Generation of diversity in combinatorial libraries
AssigneeCosmix Molecular Biologicals GmbH
InventorsJohn Collins, Peter Röttgen
Life of this patent
Estimating the term…
Abstract
The invention concerns gene banks and combinatorial derivatives thereof, prepared using phagemid- or phage-display in combination with type IIS restriction enzymes and cosmid packaging; their use for the isolation of ligands, including enzyme inhibitors, agonists and antagonists for receptors, competitive binding peptides to a defined target, diagnostic ligands for diseases and autoimmune syndromes, including surveillance tools for immune status, post-translationally modified peptides, and such ligands generated by this technology.
Claims14
A protein or peptide comprising a peptide sequence encoded by a DNA sequence from a bank of genes, wherein said genes comprise a double-stranded DNA sequence which is represented by the following formula of one of their strands:
- 5′B1B2B3 . . . BnXn+1 . . . Xn+aZn+a+1Zn+a+2Xn+a+3 . . . Xn+a+bQn+a+b+1 . . . Qn+a+b+j3′
- wherein n, a, b, and j are integers and are defined as:
- n>3, a>1, b>3 and j>1,
- wherein Xn+1 . . . Xn+a+b is a hypervariable sequence and B represents any of bases adenine (A), cytosine (C), guanine (G) or thymine (T), X represents any of bases A, C, G or T, Z represents any of bases A, C, G or T, and Q represents any of bases A, C, G or T, wherein,
- (i) Z represents G or T at a G:T ratio of 1:1, and/or
- (ii) Z represents C or T at a C:T ratio of 1:1, and/or
- (iii) Z represents A or G at a A:G ratio of 1:1, and/or
- (iv) Z represents A or C at a A:C ratio of 1:1, and
- wherein
- subsequences B1 . . . Bn and/or Qn+a+b+1 . . . Qn+a+b+j represent recognition sites for restriction enzymes, and wherein the recognition sites are oriented such that their cleavage site upon cleavage generates a cohesive end including the two bases designated Z.
A protein or peptide comprising a peptide sequence encoded by a DNA sequence from a bank of genes, wherein said genes comprise a double stranded DNA sequence which is represented by the following formula of one of their strands:
- 5′B1B2B3 . . . BnXn+1 . . . Xn+aZn+a+1Zn+a+2Xn+a+3 . . . Xn+a+bQn+a+b+1 . . . Qn+a+b+j3′
- wherein n, a, b and j are integers and
- n>3, a>1, b >3 and j>1,
- wherein Xn+1 . . . Xn+a+b is a hypervariable sequence and B represents any one of bases A, C, G or T, X represents any one of bases A, C, G or T, Z represents any one of bases A, C, G or T, and Q represents any one of bases A, C, G or T, and wherein
- four set of oligonucleotide sequences comprising Zn+a+1 and Zn+a+2 are present, wherein the four sets are characterized as follows:
- (i) a first set in which Zn+a+1 represents G and Zn+a+2 also represents G;
- (ii) a second set in which Zn+a+1 represents C and Zn+a+2 represents T;
- (iii) a third set in which Zn+a+1 represents A and Zn+a+2 represents A or C, and
- (iv) a fourth set:
- Zn+a+1 represents T and Zn+a+2 represents C or G, and wherein
- sequences B1 . . . Bn and/or Qn+a+b+1 . . . Qn+a+b+j represent recognition sites for restriction enzymes, wherein the recognition sites are oriented such that their cleavage site upon cleavage generates a cohesive end including the two bases designated Z.
A protein or peptide comprising a peptide sequence encoded by a DNA sequence from a bank of genes wherein said genes comprise a double stranded DNA sequence which is represented by the following formula of one of their strands:
- 5′B1B2B3 . . . BnXn+1 . . . Xn+aZn+a+1Zn+a+2Xn+a+3 . . . Xn+a+bQn+a+b+1 . . . Qn+a+b+j3′
- wherein n, a, b and j are integers and
- n>3, a>1, b>3 and j>1,
- wherein Xn+1 . . . Xn+a+b is a hypervariable sequence and B represents any one of bases A, C, G or T, X represents any one of bases A, C, G or T, Z represents any one of bases A, C, G or T, and Q represents any one of bases A, C, G or T, and wherein
- the following six sets of oligonucleotide sequences comprising Xn+a, Zn+a+1 and Zn+a+2 are present, wherein the six sets are characterized as follows:
- (i) a first set in which Xn+a represents A, G and/or T, or Xn+a represents C, G and/or T, Zn+a+1 represents G and Zn+a+2 represents G;
- (ii) a second set in which Xn+a represents A, C, G and/or T, Zn+a+1 represents C and Zn+a+2 represents T;
- (iii) a third set in which Xn+a represents A, C and/or G, Zn+a+1 represents A and Zn+a+2 represents A;
- (iv) a fourth set in which Xn+a represents A, C, G and/or T, Zn+a+1 represents A and Zn+a+2 represents C;
- (v) a fifth set in which Xn+a represents A, C, G and/or T, Zn+a+1 represents T and Zn+a+2 represents C;
- (vi) a sixth set in which Xn+a represents A, Zn+a+1 represents T and Zn+a+2 represents G.
- wherein B1 . . . Bn and/or Qn+a+b+1 . . . Qn+a+b+j represent recognition sites for restriction enzymes and wherein the recognition sites are oriented such that their cleavage site upon cleavage generates a cohesive end including the two bases designated Z.
Dependencies are inferred from the claim text.