CN113564191A - Biotin binding protein and affinity column preparation method - Google Patents
Biotin binding protein and affinity column preparation method Download PDFInfo
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- 108700021042 biotin binding protein Proteins 0.000 title abstract description 13
- 102000043871 biotin binding protein Human genes 0.000 title abstract description 13
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 43
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 43
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims abstract description 34
- 229960002685 biotin Drugs 0.000 claims abstract description 17
- 235000020958 biotin Nutrition 0.000 claims abstract description 17
- 239000011616 biotin Substances 0.000 claims abstract description 17
- 238000000034 method Methods 0.000 claims abstract description 10
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- 238000003199 nucleic acid amplification method Methods 0.000 claims description 6
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 claims description 5
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- 125000003275 alpha amino acid group Chemical group 0.000 claims description 3
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- 108010090804 Streptavidin Proteins 0.000 abstract description 16
- 108091006004 biotinylated proteins Proteins 0.000 abstract description 8
- 238000004925 denaturation Methods 0.000 abstract description 2
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- 239000000126 substance Substances 0.000 abstract 1
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- 239000007983 Tris buffer Substances 0.000 description 15
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
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Abstract
In biochemistry, biotin-avidin is at least a million times more avidity than antigen-antibody binding and is the substance found to have the strongest affinity in nature. Therefore, the biotin-avidin system has been widely used in immunodiagnostic techniques. Furthermore, due to the small size of biotin (MW 244.31g/mol), it does not much affect the native function of the protein itself. Therefore, it has the advantages of high affinity, high specificity and high sensitivity, and is widely applied to many fields of biotechnology at present. However, in this system, the biotinylated protein is too strongly bound to streptavidin and is difficult to separate without denaturation, and the present inventors have found that a biotin-binding protein can specifically bind to biotinylated protein and can separate the biotinylated protein and the streptavidin by increasing the concentration of biotin, thereby preparing a corresponding affinity column to purify the biotin protein.
Description
Technical Field
The method belongs to the field of biotechnology, and particularly relates to a biotin-binding protein and a preparation method of an affinity column.
Background
In biochemistry, biotinylated proteins are products of covalent binding of biotin to macromolecular substances such as proteins. The Biotin-Avidin System (BAS) is a new type of amplification System for biological reactions developed in the late 70 s. With the advent of various biotin derivatives, BAS is rapidly becoming widely used in various fields of medicine. Numerous studies in recent years have demonstrated that the biotin-avidin system can bind almost every marker that has been successfully studied. The strong binding with high affinity between biotin and avidin and the multi-stage amplification effect make BAS immune labeling and related tracing analysis more sensitive. It has become a new technology widely used for qualitative and quantitative detection and positioning observation research of trace antigens and antibodies.
Since biotin has a small molecular weight (molecular weight of 244.31), biotinylation reactions are fast, efficient and not easily disturbed. The biotinylated molecules can interact with streptavidin and avidin through biotin, and are not affected by high heat, pH and proteolysis. The binding of biotin to streptavidin or avidin is specific and efficient, and thus the interaction has wide application in many fields of biotechnology. In addition, a plurality of biotinylated molecules can be crosslinked to form a protein of interest to scientific researchers, and the protein can be combined with a plurality of streptavidin, avidin and neutravidin proteins, so that the detection sensitivity of the protein is improved. In addition, there are a number of applications for biotinylated molecules to be developed.
However, the development of this technique is limited by the fact that biotinylated proteins bind streptavidin too strongly and are difficult to separate without denaturation. By understanding, there are also some corresponding affinity column products on the market, but the effect is not satisfactory. By consulting the literatures 2010/0311076 Al 12/2010 Takakura et Al and 2010/0330701 Al 12/2010 Takakura et Al, we found a biotin-binding protein which not only has high specificity and efficiency in binding with biotinylated protein, but also can be well separated by increasing the concentration of biotin, and finally can purify the protein with 100% of biotinylation level by preparing a corresponding affinity column.
Disclosure of Invention
The invention aims to prepare a corresponding biotin-binding protein and use the protein to prepare a corresponding affinity column, so that the protein with the biotinylation level reaching 100 percent can be purified through the column.
The method comprises the following specific steps:
1. the base sequence of the biotin-binding protein is optimized and then synthesized to a corresponding carrier.
2. Transforming the vector in the step 1 into competent cells of escherichia coli, performing expression identification after culture, selecting expressed strains for amplification culture, performing induced expression under optimized conditions, collecting bacteria, and purifying.
3. And (3) dialyzing the protein obtained after the purification in the last step, replacing a corresponding Buffer, chelating with the soaked filler, and performing a series of operations to obtain a corresponding avidin column.
4. And (3) purifying the protein with the biotinylation level less than 100% by using the avidin column obtained in the previous step, detecting the purified protein by using a streptavidin column, and detecting to show that all the protein is combined with the streptavidin column, namely, the prepared avidin column is proved to be effective.
5. According to the technical scheme, the biotin-binding protein is provided, and the prepared avidin column can be used for purifying the protein with the biotinylation level of 100%.
Drawings
FIG. 1 shows biotin-binding protein obtained by purification in the present invention. The protein is soluble protein expressed by escherichia coli.
FIG. 2 shows that the transthyretin of the present invention is obtained by conventional purification, and the protein is soluble protein expressed by Escherichia coli.
FIG. 3 shows that the inventive transthyretin was tested on a streptavidin column and grey-scale analysis gave a biotinylation level of about 60%.
FIG. 4 shows that the transthyretin of the present invention was purified by a corresponding avidin column to obtain a final protein with 100% biotinylation.
FIG. 5 is a verification test of the biotinylation level of transthyretin obtained by purification through an avidin column by a streptavidin column according to the present invention.
Detailed Description
The invention is further illustrated with reference to the following examples:
the biotin-binding protein base sequence is optimized and cloned to a corresponding vector, and then is transformed, expressed, identified, expanded and cultured, and finally chelated with a filler after being collected and purified to finally prepare a corresponding affinity column. The experimental protein used in the invention is transthyretin, the base sequence of the protein is optimized, His and AVI labels are added, the protein is cloned to a corresponding carrier, biotin is added when the protein is induced and expressed in escherichia coli, partial protein can be biotinylated, the transthyretin prepared by conventional purification can only obtain a mixture of biotinylated protein and non-biotinylated protein through streptavidin verification, in order to obtain the protein with the biotinylation level reaching 100%, an avidin column prepared by the biotin-binding protein is used for purifying the mixture, and the prepared protein is verified with the streptavidin column to have the biotinylation level reaching 100%.
1. The amino acid sequence of the biotin-binding protein is optimized by an escherichia coli codon to obtain a corresponding base sequence, and then the base sequence is synthesized into a corresponding vector to finally obtain a corresponding plasmid.
2. The obtained plasmid is transformed into an escherichia coli host cell RosettaPlusS (DE3), a plate is coated, and then a clone is picked on the next day for small induction and induction condition optimization to obtain a strain with better expression quantity and induction condition.
3. Inoculating the obtained strain into a test tube for activation, transferring into a shake flask for amplification culture, adding an inducer IPTG (isopropyl-beta-D-thiogalactoside) until the final concentration of the strain in the large flask is 0.1mM when the OD of the strain in the large flask is 0.6-0.8, and culturing at 18 ℃ for 16h after induction.
4. Centrifuging the induced bacterial liquid at 7000rpm/min for 5min, collecting thalli, resuspending the thalli by using lysate, and then carrying out ultrasonic crushing on ice bath, wherein the lysate comprises the following components: 50mM Tris, PH8.0,500mM NaCl, 12000rpm/min, 20min centrifugation to remove the precipitate.
5. Purifying the supernatant obtained in the step 4, incubating the supernatant with a Ni column for 2h, washing the supernatant, and respectively using 50mM Tris, 500mM NaCl, pH8.0, and 20mM imidazole; 50mM Tris, 500mM NaCl, pH8.0,50 mM imidazole; the Ni column was eluted with 50mM Tris, 500mM NaCl, pH8.0, 250mM imidazole, and the fractions were identified by reducing SDS-PAGE electrophoresis.
6. Taking the components with better purity, combining and dialyzing the components until the working solution: 0.1M NaHCO3，0.5M NaCl, pH8.3, collecting protein, determining protein concentration by BCA method, and performing reductive SDS-PAGE electrophoretic identification.
7. Soaking appropriate amount of filler dry powder in 1mM HCl at 4 deg.C for 2-3h, washing with primary water for 5 times of column volume after soaking, washing with working solution for 5 times of column volume, and chelating with the protein obtained in step 6 at 4 deg.C overnight according to 1ml filler per 8mg protein.
8. After the filler in step 7 is intercepted, washing the filler with primary water for 5 times of the column volume, and then using confining liquid: washing with 0.1M Tris-HCl, pH8.0,5 times of column volume, and finally immersing the filler in the sealing liquid at 4 ℃ for 2-3h, wherein the volume ratio of the filler to the working liquid is 1: 1.
9. after the sealing was completed, the packing was washed 5 column volumes with primary water, with acid: 0.1M sodium acetate buffer, 0.5M NaCl, pH4.0, 5 column volumes washed with primary water, then with alkaline solution: 0.1M Tris-HCl, 0.5M NaCl, pH8.0, washing the packing for 5 column volumes, and finally, washing with a preservative solution: PBS, 50% glycerol, pH7.4, 5 column volumes of packing washed and stored. Thus, the preparation of the affinity column for biotin-binding protein was completed.
10. Optimizing a base sequence of transthyretin, adding His and AVI tags, synthesizing the base sequence into a prokaryotic expression vector, transforming the prokaryotic expression vector into an escherichia coli host cell Rosetta plus system S (DE3), coating a flat plate, selecting clone on the next day, carrying out small-amount induction and optimizing induction conditions to obtain a strain with better expression quantity and induction conditions.
11. Inoculating the obtained strain into a test tube for activation, transferring into a shake flask for amplification culture, adding an inducer IPTG (isopropyl-beta-D-thiogalactoside) until the final concentration of the strain is 0.2mM when the OD of the strain in the large flask is 0.6-0.8, and culturing at 18 ℃ for 16h after induction.
12. Centrifuging the induced bacterial liquid at 7000rpm/min for 5min, collecting thalli, resuspending the thalli by using lysate, and then carrying out ultrasonic crushing on ice bath, wherein the lysate comprises the following components: 50mM Tris, PH8.0,500mM NaCl, 12000rpm/min, 20min centrifugation to remove the precipitate.
13. Purifying the supernatant obtained in the step 12, incubating the supernatant with a Ni column for 2h, washing the supernatant, and then respectively using 50mM Tris, 500mM NaCl, pH8.0, and 20mM imidazole; 50mM Tris, 500mM NaCl, pH8.0,50 mM imidazole; the Ni column was eluted with 50mM Tris, 500mM NaCl, pH8.0, 250mM imidazole, and the fractions were identified by reducing SDS-PAGE electrophoresis.
14. Selecting the components with better purity, dialyzing to 50mM Tris, 500mM NaCl, pH8.0, 10% glycerol, changing the dialysate once after 3h, and collecting the protein after 3 h. Taking 120ul protein, mixing with 120ul PBS uniformly, taking 200ul diluted protein, testing the biotinylation level of the protein by using a 200ul streptavidin column, after sampling for 2 times by gravity, washing the streptavidin column by using 50mM Tris, 500mM NaCl, 10% glycerol and pH8.0 for one time of column volume, one time of column volume and three times of column volume respectively, carrying out reductive SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) identification on each component, and obtaining the biotinylation level of the protein to be 60% by software Image J gray scale analysis.
15. Incubating 1ml transthyretin with biotinylation level of 60% with 200ul avidin column for 1h, washing with 50mM Tris, 500mM NaCl, 10% glycerol, pH8.0, and eluting with 50mM Tris, 500mM NaCl, pH8.0, 5mM biotin, respectively; the avidin column was eluted with 50mM Tris, 500mM NaCl, pH8.0,10 mM biotin, and the fractions were identified by reducing SDS-PAGE electrophoresis.
16. Dialyzing the protein of the eluted component into 50mM Tris, 500mM NaCl, 10% glycerol and pH8.0, changing the dialyzate after 3h, removing biotin, collecting the protein, uniformly mixing 120ul transthyretin and 120ul PBS, performing gravity sample loading by using a 200ul streptavidin column, washing one time of column volume, one time of column volume and three times of column volume by using 50mM Tris, 500mM NaCl, 10% glycerol and pH8.0 respectively, and finally performing reductive SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) identification on each component, wherein the transthyretin is completely combined with the streptavidin column by using software Image J gray scale analysis, namely the biotinylation level reaches 100%.
Claims (6)
1. The preparation of the biotin protein and the avidin column thereof comprises the following contents:
(1) optimizing the amino acid sequence of the corresponding protein and then designing the optimized amino acid sequence to a corresponding vector to synthesize a corresponding plasmid;
(2) transforming, expressing and identifying, carrying out amplification culture, collecting and purifying the plasmid obtained in the step (1) to obtain biotin protein;
(3) chelating the protein obtained in the step (2) with a corresponding filler to obtain a corresponding avidin column.
2. The method according to claim 1, wherein in step (1), the base sequence of the biotin protein is:
ATGCATCACCATCACCATCACGCGATCGCCATGTCAGACGTTCAATCTTCACTCACCGGAACCTGGTACAATGAACTCAACTCCAAGATGGAATTGACTGCAAACAAAGACGGTACTCTCACTGGAAAGTACCTCGCGAAAGTTGGGGATGTCTACGTGCCCTACCCACTCTCTGGTCGCTATAACCTCCAACCCCCCGCGGGACAAGGCGTCGCTCTTGGGTGGGCGGTATCCTGGGAGAACAGTAAAATTCATTCCGCTACGACATGGAGCGGACAGTTCTTCTCTGAGTCGTCTCCAGTGATTCTTACTCAGTGGTTGTTGTCATCGAGCACTGCGCGTGGGGACGTATGGGAATCCACACTTGTGGGGAATGATTCGTTTACAAAGACGGCGCCGACTGAGCAGCAGATCGCTCATGCTCAACTCCATTGTCGCGCACCGAGGTTGAAGTAATAAACGCGT
3. the method of claim 1, wherein in step (1), the full sequence of the corresponding vector bases is:
TGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGA AGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTG ATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGGCCAGGACCCAACGCTGCCCGAGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGCATCACCATCACCATCACGAAAACCTGTATTTTCAGGGCGCGATCGCCGGCGCGCCAGATCTCAAGCTTAACTAGCTAGCGGACCGACGCGTTAAACGCGGCCGCTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT
4. the process as claimed in claim 1, wherein in step (2), the competent E.coli strain used for the transformation is Rosetta (DE3) pLysS.
5. The method according to claim 1, wherein in the step (2), the condition for the scale-up culture is: the culture temperature is as follows: 18 ℃, IPTG induction concentration: 0.1 mM.
6. The method of claim 1, wherein in step (3), the chelating ratio of the filler is: every 8mg biotin protein sequestered 1ml of the filler.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001005977A1 (en) * | 1999-07-15 | 2001-01-25 | Yeda Research And Development Co. Ltd. | Generation of stable dimers with reversible biotin-binding activity |
| US20100022401A1 (en) * | 2004-10-29 | 2010-01-28 | Licentia Oy | Avidin-like proteins from symbiotic bacteria |
| CN102803485A (en) * | 2009-06-24 | 2012-11-28 | 日本烟草产业株式会社 | Modified biotin-conjugated protein |
| US20140378657A1 (en) * | 2011-09-13 | 2014-12-25 | Uti Limited Partnership | Streptavidin mutein exhibiting reversible binding for biotin and streptavidin binding peptide tagged proteins |
-
2021
- 2021-07-26 CN CN202110834759.XA patent/CN113564191A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001005977A1 (en) * | 1999-07-15 | 2001-01-25 | Yeda Research And Development Co. Ltd. | Generation of stable dimers with reversible biotin-binding activity |
| US20100022401A1 (en) * | 2004-10-29 | 2010-01-28 | Licentia Oy | Avidin-like proteins from symbiotic bacteria |
| CN102803485A (en) * | 2009-06-24 | 2012-11-28 | 日本烟草产业株式会社 | Modified biotin-conjugated protein |
| US20140378657A1 (en) * | 2011-09-13 | 2014-12-25 | Uti Limited Partnership | Streptavidin mutein exhibiting reversible binding for biotin and streptavidin binding peptide tagged proteins |
Non-Patent Citations (1)
| Title |
|---|
| YOSHIMITSU TAKAKURA 等: "Tamavidin 2-REV: an engineered tamavidin with reversible biotin-binding capability" * |
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