Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-12-15
2003-11-25
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S004000, C435S183000, C435S069100, C536S023100, C536S023200
Reexamination Certificate
active
06653075
ABSTRACT:
FIELD OF THE INVENTION
This invention relates generally to the field of molecular biology. More particularly, the invention relates to methods and constructs useful for identifying important and/or essential regions of a protein, whether or not the function or activity of the protein is already known.
BACKGROUND OF THE INVENTION
Current technology enables one to sequence vast amounts of nucleic acids at high speed. However, sequencing alone does not describe the activity of any of the genes sequenced. One can make predictions based on sequence homology that a given gene encodes a protein that exhibits immunoglobulin folds, or may have kinase activity, and the like, but one is limited to identifying features common to known proteins.
If a protein has a known or demonstrable activity, and is not too toxic to express, one can conduct mutagenesis experiments to determine which portion or portions of the protein are responsible for its activity. In general, one prepares a series of mutant versions of the protein in question, typically by a technique such as site-specific mutagenesis, and compares the activity of the mutants with that of the wild type protein. Mutants in which the active portion of the molecule is absent are expected to exhibit little or no activity, while mutants in which an irrelevant part of the molecule is altered are expected to exhibit little difference from the wild type. Due to the number of mutagenesis steps required, one generally selects a few likely spots in the sequence to experiment with, and rarely seeks to alter every residue in turn. Thus, the approach is both time-consuming and incomplete.
SUMMARY OF THE INVENTION
We have now invented a method for systematically and quickly examining substantially every position of a protein sequence, and determining whether or not it is essential to the activity of the protein. The method is effective even if the protein has no known activity, and/or is too toxic to express in its active form.
One aspect of the invention is a method for identifying a mutation-sensitive active region of a test protein, by providing a test nucleic acid construct comprising a regulatable promoter polynucleotide and a fusion polynucleotide comprising a test polynucleotide encoding the test protein fused to a reporter polynucleotide encoding a detectable label, wherein said fusion polynucleotide is operably associated with the promoter polynucleotide, wherein expression of the fusion polynucleotide in a selected host cell results in a specific phenotype and the presence of the detectable label; mutagenizing the test nucleic acid construct to provide a mutagenized construct; transforming a selected host cell with the mutagenized construct to provide a transformed host cell; selecting a transformed host cell that exhibits the detectable label, but which does not exhibit the specific phenotype; and sequencing a portion of the mutagenized construct from the selected transformed host cell to determine the alteration of the polynucleotide(s).
Another aspect of the invention is a population of host cells, comprising a plurality of host cells, each host cell having a test nucleic acid construct which comprises a regulatable promoter polynucleotide and a fusion polynucleotide comprising a mutagenized test polynucleotide encoding a mutagenized test protein fused to a reporter gene encoding a detectable label, wherein the fusion polynucleotide is operably associated with the promoter polynucleotide, and expression of the fusion polynucleotide in the host cell results in expression of said detectable label, wherein the plurality of host cells comprises a plurality of different mutagenized test polynucleotides.
DETAILED DESCRIPTION
Definitions
The term “reporter gene” refers to a polynucleotide that encodes a molecule that can be detected readily, either directly or by its effect on host cell characteristics. Exemplary reporter genes encode enzymes, for example &bgr;-galactosidase and URA3, luminescent or fluorescent proteins, such as Green Fluorescent Protein (GFP) and variants thereof, antigenic epitopes (for example Histidine-tag or influenza hemagluttinin tag), mRNA of distinct sequences, and the like. The term “detectable label” refers to a reporter gene or protein that can be detected directly by visual, optical, or spectroscopic methods, such as, for example, GFP, GFP variants, pigments, chromogenic enzymes such as horseradish peroxidase and &bgr;-galactosidase, and the like. The terms “selectable label” and “selectable marker” refers to an enzyme reporter gene or protein that facilitates separation of cells that express the label from cells that do not express the label, or to separate cells that express the label to different degrees. Such separation can be by any convenient means, such as, for example, survival of one group or the other, dependence upon a selected nutrient or lack thereof, sensitivity to a given compound, adherence to a solid surface, and the like.
The term “regulatable promoter” refers to a portion of a polynucleotide that is capable of controlling the transcription of nearby DNA, and that responds to the presence or activity of one or more proteins by increasing or decreasing transcription of the affected DNA. A variety of suitable promoters are known, for example GAL, TET, hybrid promoters, and the like.
The term “specific phenotype” as used herein refers to an alteration in one or more characteristics of the host cell distinct from the label, as a result of the heterologous gene or protein presence, for example, death, survival (in the presence of normally lethal conditions or agents), adherence or lack of adherence, morphology, color and appearance, and the like. The specific phenotype excludes any characteristic conferred by the label, which is independent of the specific phenotype: the specific phenotype is preferably observable regardless of the presence or absence of the detectable label as a fusion partner.
The term “mutagenizing” refers to a process for altering the nucleotide sequence of a polynucleotide, for example using PCR, radiation, chemical agents, enzymes, and the like.
The term “fluorescent protein” refers to a protein capable of fluorescing when illuminated. Exemplary fluorescent proteins include, without limitation, the
Aequorea victoria
“Green Fluorescent Protein” (“GFP”: see for example D. C. Prasher et al.,
Gene
(1992) 111:229-33; M. Chalfie et al.,
Science
(1994) 263:802-05, both incorporated herein by reference), and fluorescent mutants thereof (“GFP variants”: see for example U.S. Pat. No. 5,625,048 and U.S. 5,777,079, both incorporated herein by reference).
The term “different host cells” refers to a group of host cells that differ genetically from each other. The host cells can be derived from different species (for example, different species of yeast, or different species of mammals), different strains (for example, yeast strains that differ from each other in their genotype but are otherwise derived from the same species, or yeast strains derived by mutagenizing one or more parent strains), different tissue types (for example, human liver cells, fibroblasts, kidney cells, lung cells, tumor cells of various types, and the like), different stages of differentiation, and the like.
General Method
Methods of the invention permit one to quickly identify regions of a protein, for example an enzyme, that are sensitive to mutation. Loss of activity following mutation of one or a few base pairs in a gene suggests that the codon affected encodes an amino acid critical for activity of the encoded protein. This loss of activity may result, for example, from mutation of an active site residue in an enzyme, or from distortion or blocking of a binding site. The resulting information suggests that the affected amino acid can be useful as the target of further drug discovery investigation.
In the practice of the subject method, a host cell is selected for the test nucleic acid such that expression of the test nucleic acid results in a heterologous protein that confers an observable phenotype in the h
Rebelo Do Couto Fernando J.
Tugendreich Stuart
Cooley & Godward LLP
Iconix Pharmaceuticals Inc.
Katcheves Konstantina
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