, expression of the enzyme, reverse transcriptase, utilized by many retroviruses including HIV, involves a naturally occurring translational frameshift. Frameshifted gene expression is also thought to play a role in some forms of colon cancer, Alzheimer's disease, and hemophilia A.
Rapid progress in sequencing genomes, as well as in genomics, proteomics and related disciplines has created a great number of targets for drug discovery and other potential treatments for various diseases. Specifically, phage libraries have proved invaluable in identifying peptide ligands of therapeutic value. For example, Yanofsky et al. (1996) described the isolation of a monomer peptide antagonistic to interleukin 1 (IL-1) with nanomolar affinity for the IL-1 receptor. Similarly, Wrighton et al. (1996) and Livnah et al. (1997) reported peptides that bind to the erythropoetin (EPO) receptor. Likewise Cwirla et al. (1997) described the identification of two families of peptides that bind to the human thrombopoietin (TPO) receptor. More particularly, there has been considerable progress in both construction and use of phage libraries over the last few years. For example, library diversity has been continually increasing, from 10
8
(Scott and Smith, 1991) to up to 10
11
more recently. The number and types of protein molecules displayed as well as the types of libraries have increased, while the selection methods have also continued to improve (reviewed by Lowman, 1997).
One such improved selection method, called biopanning, is the selection of peptides or proteins with specific desired binding properties. In contrast to traditional sequence based discovery methods, biopanning enables screening rates that are 10,000 times faster. This technique involves the immobilization of a target protein on a solid phase, incubation of a phage library with the solid phase to allow phage binding to the target, followed by washes of unbound phage and elution of the bound phage. The eluted phage is grown in
Escherichia coli
. Typically, several rounds of biopanning (2 to 6) are performed to identify clones of interest. These clones of interest are capable of producing or expressing the protein which may have significant utility in the production of commercial therapeutic products, industrial proteins, research reagents or consumer protein enriched products.
Though several successes in drug discovery have arisen through use of a phage library, there are different types of libraries suitable for this type of search. For example, other than the phage library, researchers often rely on combinatorial libraries. The term “combinatorial library” typically refers to libraries of biomolecules. Each element of the combinatorial library is composed of a string of several building blocks. The number of building blocks can be anywhere from 2 to 100 or even much more. Varying the building blocks at different positions within the string generates the diversity of the combinatorial library. Thus, if the string length is 5, and the number of building blocks is 10, the number of possible biomolecules in the combinatorial library is 10
5
. This is the potential diversity of the library. The observed diversity, which is the number of biomolecules actually constructed, can be less or be equal to the potential diversity.
An example of a combinatorial library is the library of random peptides on filamentous phage. The typical length of the peptide in such a library is 15 amino acid residues, and the number of building blocks (amino acids) is 20. Thus, the potential diversity of the library is 20
15
=3×10
19
. In practice, the number of
Escherichia coli
cells used to construct such a library acts as a limit on the actual diversity of the library. Thus, the observed diversity of the library is rarely above 10
11
.
There are many other examples of combinatorial libraries, including libraries of small organic molecules, phage libraries of antibodies, or libraries of genes obtained by DNA shuffling. Yet in all cases of gene libraries, be the library a phage library, a combinatorial library or another type of library, expression of a single gene in the library results in only one type (sequence) of protein being translated or produced.
Despite many successes in using phage display via libraries and biopanning, researchers still strive for even larger libraries and faster protocols in their search for a peptide or protein ideally suited for an envisioned task. Often, as part of this search, there is a need to screen a variety of proteins and ultimately select only those with the most desirable properties. As stated earlier, one problem with conventional libraries is that each gene encodes only one type of a protein, or corresponds to only one protein sequence. The following example illustrates this problem.
Escherchia coli
can make and store up to 10
7
protein molecules in a single cell. Researchers can engineer an
Escherichia coli
cell for expression of a single heterologous gene and that cell can then produce up to 70% (or even more) of that number of proteins (10
7
) as identical copies of the protein encoded by that gene. This is highly desirable when large quantities of a protein are needed. However, if the researcher is attempting to make a large number of varied proteins for the purpose of screening the proteins to find the ones with the most desired properties, then having huge amounts of a single type of protein is inefficient and wasteful. As a result, there is a critical need in the art for a way to synthesize larger libraries of more varied proteins.
The present invention overcomes this difficulty by inserting ribosomal frameshifting sequences into the gene sequence of interest to cause combinatorial translation of the gene sequence so that the single sequence yields a multitude of different peptides. By causing the reading frame switch, ribosomal frameshifting sequences affect the amino acid sequence of the protein made by the ribosome. Therefore, insertion of a ribosomal frameshifting sequence into a gene causes that gene to code for significantly more than the traditional one peptide or protein.
The initial and most widely recognized presumption in considering DNA sequences for expression potential is the requirement for an open reading frame. Surprisingly, in a previous drug discovery protocol by the inventor, a large number of sequences expressed in a random peptide library were found to contain non-open reading frame (non-ORF) and frameshifted sequences (Carcamo et al., 1998). The study was designed to isolate peptides capable of binding to growth hormone binding protein (GHBP). Originally, in biopanning experiments for the specific protein targets, namely GHBPS, the inventor expected an open reading frame (ORF) corresponding to the full length of the peptide and an epitope tag that followed. However, the inventor was surprised to observe this class of sequence in only about 50% of all sequences identified in biopanning as binding to the target (Ravera et al., 1998). Even more surprisingly, the inventor observed two other types of sequences that were, qualitatively, very different from the sequence originally expected. These two sequences contained a frameshift in the +1 or −1 direction but were, also unexpectedly, capable of expression.
One non-ORF phage clone, known as H10, which is capable of binding to the rat growth hormone binding protein (GHBP), was studied further. More specifica
Katcheves Konstantha
Yucel Remy
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