US5198346A — Generation and selection of novel DNA-binding pro…

Claim 1

Independent Claim 244 words

A method of obtaining first and second genes encoding first and second homooligomeric DNA binding proteins which hybridize to form a hybrid heteroologomeric DNA binding protein which binds to a predetermined ultimate target double stranded DNA sequence, said sequence being nonpalindromic, said sequence comprising a left target-sequence and right target subsequence each of at least 4 base pairs length, said method comprising: producing a first gene encoding a first DNA-binding oligomeric protein binding to a first target sequence and a second gene encoding a second DNA-binding oligomeric protein binding to a second target sequence, wherein said first and second DNA-binding proteins each have at least two essentially dyad-symmetric DNA-binding domains, where said first target sequence is a palindrome or gapped palindrome and comprises said left target subsequence and a palindrome-completing subsequence, and where said second target sequence is a palindrome or gapped palindrome and comprises said right target subsequence and a palindrome-completing subsequence, whereby one of the DNA-binding domains of the second DNA-binding protein binds to the right target subsequence, said genes being produced by a process of at least partially random mutation followed by selection for the binding of the corresponding protein to the corresponding target sequence, wherein said first and second proteins can hybridize so as to obtain a heterooligomeric DNA-binding protein comprising a DNA-binding domain recognizing the left target subsequence and a DNA-binding domain recognizing the right target subsequence whereby said heterooligomeric protein has an affinity for the ultimate target DNA.

Claim 2

Independent Claim 244 words

A method of obtaining first and second genes encoding first and second homooligomeric DNA binding proteins which hybridize to form a hybrid heteroologomeric DNA binding protein which binds to a predetermined ultimate target double stranded DNA sequence, said sequence being nonpalindromic, said sequence comprising a left target-sequence and right target subsequence each of at least 4 base pairs length, said method comprising: producing a first gene encoding a first DNA-binding oligomeric protein binding to a first target sequence and a second gene encoding a second DNA-binding oligomeric protein binding to a second target sequence, wherein said first and second DNA-binding proteins each have at least two essentially dyad-symmetric DNA-binding domains, where said first target sequence is a palindrome or gapped palindrome and comprises said left target subsequence and a palindrome-completing subsequence, and where said second target sequence is a palindrome or gapped palindrome and comprises said right target subsequence and a palindrome-completing subsequence, whereby one of the DNA-binding domains of the second DNA-binding protein binds to the right target subsequence, said genes being produced by a process of at least partially random mutation followed by selection for the binding of the corresponding protein to the corresponding target sequence, wherein said first and second proteins can hybridize so as to obtain a heterooligomeric DNA-binding protein comprising a DNA-binding domain recognizing the left target subsequence and a DNA-binding domain recognizing the right target subsequence whereby said heterooligomeric protein has an affinity for the ultimate target DNA.

Claim 3

Dependent Claim 197 words

The method of claim 1 wherein at least one of said first and second genes is obtained by (a) providing a cell culture, said cell culture comprising a plurality of cells, each cell bearing a selection vector, said selection vector comprising a first and a second operon, each comprising at least one expressible gene, the genes of said first and second operons being different, a copy of the target DNA sequence being included in each operon and positioned therein so that under forward selection conditions the transformed cells enjoy a selective advantage if they express a protein or polypeptide which binds to said copies of the target DNA sequence, said cell culture being transformed with a variegated gene encoding potential DNA-binding proteins or polypeptides, where said cells collectively can express a plurality of different but sequence-related potential DNA-binding proteins or polypeptides, (b) causing the cells of such culture to express said potential DNA-binding proteins or polypeptides; (c) exposing the cells to forward selection conditions to select for cells which express a protein or polypeptide which preferentially binds to said target DNA sequence; and (d) recovering the selected cells bearing a gene coding for such protein or polypeptide.

Claim 4

Dependent Claim 26 words

The method of claim 2 wherein the level of variegation is such that from 10 6 to 10 9 different potential DNA-binding proteins can be expressed.

Claim 5

Dependent Claim 23 words

The method of claim 2 wherein a gene coding for a known DNA binding protein having a helix-turn-helix DNA binding motif is variegated.

Claim 6

Dependent Claim 89 words

The method of claim 2 wherein a gene encoding a known DNA binding protein picked from the group consisting of Cro from phage λ, cI repressor from phage λ, Cro from phage 434, cI repressor from phage 434, P22 repressor, E. coli tryptophan repressor, E. coli CAP, P22 Arc, P22 Mnt, E. coli lactose repressor, MAT-a1-alpha2 from yeast, Polyoma Large T antigen, SV40 Large T antigen, Adenovirus E1A, and TFIIIA from Xenopus laevis is variegated to obtain genes coding on expression for a plurality of potential target DNA-binding proteins.

Claim 7

Dependent Claim 50 words

The method of claim 2 wherein said variegated gene comprises at least one variegated codon, said codon having three base positions, each variegated codon being characterized by a mixture of bases at at least one base position wherein the mixture of bases for at least one base position is non-equimolar.

Claim 8

Dependent Claim 17 words

The method of claim 2 wherein the ultimate target double stranded DNA sequence is an HIV sequence.

Claim 9

Dependent Claim 26 words

The method of claim 7 wherein the ultimate target doublet stranded DNA sequence is HIV 353-369 or a subsequence thereof comprising at least eight base paris.

Claim 10

Dependent Claim 72 words

The method of claim 2 wherein at least one of said operons comprises a selectable beneficial gene, an occludible promoter operably linked to said beneficial gene and directing its transcription, an occluding promoter occluding transcription of said beneficial gene, and a copy of the target DNA sequence positioned so that the binding of said protein or polypeptide to said copy represses said occluding promoter and thereby facilitates transcription of said beneficial gene.

Claim 11

Dependent Claim 11 words

The method of claim 9 wherein the beneficial gene is aadA.

Claim 12

Dependent Claim 19 words

The method of claim 10 wherein the occludible promoter is the aadA promoter and the occluding promoter is Pcon.

Claim 13

Dependent Claim 177 words

The method of claim 2 wherein said selection vector comprises: a) a first operon, which operon comprises: i) a first binding marker gene(s), ii) a first promoter directing expression of said binding marker gene(s), and iii) a first copy of the target DNA sequence, where said target DNA sequence interferes substantially with expression of the first gene(s) if and only if a protein expressed by the transformed cell binds to the target DNA sequence, (b) a second operon, which operon comprises: i) a second binding marker gene(s), ii) a second promoter directing expression of said binding marker gene(s); and iii) a second copy of the target DNA sequence, where said target DNA sequence interferes substantially with expression of said gene(s) if and only if a protein expressed by the transformed cell binds to the target DNA sequence, where the binding marker genes of said first and second operons are different, and where, when said cells are exposed to forward selection conditions the gene products of said first and second binding marker genes are deleterious to the cell.

Claim 14

Dependent Claim 13 words

The method of claim 12 wherein the binding marker genes are functionally unrelated.

Claim 15

Dependent Claim 16 words

The method of claim 12 wherein the promoters of said first and second operons are different.

Claim 16

Dependent Claim 69 words

The method of claim 12 wherein a plurality of genetic elements essential to the maintenance of the vector or the survival of the transformed cells under conditions that select for presence of said vector, said operons and said genetic elements being positioned on said vector so no single deletion even can render nonfunctional more than one of said operons without also rendering nonfunctional one of said essential genetic elements.

Claim 17

Dependent Claim 62 words

The method of claim 12, said vector further comprising a gene (pdbp) coding for a potential DNA-binding protein or polypeptide, said gene comprising: a) a coding region that codes for a polypeptide, each domain of said polypeptide having at least 50% sequence identity to a known DNA-binding domain, and b) a promoter operably linked to said coding region for controlling its expression.

Claim 18

Dependent Claim 42 words

The method of claim 12 wherein at least one of said genetic elements comprises a beneficial gene, and a control promoter operably linked to said beneficial gene, but where no instance of said target DNA sequence is associated with said genetic element.

Claim 19

Dependent Claim 46 words

The method of claim 17 wherein the control promoter is essentially identical to the promoter of one of said selectable binding marker operons, so that proteins binding to the latter promoter will also bind to the control promoter and thereby inhibit expression of said beneficial gene.

Claim 20

Dependent Claim 24 words

The method of claim 12 wherein under reverse selection conditions the gene products of said binding marker genes are beneficial to the transformed cells.

Claim 21

Dependent Claim 87 words

The method of claim 19 wherein each of the first and second operons confers a phenotype selected independently but not-identically from the group consisting of: galT,K + , tetA + , lacZ + , pheS + , argP + , thyA + , crp + , pyrF + , ptsM + , secA + /malE + /lacZ + , ompA + , btuB + , lamB + , tonA + , cir + , tsx + , aroP + , cysK + , and dctA + .

Claim 22

Dependent Claim 35 words

The method of claim 12 wherein the vector comprises a plurality of codons, each variegated codon has a root mean square deviation from a flat distribution over the allowed amino acids of less than 0.08.

Claim 23

Dependent Claim 18 words

The method of claim 21 wherein the variation at each variegated codon allows all twenty possible amino acids.

Claim 24

Dependent Claim 25 words

The method of claim 22 wherein at any variegated codon the expected ratio of occurrence of (Lys+Arg) codons to (Asp+Glu) codon is 0.8 to 1.25.

Claim 25

Independent Claim 369 words

A method of obtaining genes encoding a heterooligomeric protein which binds to a predetermined ultimate target double stranded DNA sequence, said sequence being nonpalindromic, said sequence comprising a left target subsequence and a right target subsequence each of at least 4 base pairs lengths, said method comprising: (a) providing a first gene encoding a first DNA-binding oligomeric protein binding to a first target sequence and a second gene encoding a second DNA-binding oligomeric protein binding to a second target sequence, wherein said first and second DNA-binding proteins each have at least two dyad-symmetric DNA-binding domains, wherein said first and second DNA-binding proteins each have a dimerization interface, where said first target sequence is a palindrome or gaped palindrome and comprises said left target subsequence and a palindrome-completing subsequence, whereby one of the dyad-symmetric DNA-binding domains of the first DNA-binding protein binds to said left target subsequence, and where said second target sequence is a palindrome or gaped palindrome and comprises said right target subsequence and a palindrome completing subsequence, whereby one of the dyad-symmetric DNA-binding domains of the second DNA-binding protein binds to the right target subsequence, (b) variegating the dimerization interface of the protein encoded by one of said first or second genes to obtain variegants thereof and reverse selecting for expression from said variegant of a first oligomerization mutant protein, encoded by a variegant of said variegated gene, which is no longer capable of forming a homooligomer that can bind to said first or second target sequence, respectively, and verifying that said oligomerization mutant protein maintains a tertiary structure similar to the protein form which is descended, (c) variegating the dimerization interface of the protein encoded by the other of said first or second genes to obtain variegants thereof, (d) providing host cells carrying the gene encoding said first oligomerization mutant protein and a variegant gene of step (c), each operably linked to a promoter functional in the host cell, and (e) forward selecting for expression from a step (c) variegant gene of a second oligomerization mutant protein which is capable of forming a heterooligomer with said first oligomerization mutant protein, said heterooligomer binding said ultimate target DNA sequence, and (f) isolating the genes encoding said heterooligomer.

Claim 26

Independent Claim 369 words

A method of obtaining genes encoding a heterooligomeric protein which binds to a predetermined ultimate target double stranded DNA sequence, said sequence being nonpalindromic, said sequence comprising a left target subsequence and a right target subsequence each of at least 4 base pairs lengths, said method comprising: (a) providing a first gene encoding a first DNA-binding oligomeric protein binding to a first target sequence and a second gene encoding a second DNA-binding oligomeric protein binding to a second target sequence, wherein said first and second DNA-binding proteins each have at least two dyad-symmetric DNA-binding domains, wherein said first and second DNA-binding proteins each have a dimerization interface, where said first target sequence is a palindrome or gaped palindrome and comprises said left target subsequence and a palindrome-completing subsequence, whereby one of the dyad-symmetric DNA-binding domains of the first DNA-binding protein binds to said left target subsequence, and where said second target sequence is a palindrome or gaped palindrome and comprises said right target subsequence and a palindrome completing subsequence, whereby one of the dyad-symmetric DNA-binding domains of the second DNA-binding protein binds to the right target subsequence, (b) variegating the dimerization interface of the protein encoded by one of said first or second genes to obtain variegants thereof and reverse selecting for expression from said variegant of a first oligomerization mutant protein, encoded by a variegant of said variegated gene, which is no longer capable of forming a homooligomer that can bind to said first or second target sequence, respectively, and verifying that said oligomerization mutant protein maintains a tertiary structure similar to the protein form which is descended, (c) variegating the dimerization interface of the protein encoded by the other of said first or second genes to obtain variegants thereof, (d) providing host cells carrying the gene encoding said first oligomerization mutant protein and a variegant gene of step (c), each operably linked to a promoter functional in the host cell, and (e) forward selecting for expression from a step (c) variegant gene of a second oligomerization mutant protein which is capable of forming a heterooligomer with said first oligomerization mutant protein, said heterooligomer binding said ultimate target DNA sequence, and (f) isolating the genes encoding said heterooligomer.

Claim 27

Dependent Claim 68 words

The method of claim 24 wherein both of said first and second genes are provided by a process comprising (i) mutation of one or more preselected codons to encode a plurality of predetermined expected amino acids at each preselected codon, in predetermined expected proportions, and thereby obtain a plurality of different potential DNA binding proteins, and (ii) selection for genes encoding proteins which bind the corresponding target sequence.

Claim 28

Dependent Claim 46 words

The method of claim 25 in which the first and second DNA binding proteins either are unable to hybridize at all and still bind DNA, or, if they do hybridize, have a substantially diminished affinity or specificity for the corresponding subsequence of the target DNA sequence.

Claim 29

Dependent Claim 200 words

The method of claim 25 wherein at least one of said first and second genes is obtained by (a) providing a cell culture, said cell culture comprising a plurality of cells, each cell bearing a selection vector, said selection vector comprising a first and a second operon, each comprising at least one expressible gene, the genes of said first and second operons being different, a copy of the target DNA sequence being included in each operon and positioned therein so that under forward selection conditions the transformed cells enjoy a selective advantage if they express a protein or polypeptide which binds to said copies of the target DNA sequence, said cell culture being transformed with a variegated gene encoding potential DNA-binding proteins or polypeptides, where said cells collectively can express a plurality of different but sequence-related potential DNA-binding proteins or polypeptides, (b) causing the cells of such culture to express said potential DNA-binding proteins or polypeptides; (c) exposing the cells to forward selection conditions to select for cells which express a protein or polypeptide which preferentially binds to said target DNA sequence; and (d) recovering the selected cells bearing said first or second gene coding for such protein or polypeptide.

Claim 30

Dependent Claim 26 words

The method of claim 27 wherein the level of variegation is such that from 10 6 to 10 9 different potential DNA-binding proteins can be expressed.

Claim 31

Dependent Claim 23 words

The method of claim 27 wherein a gene coding for a known DNA binding protein having a helix-turn-helix DNA binding motif is variegated.

Claim 32

Dependent Claim 89 words

The method of claim 27 wherein a gene encoding a known DNA binding protein picked from the group consisting of Cro from phage λ, cI repressor from phage λ, Cro from phage 434, cI repressor from phage 434, P22 repressor, E. coli tryptophan repressor, E. coli CAP, P22 Arc, P22 Mnt, E. coli lactose repressor, MAT-a1-alpha2 from yeast, Polyoma Large T antigen, SV40 Large T antigen, Adenovirus E1A, and TFIIIA from Xenopus laevis is variegated to obtain genes coding on expression for a plurality of potential target DNA-binding proteins.

Claim 33

Dependent Claim 50 words

The method of claim 27 wherein said variegated gene comprises at least one variegated codon, said codon having three base positions, each variegated codon being characterized by a mixture of bases at at least one base position wherein the mixture of bases for at least one base position is non-equimolar.

Claim 34

Dependent Claim 17 words

The method of claim 27 wherein the ultimate target double stranded DNA sequence is an HIV sequence.

Claim 35

Dependent Claim 26 words

The method of claim 32 wherein the ultimate target double stranded DNA sequence is HIV 353-369 or a subsequence thereof comprising at least eight base paris.

Claim 36

Dependent Claim 72 words

The method of claim 27 wherein at least one of said operons comprises a selectable beneficial gene, an occludible promoter operably linked to said beneficial gene and directing its transcription, an occluding promoter occluding transcription of said beneficial gene, and a copy of the target DNA sequence positioned so that the binding of said protein or polypeptide to said copy represses said occluding promoter and thereby facilitates transcription of said beneficial gene.

Claim 37

Dependent Claim 11 words

The method of claim 34 wherein the beneficial gene is aadA.

Claim 38

Dependent Claim 19 words

The method of claim 35 wherein the occludible promoter is the aadA promoter and the occluding promoter is Pcon.

Claim 39

Dependent Claim 177 words

The method of claim 27 wherein said selection vector comprises: a) a first operon, which operon comprises: i) a first binding marker gene(s), ii) a first promoter directing expression of said binding marker gene(s), and iii) a first copy of the target DNA sequence, where said target DNA sequence interferes substantially with expression of the first gene(s) if and only if a protein expressed by the transformed cell binds to the target DNA sequence, (b) a second operon, which operon comprises: i) a second binding marker gene(s), ii) a second promoter directing expression of said binding marker gene(s); and iii) a second copy of the target DNA sequence, where said target DNA sequence interferes substantially with expression of said gene(s) if and only if a protein expressed by the transformed cell binds to the target DNA sequence, where the binding marker genes of said first and second operons are different, and where, when said cells are exposed to forward selection conditions the gene products of said first and second binding marker genes are deleterious to the cell.

Claim 40

Dependent Claim 13 words

The method of claim 37 wherein the binding marker genes are functionally unrelated.

Claim 41

Dependent Claim 16 words

The method of claim 37 wherein the promoters of said first and second operons are different.

Claim 42

Dependent Claim 69 words

The method of claim 37 wherein a plurality of genetic elements essential to the maintenance of the vector or the survival of the transformed cells under conditions that select for presence of said vector, said operons and said genetic elements being positioned on said vector so no single deletion event can render nonfunctional more than one of said operons without also rendering nonfunctional one of said essential genetic elements.

Claim 43

Dependent Claim 62 words

The method of claim 37, said vector further comprising a gene (pdbp) coding for a potential DNA-binding protein or polypeptide, said gene comprising: a) a coding region that codes for a polypeptide, each domain of said polypeptide having at least 50% sequence identity to a known DNA-binding domain, and b) a promoter operably linked to said coding region for controlling its expression.

Claim 44

Dependent Claim 42 words

The method of claim 37 wherein at least one of said genetic elements comprises a beneficial gene, and a control promoter operably linked to said beneficial gene, but where no instance of said target DNA sequence is associated with said genetic element.

Claim 45

Dependent Claim 46 words

The method of claim 42 wherein the control promoter is essentially identical to the promoter of one of said selectable binding marker operons, so that proteins binding to the latter promoter will also bind to the control promoter and thereby inhibit expression of said beneficial gene.

Claim 46

Dependent Claim 24 words

The method of claim 37 wherein under reverse selection conditions the gene products of said binding marker genes are beneficial to the transformed cells.

Claim 47

Dependent Claim 87 words

The method of claim 44 wherein each of the first and second operons confers a phenotype selected independently but not-identically from the group consisting of: galT,K + , tetA + , lacZ + , pheS + , argP + , thyA + , crp + , pyrF + , ptsM + , secA + /malE + /lacZ + , ompA + , btuB + , lamB + , tonA + , cir + , tsx + , aroP + , cysK + , and dctA + .

Claim 48

Dependent Claim 35 words

The method of claim 37 wherein the vector comprises a plurality of codons, each variegated codon has a root mean square deviation from a flat distribution over the allowed amino acids of less than 0.08.

Claim 49

Dependent Claim 18 words

The method of claim 46 wherein the variation at each variegated codon allows all twenty possible amino acids.

Claim 50

Dependent Claim 25 words

The method of claim 47 wherein at any variegated codon the expected ratio of occurrence of (Lys+Arg) codons to (Asp+Glu) codon is 0.8 to 1.25.

Annuity Stage	Window Opens	Payment Deadline	Surcharge Period Until	Estimated Fee	Status
3.5 Years (Year 4)	1996-03-29	1996-09-28	1997-03-30	$2000.00	Window Closed / Lapsed
7.5 Years (Year 8)	2000-03-29	2000-09-28	2001-03-30	$3760.00	Window Closed / Lapsed
11.5 Years (Year 12)	2004-03-29	2004-09-28	2005-03-30	$7700.00	Window Closed / Lapsed

Generation and selection of novel DNA-binding proteins and polypeptides

Abstract

Inventors

Bibliographic Details

Claims Analysis

References Cited (Backward Citations)

USPTO Annuity & Maintenance Windows

Assigned Categories & Tags

Assign Category Tag