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US 5,658,754 AGranted
Cell-free synthesis and isolation of novel genes and polypeptides
AssigneeOptein, Inc.
InventorGlenn H. Kawasaki
Life of this patent
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Abstract
A method for the cell-free synthesis and isolation of novel genes and polypeptides is provided. Within one embodiment, an expression unit is constructed onto which semi-random nucleotide sequences are attached. The semi-random nucleotide sequences are first transcribed to produce RNA, and then translated under conditions such that polysomes are produced. Polysomes which bind to a substance of interest are then isolated and disrupted; and the released mRNA is recovered. The mRNA is used to construct cDNA which is expressed to produce novel polypeptides.
Claims50
A method for producing novel polypeptides, comprising:
- (a) constructing an in vitro expression unit comprising a 5' untranslated region containing an RNA polymerase binding site, a ribosome binding site, and a translation initiation signal, said expression unit being capable of producing mRNA;
- (b) attaching one or more semi-random polynucleotides to said expression unit;
- (c) transcribing or replicating from the RNA polymerase binding site the polynucleotides associated with the expression unit and semi-random polynucleotides to produce mRNA;
- (d) translating said mRNA to produce polysomes under conditions sufficient to maintain said polysomes;
- (e) binding said polysomes to a substance of interest;
- (f) isolating said polysomes that bind to said substance of interest;
- (g) disrupting said isolated polysomes to release mRNA;
- (h) recovering said mRNA;
- (i) constructing cDNA from said recovered mRNA;
- and
- (j) expressing said cDNA to produce novel polypeptides.
8 more elements
A method for producing novel polypeptides, comprising:
- (a) constructing an in vitro expression unit comprising a 5' untranslated region containing an RNA polymerase binding site, a ribosome binding site, and a translation initiation signal, said expression unit being capable of producing mRNA;
- (b) attaching one or more semi-random polynucleotides to the expression unit;
- (c) transcribing from the RNA polymerase binding site the polynucleotides associated with the expression unit and semi-random polynucleotides to produce mRNA;
- (d) translating said mRNA in vitro to produce biologically active polypeptides;
- (e) subdividing the mRNA encoding said biologically active polypeptides;
- (f) transcribing, translating, and subdividing as set forth in steps (c)-(e), such that the gene of interest is isolated;
- (g) constructing cDNA from said isolated gene;
- and
- (h) expressing said cDNA to produce novel polypeptides.
6 more elements
A method for producing novel polypeptides, comprising:
- (a) constructing an in vitro expression unit comprising a 5' untranslated region containing an RNA polymerase binding site, a ribosome binding site, and a translation initiation signal, said expression unit being capable of producing mRNA;
- (b) attaching one or more semi-random polynucleotides to the expression unit;
- (c) replicating from the RNA polymerase binding site the polynucleotides associated with the expression unit and semi-random polynucleotides to produce mRNA;
- (d) translating said mRNA in vitro to produce biologically active polypeptides;
- (e) subdividing the mRNA encoding said biologically active polypeptides;
- (f) translating and subdividing as set forth in steps (d)-(e) such that the gene of interest is isolated;
- (g) constructing cDNA from said isolated gene;
- and
- (h) expressing said cDNA to produce novel polypeptides.
6 more elements
A method for producing novel polypeptides, comprising:
- (a) constructing an in vitro expression unit comprising a 5' untranslated region containing an RNA polymerase binding site, a ribosome binding site, and a translation initiation signal, said expression unit being capable of producing mRNA;
- (b) attaching one or more semi-random polynucleotides to said expression unit adjacent to a polynucleotide sequence encoding a fixed amino acid sequence;
- (c) transcribing or replicating from the RNA polymerase binding site the polynucleotides associated with the expression unit and semi-random polynucleotides to produce mRNA which encodes a polypeptide of at least about fifty amino acids;
- (d) translating said mRNA to produce polysomes under conditions sufficient to maintain said polysomes;
- (e) binding said polysomes to a substance of interest;
- (f) isolating said polysomes that bind to said substance of interest;
- (g) disrupting said isolated polysomes to release mRNA;
- (h) recovering said mRNA;
- (i) constructing cDNA from said recovered mRNA;
- and
- (j) expressing said cDNA to produce novel polypeptides.
8 more elements
Dependencies are inferred from the claim text.