Yeast cells engineered to produce pheromone system protein surrogates, and uses therefor
First Claim
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1. A yeast cell having a pheromone system, which cell comprises:
- (a) a first heterologous gene encoding a heterologous surrogate of a yeast pheromone receptor, said surrogate performing in the pheromone system of the yeast cell a function naturally performed by said yeast pheromone receptor, and (b) a second heterologous gene encoding a heterologous peptide, wherein said heterologous peptide modulates the interaction of said surrogate with said pheromone system in the yeast cell, and said modulation is a selectable or screenable event, and wherein said heterologous peptide is selected from the group consisting of agonists for the surrogate receptor and antagonists of the surrogate receptor.
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Abstract
Yeast cells are engineered to express both a surrogate of a pheromone system protein (e.g., enzymes involved in maturation of α-factor, transporters of a-factor, pheromone receptors, etc.) and a potential peptide modulator of the surrogate, in such a manner that the inhibition or activation of the surrogate affects a screenable or selectable trait of the yeast cells. Various additional features improve the signal-to-noise ratio of the screening/selection system.
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Citations
60 Claims
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1. A yeast cell having a pheromone system, which cell comprises:
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(a) a first heterologous gene encoding a heterologous surrogate of a yeast pheromone receptor, said surrogate performing in the pheromone system of the yeast cell a function naturally performed by said yeast pheromone receptor, and (b) a second heterologous gene encoding a heterologous peptide, wherein said heterologous peptide modulates the interaction of said surrogate with said pheromone system in the yeast cell, and said modulation is a selectable or screenable event, and wherein said heterologous peptide is selected from the group consisting of agonists for the surrogate receptor and antagonists of the surrogate receptor. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51)
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52. A yeast cell having a pheromone system, which cell comprises:
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(a) a first heterologous gene encoding a heterologous surrogate of a yeast pheromone receptor, said surrogate performing in the pheromone system of the yeast cell a function naturally performed by said yeast pheromone receptor;
(b) a second heterologous gene encoding a heterologous peptide, wherein said heterologous peptide modulates the interaction of said surrogate with said pheromone system in the yeast cell, and said modulation is a selectable or screenable event, and wherein said heterologous peptide is selected from the group consisting of agonists for the surrogate receptor and antagonists of the surrogate receptor; and
(c) a chimeric Gα
subunit, wherein the amino terminal portion of the Gα
subunit is substantially homologous with the Gα
subunit of a yeast G protein and the remainder is substantially homologous with the corresponding portion of a Gα
subunit of a heterologous G protein.
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53. A yeast cell having a pheromone system, which cell comprises:
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(a) a first heterologous gene encoding a heterologous surrogate of a yeast pheromone receptor, said surrogate performing in the pheromone system of the yeast cell a function naturally performed by said yeast pheromone receptor;
(b) a second heterologous gene encoding a heterologous peptide, wherein said heterologous peptide modulates the interaction of said surrogate with said pheromone system in the yeast cell; and
(c) a G protein comprising a Gα
subunit, wherein said Gα
subunit is chimeric and wherein the amino terminal portion of the Gα
subunit is substantially homologous with the Gα
subunit of a yeast G protein and the remainder is substantially homologous with the corresponding portion of a Gα
subunit of a heterologous G protein;
and said modulation is a selectable or screenable event.
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54. A yeast cell having a pheromone system, which cell comprises:
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(a) a first heterologous gene encoding a heterologous surrogate of a yeast pheromone receptor, said surrogate performing in the pheromone system of the yeast cell a function naturally performed by said yeast pheromone receptor; and
(b) a second heterologous gene encoding a heterologous peptide, wherein said heterologous peptide modulates the interaction of said surrogate with said pheromone system in the yeast cell, and said modulation is a selectable or screenable event; and
wherein the yeast cell responds to a factor and fails to grow on galactose.
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55. A yeast cell having a pheromone system, which cell comprises:
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(a) a first heterologous gene encoding a heterologous surrogate of a yeast pheromone receptor, said surrogate performing in the pheromone system of the yeast cell a function naturally performed by said yeast pheromone receptor;
(b) a second heterologous gene encoding a heterologous peptide, wherein said heterologous peptide modulates the interaction of said surrogate with said pheromone system in the yeast cell; and
(c) a mutated form of Gal7 or Gal10;
wherein said modulation is a selectable or screenable event; and
wherein the yeast cell responds to a factor, expresses Ste3p, and expresses Gal1 under the control of a pheromone responsive promoter.
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56. A yeast cell having a pheromone system, which cell comprises
(a) a first heterologous gene encoding a heterologous surrogate of a yeast pheromone receptor, said surrogate performing in the pheromone system of the yeast cell a function naturally performed by said yeast pheromone receptor, and (b) a second heterologous gene encoding a heterologous peptide, wherein said heterologous peptide modulates the interaction of said surrogate with said pheromone system in the yeast cell, and said modulation is a selectable or screenable event, and wherein the heterologous peptide is 2 to 200 amino acids in length.
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57. A yeast cell having a pheromone system, which cell comprises:
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(a) a first heterologous gene encoding a heterologous surrogate of a yeast pheromone receptor, wherein said surrogate is a C5a receptor and performs in the pheromone system of the yeast cell a function naturally performed by said yeast pheromone receptor; and
(b) a second heterologous gene encoding a heterologous peptide, wherein said heterologous peptide modulates the interaction of said surrogate with said pheromone system in the yeast cell;
and said modulation is a selectable or screenable event.
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58. A method of assaying a peptide for modulation of the activity of a non-yeast surrogate for a pheromone system protein, which comprises:
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(a) providing yeast cells, wherein each of the yeast cells has a pheromone system and comprises;
(i) a first heterologous gene encoding a heterologous surrogate of a yeast pheromone receptor, in which the surrogate is human Mdr1 and performs in the pheromone system of the yeast cell a function naturally performed by said yeast pheromone receptor;
(ii) a second heterologous gene encoding a heterologous peptide, wherein said heterologous peptide modulates the interaction of said surrogate with said pheromone system in the yeast cell; and
(iii) a pheromone-responsive selectable marker;
wherein said modulation is a selectable or screenable event; and
wherein the cells grow on histidine-free media only if the surrogate transports α
-factor, the cells are galactose-sensitive only if the surrogate transports α
-factor, and endogenous pleiotropic drug resistance genes have been inactivated;
(b) determining by detecting a change in said selectable or screenable event whether the pheromone signal pathway is activated or inhibited by the interaction; and
(c) selecting said cells selected for expression of a peptide having a desired activating or inhibiting effect, thereby assaying a peptide for modulation of the activity of a non-yeast surrogate for a pheromone system protein.
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59. A yeast cell having a pheromone system, which cell comprises:
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(a) a first heterologous gene encoding a heterologous surrogate of a yeast pheromone receptor, said surrogate performing in the pheromone system of the yeast cell a function naturally performed by said yeast pheromone receptor; and
(b) a second heterologous gene encoding a heterologous peptide, wherein said heterologous peptide modulates the interaction of said surrogate with said pheromone system in the yeast cell, and said modulation is a selectable or screenable event; and
wherein the yeast cell lacks ras function in the presence of cAMP. - View Dependent Claims (60)
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Specification