Just parking. (Interesting that Kawasaki's next patent was at ZymoGenetics.)
United States Patent 5,658,754
Kawasaki August 19, 1997
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Cell-free synthesis and isolation of novel genes and polypeptides
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.
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SUMMARY OF THE INVENTION
Briefly stated, the present invention relates to methods for synthesizing, screening, and selecting high numbers of novel genes and polypeptides. The methods generally comprise the steps of (a) constructing an in vitro expression unit comprising a 5' untranslated region containing an RNA polymerase binding sequence, a ribosome binding sequence, and a translation initiation signal, the expression unit being capable of producing mRNA; (b) attaching one or more semi-random nucleotide sequences to an expression unit; (c) transcribing or replicating the sequences associated with the expression unit and semi-random nucleotide sequences to produce RNA; (d) translating the RNA to produce polysomes under conditions sufficient to maintain the polysomes;
(e) binding the polysomes to a substance of interest; (f) isolating the polysomes that bind to the substance of interest; (g) disrupting the isolated polysomes to release mRNA; (h) recovering and constructing cDNA from the released mRNA; and (i) expressing the gene to produce novel polypeptides.
In one embodiment of the method described above, the process may be repeated on mRNA that has been enriched for desirable sequences by amplifying the RNA or respective cDNA. Subsequently, this amplified subset of genes may be cycled through the various steps outlined above to further enrich for desirable novel genes until desirable sequences represent a significant (>10.sup.-3) fraction of the truncated population. In principle, the method may be repeated until the population of genes is nearly homogeneous.
Within a second aspect of the present invention, a method for producing novel polypeptides is provided, comprising the steps of (a) constructing an in vitro expression unit comprising a 5' untranslated region containing an RNA polymerase binding sequence, a ribosome binding sequence, and a translation initiation signal, the expression unit being capable of producing mRNA; (b) attaching one or more semi-random nucleotide sequences to the expression unit; (c) transcribing sequences associated with the expression unit and semi-random nucleotide sequences to produce RNA; (d) translating the RNA to produce biologically active polypeptides; (e) subdividing the RNA encoding the biologically active polypeptides; (f) transcribing, translating, and subdividing as set forth in steps (c)-(e) so that the gene of interest is isolated; (g) constructing cDNA from the isolated gene; and (h) expressing the cDNA to produce novel polypeptides.
In yet another aspect of the present invention, a method of producing novel polypeptides is provided comprising the steps of (a) constructing an in vitro expression unit comprising a 5' untranslated region containing an RNA polymerase binding sequence, a ribosome binding sequence, and a translation initiation signal, the expression unit being capable of producing mRNA; (b) attaching one or more semi-random nucleotide sequence to the expression unit; (c) replicating the sequences associated with the expression unit and semi-random sequences to produce RNA; (d) translating the RNA to produce biologically active polypeptides; (e) subdividing the RNA encoding the 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 the isolated gene, and (h) expressing the cDNA to produce novel polypeptides.
The expression unit described above comprises an RNA polymerase binding sequence, a ribosome binding site, and a translation initiation signal. The expression unit may further comprise a translation enhancer or "activator" sequences, a 3' tail of a selected sequence and appropriate restriction sites. The semi-random DNA sequences may be generated by mechanically, chemically, or enzymatically fragmenting naturally-occurring DNA, by chemically synthesizing the DNA, or by polymerizing the DNA directly onto the expression unit. The substance of interest may be a surface antigen, receptor protein, toxin, organic polymer, active site of a protein molecule, metabolite, antibody, metal, hormone, or other compound.
These and other aspects will become evident upon reference to the following detailed description.... |
