The invention relates to methods for identifying genes encoding signal sequences.
The demonstrated clinical utility of certain growth factors and cytokines, for example, insulin, erythropoietin, granulocyte-colony stimulating factor, granulocyte-macrophage colony stimulating factor, human growth hormone, interferon-beta, and interleukin-2 in the treatment of human disease has generated considerable interest in identifying novel proteins of this class.
Since growth factors and cytokines are secreted proteins, they often possess xe2x80x9csignal sequencesxe2x80x9d at their amino terminal end. The signal sequence directs a secreted or membrane protein to a sub-cellular membrane compartment, the endoplasmic reticulum, from which the protein is dispatched for secretion from the cell or presentation on the cell surface. Techniques that detect signal sequences or nucleic acid sequences encoding a signal sequence have been employed as tools in the discovery of novel cytokines and growth factors.
Among the methods that have been used to identify secreted proteins are methods that rely on the homology between some secreted proteins. For example, DNA probes or PCR oligonucleotides that recognize sequence motifs present in genes encoding known secreted proteins have been used in screening assays to identify novel secreted proteins. In a related approach, homology-directed sequence searching of Expressed Sequence Tag (EST) sequences generated by high-throughput sequencing of specific cDNA libraries has been used to identify genes encoding secreted proteins. Both of these approaches can identify a signal sequence when there is a high degree of similarity between the DNA sequence used as a probe and the putative signal sequence.
xe2x80x9cSignal peptide trappingxe2x80x9d has also been used to identify secreted proteins (Tashiro et al., 1993, Science 261:600-603; Honjo et al., 1996; U.S. Pat. No. 5,525,486, and U.S. Pat. No. 5,536,637). Generically, this technique involves the ligation of cDNA, prepared from various mRNA sources, to a reporter gene lacking a signal sequence. The resulting chimeric constructs are introduced into an appropriate host cell. Depending upon the nature of the reporter gene, host cells are scored for either the presence of reporter protein at the cell surface or secretion of the reporter protein from cells. In both cases, a positive score indicates that the cell harbors a chimeric construct having a cDNA encoding a signal sequence which directs the export of the reporter protein to the cell surface or into the extracellular medium.
In a related method (Klein et al., 1996, Proc. Nat. Acad. Sci. USA 93:7108-7113; Jacobs, 1996, U.S. Pat. No. 5,536,637) the Saccharomyces cerevisiae gene, SUC2, which encodes a secreted invertase protein, is used as a reporter. Invertase catalyzes the hydrolysis of sucrose into glucose and fructose, sugars which, unlike sucrose, can be readily utilized by S. cerevisiae as a carbon source. Strains of S. cerevisiae that cannot secrete SUC2 protein are unable to grow on media with sucrose as the sole carbon source. Thus, a mutant SUC2 gene which does not encode a signal peptide can be used as a reporter in signal sequence trapping. Chimeric constructs composed of random cDNAs fused to DNA encoding SUC2 lacking a signal sequence are transformed into S. cerevisiae, and transformants secreting chimeric SUC2 are selected by growing the transformants under conditions where sucrose is the sole carbon source. This method offers a genetic selection for cDNAs encoding signal peptides.
The invention features a method for identifying nucleic acid sequences encoding signal sequences. Most secreted and membrane-associated proteins possess such signal sequences composed of 15-30 hydrophobic amino acid residues at their amino termini. Because signal sequences are present in secreted proteins and membrane-associated proteins, the identified nucleic acid sequences, which will encode at least a portion of a secreted or membrane-associated protein, can be used to isolate additional nucleic acid molecules encoding the entirety of the secreted or membrane-associated protein.
KRE9 is an example of a yeast secreted protein. Yeast KRE9 null mutants show severe growth retardation (essentially no growth) when glucose is the sole carbon source. Growth of a KRE9 null mutant on glucose can be restored by transformation with DNA encoding wild type KRE9 protein, but not by transformation with DNA encoding a mutant KRE9 protein lacking a signal sequence. Thus, secretion of KRE9 protein via its signal sequence is required for its normal function. Importantly, the presence of extracellular KRE9 protein does not rescue the KRE9 null phenotype. This result suggests that KRE9 protein must pass through the secretory pathway in order to exert its normal function. Although yeast KRE9 null mutants show essentially no growth when glucose is used as the carbon source, they can be maintained on galactose because of induction of the KNH1, a functional homolog of KRE9.
The invention features a method for identifying secreted and membrane-associated proteins using yeast that lack functional KRE9 protein and are transformed with a chimeric DNA molecule in which a mutant KRE9 gene lacking its signal sequence encoding portion is fused to a test sequence. The transformed yeast are grown on a selective medium that is designed permit (or prevent) growth of cells which produce functional, secreted KRE9. If the test sequence encodes a signal sequence (fused in-frame to the sequence encoding mature KRE9 protein), the yeast cell will grow (or not grow in the case of a selective medium which is designed to prevent growth of cells expressing functional, secreted KRE9) on the selective medium. Thus, the invention features a novel selection method utilizing DNA constructs containing a chimeric KRE9 gene in which the part of the KRE9 gene encoding the native KRE9 signal sequence is replaced with a candidate signal sequence encoding sequence. The ability of these chimeric constructs to rescue KRE9 null mutants grown on glucose is tested as follows. The chimeric constructs are used to transform KRE9 null mutants. The transformed cells are transferred to plates having glucose as the sole carbon source. Those chimeric constructs that allow a transformed KRE9 null mutant to grow on glucose contain candidate signal sequence encoding sequences.
Since growth factors and cytokines are secreted proteins, possessing signal sequences at their amino termini, signal sequence trapping can be employed as a tool in the discovery of novel proteins of this class.
One embodiment of the methods of the invention includes the following steps:
(a) obtaining a nucleic acid molecule which includes a chimeric gene, the chimeric gene including a first portion and a second portion, the first portion encoding a KRE9 lacking a functional signal sequence and the second portion being a heterologous nucleic acid sequence;
(b) transforming a yeast cell lacking a functional KRE9 gene with the nucleic acid molecule; and
(c) determining whether the transformed yeast cell grows when supplied with a medium that permits growth of a yeast cell expressing KRE9 having a functional signal sequence, but does not permit growth of a yeast cell that does not express KRE9 having a functional signal sequence, wherein growth on the medium indicates that the heterologous nucleic acid sequence present in the yeast cell encodes a signal sequence.
In another embodiment the method, step (a) includes:
(i) obtaining double-stranded DNA; and
(ii) ligating the double-stranded DNA to a DNA molecule encoding KRE9 lacking a functional signal sequence to create a chimeric gene.
In another embodiment of the invention step (a) includes:
(i) obtaining double-stranded DNA;
(ii) ligating the double-stranded DNA to a DNA molecule encoding KRE9 lacking a functional signal sequence to create a chimeric gene;
(iii) transforming a bacterium with a nucleic acid molecule that includes the chimeric gene;
(iv) growing the transformed bacterium; and
(v) isolating the nucleic acid molecule which includes the a chimeric gene from the transformed bacterium.
In another embodiment of the invention the method, in order to identify the signal sequence, the method includes: isolating and sequencing a portion of the chimeric gene contained within a yeast cell that grows when supplied with a medium that permits growth of a yeast cell expressing KRE9, but does not permit growth of a yeast cell that does not express KRE9 having a functional signal sequence.
In various preferred embodiments, first portion of the nucleic acid molecule is pBOSS1; second portion of the nucleic acid molecule is cDNA; the yeast strain is Yscreen2; the medium contains glucose as the sole carbon source; the medium contains a calcineurin inhibitor; and the method includes using a nucleic acid molecule encoding the signal sequence to screen an eukaryotic library for a full-length gene or cDNA encoding a protein comprising the identified signal sequence.
The invention also features a yeast cell transformed with a nucleic acid molecule comprising a chimeric gene, the chimeric gene comprising a first portion and a second portion, the first portion encoding a KRE9 lacking a functional signal sequence and the second portion being a heterologous nucleic acid sequence.
The invention also features a method that includes:
(a) obtaining a nucleic acid molecule which includes a chimeric gene, the chimeric gene including a first portion and a second portion, the first portion encoding a KRE9 lacking a functional signal sequence and the second portion being a heterologous nucleic acid sequence;
(b) transforming a yeast cell lacking a functional KRE9 gene with the nucleic acid molecule; and
(c) determining whether the transformed yeast cell grows when supplied with a medium that does not permit growth of a yeast cell expressing KRE9 having a functional signal sequence, but does permit growth of a yeast cell that does not express KRE9 having a functional signal sequence, wherein lack of growth on the medium indicates that the heterologous nucleic acid sequence present in the yeast cell encodes a signal sequence. In a preferred embodiment the medium contains K1 killer toxin.
In another preferred embodiment step (a) includes: (i) obtaining a double-stranded DNA; and (ii) ligating the double-stranded DNA to a DNA molecule encoding KRE9 lacking a functional signal sequence to create a chimeric gene.
In a another preferred embodiment the method, in order to identify the signal sequence, includes: isolating and sequencing a portion of the chimeric gene contained within the yeast cell that does not grow when supplied with a medium that does not permit growth of a yeast cell expressing KRE9, but does permit growth of a yeast cell that does not express KRE9 having a functional signal sequence.
The invention also features the expression vector pBOSS-1 and a genetically engineered host cell which harbors pBOSS-1.
A xe2x80x9cnonfunctional KRE9 genexe2x80x9d is a KRE9 gene having a mutation or deletion in its signal sequence encoding portion such that the gene does not encode a functional signal sequence and thus does not produce a functional KRE9 protein. Cells which fail to produce functional KRE9 protein exhibit slow vegetative growth and are effectively unable to grow on glucose. In the case where the nonfunctional KRE9 gene is produced by a point mutation, it is preferable that there be more than one mutation to decrease the chance of reversion to the wild type.
The KRE9-based signal sequence trap of the invention includes a positive selection method to screen for putative signal sequence encoding sequences. The selection strategy permits screening of a large number putative signal sequence encoding sequences because those cells that do not contain such a sequence essentially do not grow. This is in contrast to most other signal trap methods such as that described in U.S. Pat. No. 5,525,486 which rely solely on the detection of a protein encoded by a reporter gene. Furthermore, because there is no cross-feeding, a relatively large number of yeast can screened on any given plate.
In an alternative selection method of the invention, a negative selection is employed using K1 killer toxin. K1 killer toxin appears to kill sensitive yeast cells following binding to cell wall xcex21,6-glucans. Thus, cells with mutations in KRE9 are resistant to killing by K1 killer toxin. This selection method confers advantages similar those of the positive selection strategy in that large numbers of putative signal sequence encoding sequences can be screened.
Without being bound by any particular theory, the KRE9 protein reportedly encodes a soluble secretory-pathway protein required for yeast cell wall synthesis and growth. Specifically, the KRE9 protein plays a significant role in synthesis of cell surface xcex21,6-glucan (Brown and Bussey, 1993, Mol. Cell. Biol. 13:6346-6356) which is necessary for normal cell growth. When glucose is present in the medium, xcex21,6-glucan synthesis is normal provided that functional, secreted KRE9 protein is present. In the absence of functional KRE9 protein, yeast cells grow slowly when glucose is provided in the medium because of abnormal cell wall synthesis.
The KRE9-based signal trap, which is based on biosynthetic requirements, contrasts with the principle of signal trap systems based on catabolic requirements, for example the SUC2 signal trap selection system (U.S. Pat. No. 5,536,637). SUC2 protein is involved in catabolism in that it cleaves certain sugars to form nutrients which can be used as a carbon and energy source. As described above, the SUC2 signal trap selection system is based on the fact that yeast cells that lack functional SUC2 protein cannot utilize sucrose or raffinose as a carbon source. Thus, SUC2 null cells cannot grow when sucrose or raffinose is the sole carbon source.
One important advantage of a KRE9-based signal sequence trap of the invention is the low number of false positives generated by this method. This is in contrast to other signal trap methods such as that based on the yeast SUC2(U.S. Pat. No. 5,536,637). SUC2 null mutants are unable to grow when the energy source is sucrose or raffinose. When presented extracellularly, SUC2 protein can rescue SUC2 null mutants grown under restrictive conditions via a phenomenon referred to as cross-feeding. This arises because extracellular SUC2 protein cleaves sucrose into diffusible nutrients on which neighboring yeast cells can grow (i.e., fructose and glucose). KRE9 null mutants are not subject to cross-feeding, because extracellular KRE9 cannot restore growth of null KRE9 mutants on glucose. Thus, a KRE9 gene engineered to lack its signal sequence can be used as a reporter in signal sequence trapping and will not be subject to the background problems (i.e., false positives) that limit can limit the success of the less tightly regulated selection systems. Because the method of the invention is not subject to background problems to any significant degree, higher throughput screening is possible.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.