Methods and compositions for the production, identification and purification of fusion proteins
First Claim
1. An isolated nucleic acid molecule comprising:
- (a) one or more recombination sites; and
(b) one or more nucleic acid sequences which encode an amino acid sequence tag.
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Abstract
The present invention provides compositions and methods for producing fusion proteins that comprise an amino acid sequence tag. The amino acid sequence tag may be an amino acid sequence that is capable of being post-translationally modified; for example, the amino acid sequence may be an amino acid sequence that is capable of being biotinylated. The amino acid sequence tag may also be an amino acid sequence that is recognized by an antibody (or fragment thereof) or other specific interacting reagent. The invention includes isolated nucleic acid molecules comprising one or more nucleic acid sequences which encode an amino acid sequence tag. The nucleic acid molecules of the invention may also comprise one or more recombination sites and/or one or more topoisomerase recognition sites and/or one or more topoisomerases. The nucleic acid molecules of the invention can be used in recombinational cloning and/or topoisomerase-mediated cloning methods in order to produce polynucleotide constructs which encode fusion proteins that comprise an amino acid sequence tag. Also provided are host cells, kits and compositions comprising the nucleic acid molecules of the invention.
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Citations
156 Claims
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1. An isolated nucleic acid molecule comprising:
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(a) one or more recombination sites; and
(b) one or more nucleic acid sequences which encode an amino acid sequence tag. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 130, 131)
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19. A method of producing a polynucleotide construct that encodes a fusion protein that comprises an amino acid sequence tag, said method comprising:
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(a) obtaining a first nucleic acid molecule comprising a nucleotide sequence of interest flanked by at least a first and at least a second recombination sites that do not recombine with each other;
(b) obtaining a second nucleic acid molecule comprising;
(i) at least a third and fourth recombination sites that do not recombine with each other; and
(ii) one or more nucleic acid sequences which encode an amino acid sequence tag; and
(c) contacting said first nucleic acid molecule with said second nucleic acid molecule under conditions favoring recombination between said first and third and between said second and fourth recombination sites, thereby producing a product polynucleotide construct;
wherein said product polynucleotide construct encodes a fusion protein comprising;
(i) said amino acid sequence tag; and
(ii) the amino acid sequence encoded by said nucleotide acid sequence of interest. - View Dependent Claims (20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 136, 139, 140, 141, 142, 143, 144)
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39. An isolated nucleic acid molecule comprising:
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(a) one or more topoisomerase recognition sites and/or one or more topoisomerases; and
(b) one or more nucleic acid sequences which encode an amino acid sequence tag. - View Dependent Claims (40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 132, 133)
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60. A method of producing a polynucleotide construct that encodes a fusion protein that comprises an amino acid sequence tag, said method comprising:
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(a) obtaining a first nucleic acid molecule comprising a nucleotide sequence of interest;
(b) obtaining a second nucleic acid molecule comprising at least two topoisomerase recognition sites, at least one topoisomerase, and at least one nucleic acid sequence which encodes an amino acid sequence tag;
(c) mixing said first nucleic acid molecule with said second nucleic acid molecule; and
(d) incubating said mixture under conditions such that said first nucleic acid molecule is inserted into said second nucleic acid molecule between said at least two topoisomerase recognition sites, thereby producing a product polynucleotide construct;
wherein said product polynucleotide construct encodes a fusion protein comprising;
(i) said amino acid sequence tag; and
(ii) the amino acid sequence encoded by said nucleotide sequence of interest. - View Dependent Claims (61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 137, 145, 146, 147, 148, 149, 150)
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80. An isolated nucleic acid molecule comprising:
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(a) one or more recombination sites;
(b) one or more topoisomerase recognition sites and/or one or more topoisomerases; and
(c) one or more nucleic acid sequences which encode an amino acid sequence tag. - View Dependent Claims (81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 134, 135)
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104. A method of producing a polynucleotide construct that encodes a fusion protein that comprises an amino acid sequence tag, said method comprising:
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(a) obtaining a first nucleic acid molecule comprising a nucleotide sequence of interest;
(b) obtaining a second nucleic acid molecule comprising (i) at least a first topoisomerase recognition site flanked by (ii) at least a first recombination site, and (iii) at least a second topoisomerase recognition site flanked by (iv) at least a second recombination site, wherein said first and second recombination sites do not recombine with each other, and (v) at least one topoisomerase;
(c) obtaining a third nucleic acid molecule comprising;
(i) at least a third and fourth recombination sites that do not recombine with each other; and
(ii) one or more nucleic acid sequences which encode an amino acid sequence tag;
(d) mixing said first nucleic acid molecule with said second nucleic acid molecule;
(e) incubating said mixture under conditions such that said first nucleic acid molecule is inserted into said second nucleic acid molecule between said at least two topoisomerase recognition sites, thereby producing a first product polynucleotide construct;
(f) contacting said first product polynucleotide construct with said third nucleic acid molecule under conditions favoring recombination between said first and third and between said second and fourth recombination sites, thereby producing a second product polynucleotide construct;
wherein said second product polynucleotide construct encodes a fusion protein comprising;
(i) said amino acid sequence tag; and
(ii) the amino acid sequence encoded by said nucleotide sequence of interest. - View Dependent Claims (105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 138, 151, 152, 153, 154, 155, 156)
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129. A vector selected from the group consisting of pET104-DEST, pET 104/GW/lacZ, pET 104/D-TOPO, pET 104/D/lacZ, pcDNA6/Biotag™
- -DEST, pcDNA6/Biotag™
-GW/lacZ, pcDNA6/Biotag™
/D-TOPO, pcDNA6/Biotag™
/lacZ, pMT/Biotag™
-DEST, and pMT/Biotag™
/GW-lacZ.
- -DEST, pcDNA6/Biotag™
Specification