EP0035384A2
Expired / Lapsed
EP
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Deoxynucleotide linkers to be attached to a cloned DNA coding sequence
Assignee: University of California
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Filing: 1981-02-27
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Granted: —
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Expires: 2001-02-27
Abstract
Abstract The present invention is based upon a general principle of providing specific olingonucleotide segments (<<linkers>>, herein) to be attached in sequence to a cloned DNA coding segment. The linkers of the present invention confer desired functional properties on the expression of the protein coded by the coding sequence. Using linkers of the present invention, the desired protein may be expressed either as a fusion or non-fusion protein. A linker coding for an additional sequence of amino acids may be attached, the sequence being chosen to provide properties exploitable in a simplified purification process. A linker coding for an amino acid sequence of the extended specific cleavage site of a proetolytic enzyme is provided, as well as specific cleavage linkers for simple specific cleavage sites.
Inventors
- 👤 William J. Rutter
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-23 17:53
CPC Classifications
C; C07; C07K; C07K14/00; C07K14/435; C07K14/575; C07K14/62; C12; C12N; C12N15/00; C12N15/09; C12N15/11; C12N15/62; C12N15/63; C12N15/66; C12N15/70; C07K2319/00; C07K2319/50; C07K2319/61; C07K2319/70; C07K2319/74; C07K2319/75
Claims Analysis
Total Claims: 22
Claim 1
Independent Claim
35 words
A specific cleavage linker comprising a deoxynucleotide sequence coding for a specific cleavage sequence comprising a sequence of one or more amino acids which is spec- ificall'y recognized and cleavable by enzymatic or chemical means.
Claim 2
Dependent Claim
35 words
A specific cleavage linker comprising a deoxynucleotide sequence coding for a specific cleavage sequence comprising a sequence of one or more amino acids which is spec- ificall'y recognized and cleavable by enzymatic or chemical means.
Claim 3
Dependent Claim
63 words
The specific cleavage linker of claim 1 which also contains a deoxynucleotide sequence containing (i) a restriction site arrd (ii) b n c m at the 5'-end of said linker, said restriction site is recognized and cleavable by a restriction endonuclease, b and c may be any deoxynucleotide and n and m are integers such that n + m = 0,1 or 2.
Claim 4
Dependent Claim
35 words
The specific cleavage linker of claim 1 or 2 wherein said linker codes for a specific cleavage sequence specifically recognized and cleavable by an enzyme selected from the group comprising enterokinase, kallikrein B and chymosin.
Claim 5
Dependent Claim
117 words
The specific cleavage linker of claim 1 wherein said linker comprises a deoxynucleotide sequence comprising a plus strand having the sequence 5'-(GAL)(n)AAJ-3' or 5'-(GAL)(n)WGZ-3', wherein L is A, T, C or G, J is A or G, W is A if Z is A or G or W is C if Z is A, G, C or T, Z is A, T, C or G if W is C or Z is A or G if W is A and n refers to the number of the triplet codons GAL in the deoxynucleotide sequence and may be 2, 3 or 4, said specific cleavage linker codes for the specific cleavage sequence specifically recognized and cleavable by enterokinase.
Claim 6
Dependent Claim
23 words
The specific cleavage linker of claim 4 wherein L is T or G, J is A, W is C and Z is T.
Claim 7
Dependent Claim
24 words
The.specific cleavage linker of claim 4 wherein said linker also contains a deoxynucleotide sequence having the restriction site for HindIII endonuclease at the 5'-end.
Claim 8
Dependent Claim
26 words
The specific cleavage linker of claim 6 wherein the deoxynucleotide sequence having the restriction site for .HindIII endonuclease comprises a plus strand having the sequence 5'-CCAAGCTTGG-3'.
Claim 9
Dependent Claim
23 words
A specific purification linker comprising a deoxynucleotide sequence coding for an amino acid sequence which is selectively bindable to a solid phase material.
Claim 10
Dependent Claim
23 words
A specific purification linker comprising a deoxynucleotide sequence coding for an amino acid sequence which is selectively bindable to a solid phase material.
Claim 11
Dependent Claim
37 words
The specific purification linker of claim 8 wherein said amino acid sequence is selected from the group comprising a polyanionic amino acid sequence, a polycationic amino acid sequence, a hydrophobic amino acid sequence and an immunogenic peptide.
Claim 12
Dependent Claim
73 words
The specific purification linker of claim 8 wherein said linker comprises a deoxynucleotide sequence comprising a plus strand having the sequence 5'-(GAL)(m)AAK(GAL)(n)AAJ-3', wherein L is A, T, C or G, K is T or C, J is A or G and m and n refer to the number of the triplet codons GAL in the deoxynucleotide sequence, m may be 1, 2, 3 or 4 and n may be 2, 3 or 4.
Claim 13
Dependent Claim
34 words
The specific purification linker of claim 8 wherein said amino acid sequence comprises amino acids selected from the group consisting of leucine, isoleucine, valine or phenylalanine, said sequence being 10-50 amino acids in length.
Claim 14
Dependent Claim
39 words
A ribosomal binding site linker comprising a first deoxynucleotide sequence which is homologous to the 3'-end of 16S ribosomal RNA and a second deoxynucleotide sequence comprising 3-11 deoxynucleotides, said second deoxynucleotide sequence is joined to said first deoxynucleotide sequence.
Claim 15
Dependent Claim
39 words
A ribosomal binding site linker comprising a first deoxynucleotide sequence which is homologous to the 3'-end of 16S ribosomal RNA and a second deoxynucleotide sequence comprising 3-11 deoxynucleotides, said second deoxynucleotide sequence is joined to said first deoxynucleotide sequence.
Claim 16
Dependent Claim
16 words
The ribosomal binding site linker of claim 12 wherein said linker also contains a termination codon.
Claim 17
Dependent Claim
16 words
The ribosomal binding site linker of claim 12 wherein said linker also contains a promoter segment.
Claim 18
Dependent Claim
61 words
The ribosomal binding site linker of claim 13 wherein said linker comprises a deoxynucleotide sequence comprising a plus strand having the sequence 5'-L(n)TAGGAGGAL (m)-3' or 5'-L(n)TAGGAGGATCAATTCC-3', wherein L is A, T, C or G, n is 0, 1 or 2 and m is any integer from 3 to 11, n and m denoting the number of L nucleotides in the sequence.
Claim 19
Dependent Claim
55 words
The ribosomal binding site linker of claim 14 wherein said linker comprises a deoxynucleotide sequence comprising a plus strand having the sequence , wherein J is A or G, L is A, T, C or G and m is any integer from 3 to 11 and denotes the number of L deoxynucleotides in the sequence.
Claim 20
Dependent Claim
118 words
A composite linker comprising a first deoxynucleotide sequence having a restriction site which is recognized and cleavable by a restriction endonuclease, a second deoxynucleotide sequence which is homologous to the 3'-end of 16S ribosomal RNA, a third deoxynucleotide sequence having 3-11 deoxynucleotides, a fourth deoxynucleotide sequence having the initiation codon, a fifth deoxynucleotide sequence coding for an amino acid sequence which is selectively bindable to a solid phase material and a sixth deoxynucleotide sequence coding for a specific cleavage sequence comprising a sequence of one or more amino acids which is specifically recognized and cleavable by enzymatic or chemical means, said first, second, third, fourth, fifth and sixth deoxynucleotide sequences are joined together in the direction of translation.
Claim 21
Dependent Claim
118 words
A composite linker comprising a first deoxynucleotide sequence having a restriction site which is recognized and cleavable by a restriction endonuclease, a second deoxynucleotide sequence which is homologous to the 3'-end of 16S ribosomal RNA, a third deoxynucleotide sequence having 3-11 deoxynucleotides, a fourth deoxynucleotide sequence having the initiation codon, a fifth deoxynucleotide sequence coding for an amino acid sequence which is selectively bindable to a solid phase material and a sixth deoxynucleotide sequence coding for a specific cleavage sequence comprising a sequence of one or more amino acids which is specifically recognized and cleavable by enzymatic or chemical means, said first, second, third, fourth, fifth and sixth deoxynucleotide sequences are joined together in the direction of translation.
Claim 22
Dependent Claim
39 words
The composite linker of claim 17 wherein said linker comprises a deoxynucleotide sequence comprising a plus strand having the sequence wherein L is A, T, C or G, J is A or G, and K is T or C.
References Cited (Backward Citations)
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