EP0286239A1
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EP
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Production and purification of a protein fused to a binding protein
Assignee: New England Biolabs Inc
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Filing: 1988-03-09
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Granted: —
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Expires: 2008-03-09
Abstract
Abstract Methods and products are provided for producing and/or purifying virtually any hybrid polypeptide molecule employing recombinant DNA techniques. More specifically, a DNA fragment coding for a protein molecule, e.g. a polypeptide or portion thereof, is fused to a DNA fragment coding for a binding protein such as the gene coding for the maltose binding protein. The fused DNA is inserted into a cloning vector and an appropriate host transformed. Upon expression, a hybrid polypeptide is produced which can be purified by contacting the hybrid polypeptide with a ligand or substrate to which the binding protein has specific affinity, e.g. by affinity chromatography. The hybrid polypeptide so purified may in certain instances be useful in its hybrid form, or it may be cleaved to obtain the protein molecule itself by, for example, linking the DNA fragments coding for the target and binding proteins with a DNA segment which codes for a peptide which is recognized and cut by a proteolytic enzyme. The present invention also relates to certain vectors useful in practing the above process as well as to a bioreactor and methods employing the bound hybrid polypeptide, e.g. where the bound target polypeptide is contacted and reacted with a sustance which interacts with the bound target polypeptide to produce a desired result.
Inventors
- 👤 Chudi Guan
- 👤 Hiroshi Inouye
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-28 11:46
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/11; C12N15/52; C12N15/62; C07; C07K; C07K2319/00; C07K2319/20; C07K2319/24; C07K2319/50
Claims Analysis
Total Claims: 36
Claim 1
Independent Claim
87 words
A method for producing and purifying a protein molecule comprising: a) constructing a DNA expression vector which expresses a hybrid polypeptide in a transformed host cell, the hybrid polypeptide comprising the protein molecule and a binding protein, or portion thereof, having a specific affinity for a substrate. b) introducing the expression vector into an appropriate host cell and expressing the hybrid polypeptide; c) contacting the hybrid polypeptide produced by the transformed cell with the substrate to which the binding protein binds; and (d) recovering the protein molecule.
Claim 2
Independent Claim
87 words
A method for producing and purifying a protein molecule comprising: a) constructing a DNA expression vector which expresses a hybrid polypeptide in a transformed host cell, the hybrid polypeptide comprising the protein molecule and a binding protein, or portion thereof, having a specific affinity for a substrate. b) introducing the expression vector into an appropriate host cell and expressing the hybrid polypeptide; c) contacting the hybrid polypeptide produced by the transformed cell with the substrate to which the binding protein binds; and (d) recovering the protein molecule.
Claim 3
Dependent Claim
33 words
The method of claim 1, wherein the DNA coding for the hybrid polypeptide contains a linking DNA fragment which links the DNA encoding the protein molecule with the DNA encoding the binding protein.
Claim 4
Dependent Claim
14 words
The method of claim 1, wherein the substrate is contained within an affinity column.
Claim 5
Dependent Claim
45 words
The method of claim 1, wherein the binding protein is selected from the group consisting of sugar binding protein, receptor protein, amino acid binding protein, sulfate binding protein, vitamin binding protein, metal binding protein, phosphate binding protein, lectin binding protein or nucleic acid binding protein.
Claim 6
Dependent Claim
14 words
The method of claim 4, wherein the sugar binding protein is maltose binding protein.
Claim 7
Dependent Claim
11 words
The method of claim 5, wherein the substrate is cross-linked amylose.
Claim 8
Dependent Claim
31 words
The method of claim 1, comprising the further step of releasing the hybrid polypeptide from the substrate by contacting the bound hybrid polypeptide with a substance which displaces the hybrid polypeptide.
Claim 9
Dependent Claim
18 words
The method of claim 1, comprising the further step of cleaving the protein molecule from the hybrid polypeptide.
Claim 10
Dependent Claim
74 words
A fusion vector for constructing an expression vector which expresses a binding protein fused to a protein molecule to be purified, comprising: (a) a DNA fragment coding for the binding protein or biologically active portion thereof, the binding protein having a specific affinity for a substrate; and (b) a DNA fragment which codes for a linking sequence for linking the DNA coding for the binding protein with the DNA coding for the protein molecule.
Claim 11
Dependent Claim
74 words
A fusion vector for constructing an expression vector which expresses a binding protein fused to a protein molecule to be purified, comprising: (a) a DNA fragment coding for the binding protein or biologically active portion thereof, the binding protein having a specific affinity for a substrate; and (b) a DNA fragment which codes for a linking sequence for linking the DNA coding for the binding protein with the DNA coding for the protein molecule.
Claim 12
Dependent Claim
45 words
The fusion vector of claim 9, wherein binding protein is selected from the group consisting of sugar binding protein, receptor protein, amino acid binding protein, sulfate binding protein, vitamin binding protein, metal binding protein, phosphate binding protein, lectin binding protein or nucleic acid binding protein.
Claim 13
Dependent Claim
15 words
The fusion vector of claim 10, wherein the sugar binding protein is maltose binding protein.
Claim 14
Dependent Claim
16 words
The fusion vector of claim 9, wherein the linking sequence comprises one or more restriction sites.
Claim 15
Dependent Claim
23 words
The fusion vector of claim 9, wherein the linking sequence codes for a polypeptide which is recognized and cleaved by a proteolytic agent.
Claim 16
Dependent Claim
29 words
The fusion vector of claim 9, wherein the linking sequence codes for a spacer polypeptide which separates the binding protein from the protein molecule expressed by the expression vector.
Claim 17
Dependent Claim
10 words
The fusion vector of claim 9, comprising the plasmid pCG150.
Claim 18
Dependent Claim
58 words
A DNA expression vector for producing a purified protein molecule, which upon expression produces a binding protein fused to the protein molecule, comprising: (a) a DNA fragment coding for the binding protein or portion thereof, the binding protein having a specific affinity for a substrate; and (b) a DNA fragment coding for the protein molecule to be purified.
Claim 19
Dependent Claim
58 words
A DNA expression vector for producing a purified protein molecule, which upon expression produces a binding protein fused to the protein molecule, comprising: (a) a DNA fragment coding for the binding protein or portion thereof, the binding protein having a specific affinity for a substrate; and (b) a DNA fragment coding for the protein molecule to be purified.
Claim 20
Dependent Claim
45 words
The expression vector of claim 9, wherein binding protein is selected from the group consisting of sugar binding protein, receptor protein, amino acid binding protein, sulfate binding protein, vitamin binding protein, metal binding protein, phosphate binding protein, lectin binding protein or nucleic acid binding protein.
Claim 21
Dependent Claim
15 words
The expression vector of claim 17, wherein the sugar binding protein is maltose binding protein.
Claim 22
Dependent Claim
31 words
The expression vector of claim 16, wherein a DNA fragment coding for a linking sequence is interposed between the DNA encoding the binding protein and the DNA ecoding the protein molecule.
Claim 23
Dependent Claim
16 words
The expression vector of claim 19, wherein the linking sequence comprises one or more restriction sites.
Claim 24
Dependent Claim
23 words
The expression vector of claim 19, wherein the linking sequence codes for a polypeptide which is recognized and cleaved by a proteolytic agent.
Claim 25
Dependent Claim
29 words
The expression vector of claim 19, wherein the linking sequence codes for a spacer polypeptide which separates the binding protein from the protein molecule expressed by the expression vector.
Claim 26
Dependent Claim
39 words
A bioreactor comprising: (a) a hybrid polypeptide comprising a protein molecule which interacts with a substance to produce a desired result and a binding protein, or portion thereof, having a specific affinity for a substrate; and (b) the substrate,
Claim 27
Dependent Claim
39 words
A bioreactor comprising: (a) a hybrid polypeptide comprising a protein molecule which interacts with a substance to produce a desired result and a binding protein, or portion thereof, having a specific affinity for a substrate; and (b) the substrate,
Claim 28
Dependent Claim
14 words
wherein the binding protein component of the hybrid polypeptide is bound to the substrate.
Claim 29
Dependent Claim
14 words
wherein the binding protein component of the hybrid polypeptide is bound to the substrate.
Claim 30
Dependent Claim
14 words
The method of claim 23, wherein the substrate is contained within an affinity column.
Claim 31
Independent Claim
89 words
A method of making a bioreactor comprising: a) constructing a DNA expression vector which expresses a hybrid polypeptide in a transformed host cell, the hybrid polypeptide comprising a protein molecule which interacts with a substance to produce a desired result and a binding protein, or portion thereof, having a specific affinity for a substrate. b) introducing the expression vector into an appropriate host cell and expressing the hybrid polypeptide; and c) contacting the hybrid polypeptide produced by the transformed cell with the substrate to which the binding protein binds.
Claim 32
Independent Claim
89 words
A method of making a bioreactor comprising: a) constructing a DNA expression vector which expresses a hybrid polypeptide in a transformed host cell, the hybrid polypeptide comprising a protein molecule which interacts with a substance to produce a desired result and a binding protein, or portion thereof, having a specific affinity for a substrate. b) introducing the expression vector into an appropriate host cell and expressing the hybrid polypeptide; and c) contacting the hybrid polypeptide produced by the transformed cell with the substrate to which the binding protein binds.
Claim 33
Dependent Claim
14 words
The method of claim 25, wherein the substrate is contained within an affinity column.
Claim 34
Independent Claim
54 words
A method of reacting a substance with a protein molecule with which it interacts to produce a desired result comprising; (a)binding a hybrid polypeptide comprising the protein molecule and a binding protein, or portion thereof, having a specific affinity for a substrate to the substrate; and (b)contacting the substance with the bound hybrid polypeptide.
Claim 35
Independent Claim
54 words
A method of reacting a substance with a protein molecule with which it interacts to produce a desired result comprising; (a)binding a hybrid polypeptide comprising the protein molecule and a binding protein, or portion thereof, having a specific affinity for a substrate to the substrate; and (b)contacting the substance with the bound hybrid polypeptide.
Claim 36
Dependent Claim
14 words
The method of claim 27, wherein the substrate is contained within an affinity column.
References Cited (Backward Citations)
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