Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-03-19
2001-05-08
Yucel, Remy (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S254110, C435S254210
Reexamination Certificate
active
06228590
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a novel method of identifying nucleic acid sequences encoding secreted and membrane-bound proteins based upon the presence of signal sequences.
BACKGROUND
Extracellular proteins are essential in the formation, differentiation and maintenance of multicellular organisms. The determination by individual cells of whether to live, proliferate, migrate, differentiate, interact with other cells or secrete are governed by information received from the cells neighbors and the immediate environment. This information is often transmitted by secreted polypeptides (e.g., mitogenic factors, survival factors, cytotoxic factors, differentiation factors, neuropeptides, and hormones) which are in turn received and interpreted by diverse cell receptors. These secreted polypeptides or signaling molecules normally pass through the cellular secretory pathway to reach their site of action in the extracellular environment.
The targeting of both secreted and transmembrane proteins to the secretory pathway is accomplished via the attachment of a short, amino-terminal sequence, known as the signal peptide or signal sequence (von Heijne (1985)
J. Mol. Biol.
184:99-105; Kaiser & Botstein, (1986),
Mol. Cell. Biol.
6:2382-2391). The signal peptide itself contains several elements necessary for optimal function, the most important of which is a hydrophobic component. Immediately preceding the hydrophobic sequence is often a basic amino acid or acids, whereas at the carboxyl-terminal end of the signal peptide are a pair of small, uncharged amino acids separated by a single intervening amino acid which defines the signal peptidase cleavage site. While the hydrophobic component, basic amino acid and peptidase cleavage site can usually be identified in the signal peptide of known secreted proteins, the high level of degeneracy within any one of these elements makes difficult the identification or isolation of secreted or transmembrane proteins solely by searching for signal peptides in DNA data bases (e.g. GeneBank, GenPept), or based upon hybridization with DNA probes designed to recognize cDNA's encoding signal peptides.
Secreted and membrane-bound cellular proteins have wide applicability in various industrial applications, including pharmaceuticals, diagnostics, biosensors and bioreactors. For example, most protein drugs commercially available at present, such as thrombolytic agents, interferons, interleukins, erythropoietins, colony stimulating factors, and various other cytokines are secretory proteins. Their receptors, which are membrane proteins, also have potential as therapeutic or diagnostic agents. Significant resources are presently being expended by both industry and academia to identify new native secreted proteins.
According to a screening method recently reported by Klein et al. (1996),
Proc. Natl. Acad. Sci.
93:7108-7113 and Jacobs (U.S. Pat. No. 5,563,637, issued Jul. 16, 1996), cDNAs encoding novel secreted and membrane-bound mammalian proteins are identified by detecting their secretory leader sequences using the yeast invertase gene as a reporter system. The enzyme invertase catalyzes the breakdown of sucrose to glucose and fructose as well as the breakdown of raffinose to sucrose and melibiose. The secreted form of invertase is required for the utilization of sucrose by yeast (
Saccharomyces cerevisiae
) so that yeast cells that are unable to produce secreted invertase grow poorly on media containing sucrose as the sole carbon and energy source. Both Klein, supra, and Jacobs, supra, take advantage of the known ability of mammalian signal sequences to functionally replace the native signal sequence of yeast invertase. DNA from a mammalian cDNA library is ligated to the 5′-end of a DNA encoding a nonsecreted yeast invertase (e.g., lacking the natural invertase signal peptide), the ligated DNA is isolated and transformed into yeast cells that do not contain an invertase gene. Recombinants containing the nonsecreted yeast invertase gene ligated to a mammalian signal sequence are identified based upon their ability to grow on a medium containing only sucrose or only raffinose as the carbon source. The mammalian signal sequences identified are then used to screen a second, full-length mammalian cDNA library to isolate the full-length clones encoding the corresponding secreted proteins.
Given the great efforts presently being expended to discover novel secreted and transmembrane proteins as potential therapeutic agents, there is a great need for an improved system which can simply and efficiently identify the coding sequences of such proteins in mammalian recombinant DNA libraries. While effective, the invertase yeast selection process described above has several disadvantages. First, it requires the use of special yeast cells in which the SUC2 gene encoding the invertase protein has been deleted or the coding sequence of the native invertase signal has been mutated so that the invertase is not secreted. Second, even invertase-deficient yeast may grow on sucrose or raffinose, albeit at a low rate, therefore, the invertase selection may need to be repeated several times to improve the selection for transformants containing the signal-less yeast invertase gene ligated to a mammalian secretory leader sequence. See, Jacobs, supra. Third, the invertase selection process is further inadequate because a certain threshold level of enzyme activity needs to be secreted to allow growth. Although 0.6-1% of wild-type invertase secretion is sufficient for growth, certain mammalian signal sequences are not capable of functioning to yield even this relatively moderate level of secretion (Kaiser et al. (1987),
Science
235:312-317). As a result, there still exists the need for an improved and simplified technique for selecting genes encoding signal sequence-containing (secreted or membrane-bound) polypeptides.
SUMMARY OF THE INVENTION
The present invention concerns a novel and improved method for identifying genes encoding secreted and membrane-bound proteins using a host phenotypic background that is deficient in post-translational translocation of siren sequences, sequences that, as discovered herein, are functionally, but not structurally similar, to authentic signal peptides. Siren sequences in their native context are not authentic signal sequences, but nonetheless direct secretion of a C-terminally attached reporter protein, resulting in false positives that lead the gene searcher astray during a search for DNA encoding novel secreted proteins. These misleading sequences, reminiscent of the mythical creatures (“sirens”) that led mariners astray, have been termed “siren sequences.” As discovered herein, the siren sequences allow secretion of the attached reporter protein via a post-translational translocation pathway, not a co-translational secretion pathway. It has been further discovered herein that when screening or selecting for heterologous-signal-peptide-directed reporter protein secretion using yeast deficient for translocating siren-sequence/reporter protein fusion constructs, a significant number of false positives are thereby eliminated. The present methods thus provide a greater relative number of correctly identified signal sequences, minimizing the cost and time required to identify and characterize non-novel or false sequences.
Yeast cells deficient in the post-translational translocation pathway, but that still retain co-translational pathway secretion, are a preferred host for transformation with DNA containing a coding sequence of a mammalian peptide ligated to DNA encoding the reporter protein lacking a functional native signal peptide. The transformed cells are selected or preferably screened for their ability to secrete the reporter protein. The DNA encoding the signal sequence/reporter protein, in the yeast cells that were identified as positive for reporter secretion, is then analyzed for novelty, by comparison to sequences in gene or protein databanks for example. The DNA encoding the signal sequence/reporter protein i
Agarwal Atulya R.
Genentech Inc.
Kresnak Mark T.
Svoboda Craig G.
Yucel Remy
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