Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues
Reexamination Certificate
2001-11-21
2002-05-28
McKelvey, Terry (Department: 1636)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
C530S324000, C530S325000, C530S326000, C530S350000
Reexamination Certificate
active
06395872
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention relates to methods for identifying genes encoding novel proteins.
There is considerable medical interest in secreted and membrane-associated mammalian proteins. Many such proteins, for example, cytokines, are important for inducing the growth or differentiation of cells with which they interact or for triggering one or more specific cellular responses.
An important goal in the design and development of new therapies is the identification and characterization of secreted proteins and the genes which encode them. Traditionally, this goal has been pursued by identifying a particular response of a particular cell type and attempting to isolate and purify a secreted protein capable of eliciting the response. This approach is limited by a number of factors. First, certain secreted proteins will not be identified because the responses they evoke may not be recognizable or measurable. Second, because in vitro assays must be used to isolate and purify secreted proteins, somewhat artificial systems must be used. This raises the possibility that certain important secreted proteins will not be identified unless the features of the in vitro system (e.g., cell line, culture medium, or growth conditions) accurately reflect the in vivo milieu. Third, the complexity of the effects of secreted proteins on the cells with which they interact vastly complicates the task of isolating important secreted proteins. Any given cell can be simultaneously subject to the effects of two or more secreted proteins. Because any two secreted proteins will not have the same effect on a given cell and because the effect of a first secreted protein on a given cell can alter the effect of a second secreted protein on the same cell, it can be difficult to isolate the secreted protein or proteins responsible for a given physiological response. In addition, certain secreted and membrane-associated proteins may be expressed at levels that are too low to detect by biological assay or protein purification.
In another approach, genes encoding secreted proteins have been isolated using DNA probes or PCR oligonucleotides which recognize sequence motifs present in genes encoding known secreted protein. In addition, homology-directed searching of Expressed Sequence Tag (EST) sequences derived by high-throughput sequencing of specific cDNA libraries has been used to identify genes encoding secreted proteins. These approaches depend for their success on a high degree of similarity between the DNA sequences used as probes and the unknown genes or EST sequences.
More recently, methods have been developed that permit the identification of cDNAs encoding a signal sequence capable of directing the secretion of a particular protein from certain cell types. Both Honjo, U.S. Pat. No. 5,525,486, and Jacobs, U.S. Pat. No. 5,536,637, describe such methods. These methods are said to be capable of identifying secreted proteins.
The demonstrated clinical utility of several secreted proteins in the treatment of human disease, for example, erythropoietin, granulocyte-macrophage colony stimulating factor (GM-CSF), human growth hormone, and various interleukins, has generated considerable interest in the identification of novel secreted proteins. The method of the invention can be employed as a tool in the discovery of such novel proteins.
SUMMARY OF THE INVENTION
The invention features a method for isolating cDNAs and identifying encode secreted or membrane-associated (e.g. transmembrane) mammalian proteins. The method of the invention relies upon the observation that the majority of secreted and membrane-associated proteins possess at their amino termini a stretch of hydrophobic amino acid residues referred to as the “signal sequence.” The signal sequence directs secreted and membrane-associated proteins to a sub-cellular membrane compartment termed the endoplasmic reticulum, from which these proteins are dispatched for secretion or presentation on the cell surface.
The invention describes a method in which cDNAs that encode signal sequences for secreted or membrane-associated proteins are isolated by virtue of their abilities to direct the export of the reporter protein, alkaline phosphatase (AP), from mammalian cells. The present method has major advantages over other signal peptide trapping approaches. The present method is highly sensitive. This facilitates the isolation of signal peptide associated proteins that may be difficult to isolate with other techniques. Moreover, the present method is amenable to throughput screening techniques and automation. Combined with a novel method for cDNA library construction in which directional random primed cDNA libraries are prepared, the invention comprises a powerful and approach to the large scale isolation of novel secreted proteins.
The invention features a method for identifying a cDNA nucleic acid encoding a mammalian protein having a signal sequence, which method includes the following steps:
a) providing library of mammalian cDNA;
b) ligating the library of mammalian cDNA to DNA encoding alkaline phosphatase lacking both a signal sequence and a membrane anchor sequence to form ligated DNA;
c) transforming bacterial cells with the ligated DNA to create a bacterial cell clone library;
d) isolating DNA comprising the mammalian cDNA from at least one clone in the bacterial cell clone library;
e) separately transfecting DNA isolated from clones in step (d) into mammalian cells which do not express alkaline phosphatase to create a mammalian cell clone library wherein each clone in the mammalian cell clone library corresponds to a clone in the bacterial cell clone library;
f) identifying a clone in the mammalian cell clone library which express alkaline phosphatase;
g) identifying the clone in the bacterial cell clone library corresponding to the clone in the mammalian cell clone library identified in step (f); and
h) isolating and sequencing a portion of the mammalian cDNA present in the bacterial cell library clone identified in step (g) to identify a mammalian cDNA encoding a mammalian protein having a signal sequence.
A cDNA library is a collection of nucleic acid molecueles that are a cDNA copy of a sample of mRNA.
In another aspect, the invention features ptrAP3 expression vector.
In another aspect, the invention features a substantially pure preparation of ethb0018f2 protein. Preferably, the ethb0018f2 protein includes an amino acid sequence substantially identical to the amino acid sequence shown in
FIG. 5
(SEQ ID NO: 5); is derived from a mammal, for example, a human.
The invention also features purified DNA (for example, cDNA) which includes a sequence encoding a ethb0018f2 protein, preferably encoding a human ethb0018f2 protein (for example, the ethb0018f2 protein of
FIG. 5
; SEQ ID NO:5); a vector and a cell which includes a purified DNA of the invention; and a method of producing a recombinant ethb0018f2 protein involving providing a cell transformed with DNA encoding ethb0018f2 protein positioned for expression in the cell, culturing the transformed cell under conditions for expressing the DNA, and isolating the recombinant ethb0018f2 protein. The invention further features recombinant ethb0018f2 protein produced by such expression of a purified DNA of the invention.
By “ethb0018f2 protein” is meant a polypeptide which has a biological activity possessed by naturally-occuring ethb0018f2 protein. Preferably, such a polypeptide has an amino acid sequence which is at least 85%, preferably 90%, and most preferably 95% or even 99% identical to the amino acid sequence of the ethb0018f2 protein of
FIG. 5
(SEQ ID NO: 5).
By “substantially identical” is meant a polypeptide or nucleic acid having a sequence that is at least 85%, preferably 90%, and more preferably 95% or more identical to the sequence of the reference amino acid or nucleic acid sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most
Gearing David Paul
Levinson Douglas Adam
McCarthy Sean Anthony
McKelvey Terry
Millennium Pharmaceuticals Inc.
Millennium Pharmaceuticals Inc.
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