Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Nucleoproteins – e.g. – chromatin – chromosomal proteins,...
Reexamination Certificate
1998-11-23
2001-02-27
Campbell, Eggerton A. (Department: 1646)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Nucleoproteins, e.g., chromatin, chromosomal proteins,...
C530S344000, C530S300000, C536S023100, C536S025400, C435S068100
Reexamination Certificate
active
06194550
ABSTRACT:
FIELD OF THE INVENTION
We describe herein novel high-affinity polypeptide ligands that specifically bind a desired target molecule. A method is presented for selecting a polypeptide ligand that specifically binds any desired target molecule. The method is termed SPERT, an acronym for Systematic Polypeptide Evolution by Reverse Translation. The method of the invention (SPERT) is useful to isolate a polypeptide ligand for a desired target molecule. The polypeptide products of the invention are useful for any purpose to which a binding reaction may be put, for example in assay methods, diagnostic procedures, cell sorting, as inhibitors of target molecule function, as probes, as sequestering agents and the like. In addition, polypeptide products of the invention can have catalytic activity. Target molecules include natural and synthetic polymers, including proteins, polysaccharides, glycoproteins, hormones, receptors and cell surfaces, nucleic acids, and small molecules such as drugs, metabolites, cofactors, transition state analogs and toxins.
BACKGROUND OF THE INVENTION
As translation of mRNA proceeds, stable complexes are formed. These complexes are made of ribosomes bound to mRNA with tRNA and nascent polypeptide encoded by the messenger RNA. Termed “ribosome complexes” herein, such complexes can be isolated by various known processes (Connolly and Gilmore (1986) J. Cell Biol. 103:2253; Perara et al. (1986) Science 232:348). Antigen-encoding mRNAs have been purified by taking advantage of the immunoreactivity of nascent polypeptides associated with ribosome complexes (Sambrook, J., Fritsch, E. F., Maniatis, T.
Molecular Cloning: A Laboratory Manual
(Cold Spring Harbor, N.Y.) (1989) ibid. sections 8.9-8.10). Such immunoreactive ribosome complexes can be immunoprecipitated from solution or separated by protein A column chromatography from non-reactive ribosome complexes (Schutz et al. (1977) Nuc. Acids Res. 4, 71; Shapiro and Young (1981) J. Biol. Chem. 256, 1495). Cyclical selection and amplification of RNAs with partitionable properties is now also possible. Historically, mRNA selection is closely tied to immunopurification of ribosome complexes, however, the partitioning of ribosome complexes according to the present invention is not restricted to immunoreactivity of the nascent polypeptides.
SUMMARY OF THE INVENTION
In its broadest aspect, the method of systematic polypeptide evolution by reverse translation (SPERT) includes a candidate mixture of polypeptides having a randomized amino acid sequence. Each member of the mixture is linked to an individualized mRNA which encodes the amino acid sequence of that polypeptide. The candidate polypeptides are partitioned according to their property of binding to a given desired target molecule. The partitioning is carried out in such a way, herein described, that each mRNA encoding a polypeptide is partitioned exactly together with that polypeptide. In this way each polypeptide is partitioned together with the means for further amplifying it by an in vitro process. Ultimately, both the desired optimal polypeptide ligand of the desired target and the mRNA encoding the polypeptide are simultaneously selected, allowing further synthesis of the selected polypeptide as desired, and further amplification of the coding sequence. It is therefore not necessary to analyze the amino acid sequence of the selected polypeptide (using protein chemistry) in order to produce it in desired quantities.
Viewed another way, the invention is the selective evolution of a nucleic acid that encodes a polypeptide ligand of a desired target. The present method is therefore a selection based upon coding properties available in a candidate nucleic acid mixture. In previously filed applications, U.S. Ser. No. 07/536,428, filed Jun. 11, 1990 and U.S. Ser. No. 07/714,131 filed Jul. 10, 1991, both of which are incorporated herein by reference, the inventors herein have taught a method for selective evolution of nucleic acids based upon binding properties of the nucleic acids themselves. The insight that cyclical selection and amplification can be a powerful tool for developing novel compounds when coupled with a partitioning system is herein adapted to evolving specific coding nucleic acids, based on the partitioning properties of polypeptide ligands binding to target molecules.
More specifically, the invention includes a method for making a polypeptide ligand of a desired target molecule which includes the following steps: First, synthesizing a mixture of translatable mRNA's, having certain sequence segment in common such as a ribosome binding site and a translation initiation codon and having a segment encoding a polypeptide at least part of which coding region is a randomized sequence. Second, employing the mRNA mixture in an in vitro translation system. Synthesis of nascent polypeptides ensues, each encoded by its own mRNA. At any time during translation, stable ribosome complexes can be isolated. It is preferred to isolate complexes in which translation has been stopped, or “stalled” by any of several known circumstances. Each isolated ribosome complex includes at least one ribosome, one nascent peptide and the coding mRNA which is now said to be translated mRNA. Although its chemical structure is unaltered, translated mRNA is bound to the ribosome complex in a different manner than it was bound prior to translation, as is known in the art. Third, the ribosome complexes are partitioned with respect to the binding of each nascent polypeptide to a desired target molecule. Some polypeptides bind weakly, some tightly, some not at all, with the target. The partitioning, however conducted, generally separates the mixture of ribosome complexes into ribosome complex-target pairs and unbound complexes. The set of ribosome complex-target pairs is thereby enriched for those polypeptides (and, necessarily their coding mRNA's) that can bind to the target. Fourth, the encoding mRNA's are separated from the complexes and amplified by conventional means for amplifying nucleic acids, such as reverse transcription and polymerase chain reaction (PCR). This amplification sets the stage for a subsequent round of transcription, polypeptide synthesis and partitioning to further enrich for target-binding polypeptide ligands. These cycles can be reiterated as many times as desired, until a desired binding affinity is achieved, or no further improvement in binding affinity is observed. The coding mRNA for any polypeptide selected in the foregoing manner can be cloned and sequenced, if desired. An individual polypeptide ligand can then be prepared in vivo from cloned coding mRNA, or by chemical or enzymatic methods in vitro.
In an alternate embodiment of the present invention, means for linking the nascent polypeptide to the translated mRNA are included in the design of the system. According to this method, a direct connection—either via covalent bonding or very tight affinity interactions—between the polypeptide and the mRNA allows for the removal of the ribosomal linkage between these two elements leaving mRNA•polypeptide copolymers. By removing the relatively large ribosome from the mRNA polypeptide copolymer, the ability to partition polypeptides based on the affinity of the randomized polypeptides to a given target may be greatly increased. In addition, the ribosome is then freed to translate additional mRNA species. The fewer ribosomes that can be utilized, the more randomized polypeptides can be generated in the process. In a specific example of this embodiment, a biotin molecule is covalently bound to the 5′ end of the mRNA sequence utilized, and the nucleic acid template includes a fixes sequence in the translated region that encodes a polypeptide that may be covalently bound to biotin.
The present invention provides a class of products which are polypeptides, each having a unique sequence, each of which has the property of binding specifically to a desired target compound or molecule. Each compound of the invention is a specific ligand of a given ta
Gold Larry
Pribnow David
Smith Jonathan Drew
Tuerk Craig
Bugaisky Gabriele
Campbell Eggerton A.
Swanson & Bratschun LLC
LandOfFree
Systematic polypeptide evolution by reverse translation does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Systematic polypeptide evolution by reverse translation, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Systematic polypeptide evolution by reverse translation will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2594082