Expression cloning processes for the discovery,...

Details Expression cloning processes for the discovery,... Expression cloning processes for the discovery,...

1998-11-17

2003-02-25

Fredman, Jeffrey (Department: 1653)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091200, C435S320100, C435S252100, C536S022100, C536S023100, C536S024300, C536S024310, C536S024320, C536S024330

Reexamination Certificate

active

06524792

ABSTRACT:

I. FIELD OF THE INVENTION
The present in invention relates, generally, to the field of expressed gene technology and expression cloning. More specifically, the present invention relates to the identification, characization, and isolation of transcribed nucleic acid sequences encoding polypeptides having a predetermined property, e.g., cellular localization, structure, enzymatic function, or affinity to other molecules, and the production of the corresponding polypeptides.
II. BACKGROUND OF THE INVENTION
General Background. Proteins are the most prominent biomolecules in living organisms; in addition to their role as structural components and catalysts, they play a crucial role in regulatory processes. Both regulation of cell proliferation and metabolic functions are largely controlled and effected by the cooperation of numerous cellular and extracellular proteins. Lehninger, A. L., 1975
, “Biochemistry
”, Worth Publishers Inc., New York, N.Y. For example, signal transduction pathways of many kinds that affect critical physiological responses operate through proteins by way of their intermolecular interactions. Metzler, D. E., 1977
, “Biochemistry
”, Academic Press Inc., London. Furthermore, the transcription of genes and the regulation of such transcription is dependent upon and controlled by the interdependence of numerous protein factors. Wainwright, S. D., “
Control Mechanisms and Protein Synthesis
”, Columbia University Press, New York and London.
Proper functioning of a multicellular organism does not only depend on the interaction of biomolecules within the cell, but individual cells must also communicate appropriately. Such intercellular communication, and interaction of cells with the environment is often realized by the actions of receptors on the extracellular surface and associated intracellular signal transduction mechanisms. Poste, G., Nicholson, G. L., 1976
, “The Cell Surface
”, Elseviere, Amsterdam. The information is communicated through the cell environment to regulate gene expression or protein activities in the cell. Secreted proteins in the extracellular environment thereby exert potent regulatory effects on certain cellular functions.
In view of the above outlined, very simplified paradigm of cell function, particular properties of a protein, including cellular localization, structure, affinity to a binding partner, or enzymatic activity under physiological conditions appear to be highly indicative of its type of function. With respect to a particular cellular localization, secreted proteins, for example, are likely to function as intercellular communicators of signals, while membrane associated receptors having an extracellular and intracellular domain most likely transmit an extracellular signal into the cell. Cytoplasmic proteins may function as intracellular signal transmitters and coordinators. Jeter, J. R., Cameron, I. L., Padilla, I. L., Padilla, G. M., Zimmerman, A. M., 1978
, “Cell Cycle Regulation
”; London. Nuclear proteins are likely to be involved in certain aspects of gene regulation. Zawel et al., 1995
, Annu. Rev. Biochem
. 64:533-561. Mature proteins found in the Golgi or the ER may have regulatory roles in the post-translational processing of protein precursors, e.g., cleavage or addition of carbohydrates. Hirschberg, 1987
, C. Annu. Rev. Biochem
. 56:63-87.
Membrane-Associated Proteins. For many years, the paradigm of cell function has motivated numerous drug discovery programs to focus on identifying membrane-associated proteins, in particular new receptors, and their respective functions. Porter, R. and O'Connor, M., 1970
, “Molecular Properties of Drug Receptors, Ciba Foundation Symposium
”, J&A Churchill, London. Many examples in fact compel the conclusion that improper function of membrane receptors is a significant source of the development of serious metabolic and proliferative diseases such as cancer. For example, a certain form of
Diabetes mellitus
, i.e., the non-insulin-dependent diabetes (NIDDM) may be caused by mutations in the insulin receptor. Ullrich et al., 1985
, Nature
313:756-761; Taira et al., 1989
, Science
245:63-66. Furthermore, 30% of all mammary carcinomas are associated with amplification of the receptor tyrosine kinase HER2. Bargman et al., 1986
, Cell
45:649-657; Slamon et al., 1989
, Science
244:707-712. In addition to traditional drug discovery programs targeting receptors, an ambitiously pursued objective has become to identify membrane-associated receptors as possible gene therapy targets using comparative genomics, which allows. determination of changes in gene expression under, e.g., pathological conditions. Wels, et al., 1995
, Gene
159(1):73-80.
Secreted Proteins. While receptors have mostly been considered as important potential therapeutic targets, secreted proteins are of particular interest as potential therapeutic agents. They often have a signalling or hormone function, and hence have a high and specific biological activity. Schoen, F. J., 1994
, “Robbins Pathologic Basis of Disease
”,. W.B. Saunders Company, Philadelphia. For example, secreted proteins control physiological reactions such as differentiation and proliferation, blood clotting and thrombolysis, somatic growth and cell death, and immune response. Schoen, F. J., 1994
, “Robbins Pathologic Basis of Disease
”, W. B. Saunders Company, Philadelphia.
Significant resources and research efforts have been expended for the discovery and investigation of new secreted proteins controlling biological functions. Many of such secreted proteins, including cytokines and peptide hormones, are manufactured and used as therapeutic agents. Zavyalov et al., 1997
, APMIS
105(3):161-186. However, of the several thousand expected secreted proteins, only a few are currently used as therapeutic compounds. It can be expected that many of the so far undiscovered secreted proteins of the human organism are effective in correcting physiological disorders and are thus promising candidates for new drugs.
In the past, novel cytokines and hormone proteins were identified by assaying a certain cell type for its response to protein fractions or purified proteins. Lauffenburger et al., 1996
, Biotechnology and Bioengineering
52(1):61-80. Other investigators have used sequence similarities on DNA level to clone novel interferons and interleukins. Nabori et al., 1992
, Analyt. Biochem
. 205(1):42-46. In again another approach, differential display techniques were used to compare the expression patterns of stimulated versus unstimulated cells. Nagata et al., 1980
, Nature
287:401-408. All these methods may yield identification and isolation of certain secreted polypeptides.
Recently, a screening method for the identification of cDNA encoding novel secreted mammalian proteins in yeast using the invertase gene as a selection marker has been described. See, U.S. Pat. No. 5,536,637 (the “'637 Patent”). The disclosed technology relies on the concept that leader sequences of mammalian cDNAs are effective in exporting the invertase protein depleted of its leader sequence. This approach yields partial cDNAs which in turn can be used to screen a full-length cDNA library. The novel protein of interest can then be manufactured by standard, but laborious, techniques, including subcloning, transforming a recombinant host, expression, development and implementation of a purification process. Furthermore, since the assays described in the '637 Patent are performed in yeast, the glycosylation pattern of the isolated products will differ significantly from the natural product produced in mammalian cells. This difference is a major impediment in view of the fact that an extremely important feature of secreted proteins (as it is true for the extracellular domain of receptors) is their glycosylation pattern and carbohydrate composition. Rademacher et al., 1988
, Annu. Rev. Biochem
. 57:785-838.
Nuclear Proteins. In the nucleus, both replication of DNA and transcription of genes is actually implemented. Many nuclear proteins are directly involved in these processes a

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