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1. 发明公开

EP1224294A2 MUTANT LUCIFERASE 有权
标题翻译：突变LUCIFERASE
公开(公告)号：EP1224294A2
公开(公告)日：2002-07-24
申请号：EP00971589.7
申请日：2000-10-26
申请人： The Secretary of State for Defence
发明人： SQUIRRELL, David, James , MURPHY, Melenie, Jane , PRICE, Rachel, Louise , WHITE, Peter, John , WILLEY, Tara, LouiseThe Babraham Institute
IPC分类号： C12N15/53 , C12N9/02 , C12Q1/66
CPC分类号： C12N9/0069 , C12Y113/12007 , G01N2333/90241
摘要： A recombinant protein having luciferase activity and at least 60 % similarity to a wild-type luciferase wherein in the sequence of the enzyme, the amino acid residue corresponding to residue 357 in Photinus pyralis luciferase is mutated as compared to the corresponding wild-type luciferase, such that the luciferase enzyme is able to emit light at a different wavelength as compared to the corresponding wild-type luciferase and/or has enhanced thermostability as compared to the corresponding wild-type luciferase. In general, the residue corresponding to 357 in Photinus pyralis luciferase is changed from an acidic amino acid to a non-acidic amino acid and preferably an uncharged polar amino acid such as tyrosine. Mutant luciferases in accordance with the invention can produce a large (50nm) wavelength shift in emitted light and have good thermostability. The resultant colour shift can be reversed by addition of coenzyme A. These properties make the mutant particularly useful in a variety of assays.
摘要翻译：一种具有萤光素酶活性并与野生型萤光素酶有至少60％相似性的重组蛋白，其中在该酶的序列中，与相应的野生型萤光素酶相比，对应于萤火虫萤光素酶中的残基357的氨基酸残基发生突变，使得萤光素酶能够与相应的野生型萤光素酶相比在不同的波长发光和/或与相应的野生型萤光素酶相比具有增强的热稳定性。一般来说，相当于紫堇属荧光素酶357的残基由酸性氨基酸变为非酸性氨基酸，优选为不带电的极性氨基酸如酪氨酸。根据本发明的突变萤光素酶可以在发射光中产生大的（50nm）波长偏移并且具有良好的热稳定性。所产生的颜色变化可以通过添加辅酶A来逆转。这些性质使突变体在多种测定中特别有用。

2. 发明授权

EP1224294B1 MUTANT LUCIFERASE 有权
标题翻译： MUTANTENLUCIFERASE
公开(公告)号：EP1224294B1
公开(公告)日：2007-10-03
申请号：EP00971589.7
申请日：2000-10-26
申请人： THE SECRETARY OF STATE FOR DEFENCE
发明人： SQUIRRELL, David, James , MURPHY, Melenie, Jane , PRICE, Rachel, Louise , WHITE, Peter, John , WILLEY, Tara, Louise Department of Bio Chemistry
IPC分类号： C12N15/53 , C12N9/02 , C12Q1/66
CPC分类号： C12N9/0069 , C12Y113/12007 , G01N2333/90241
摘要： A recombinant protein having luciferase activity and at least 60 % similarity to a wild-type luciferase wherein in the sequence of the enzyme, the amino acid residue corresponding to residue 357 in Photinus pyralis luciferase is mutated as compared to the corresponding wild-type luciferase, such that the luciferase enzyme is able to emit light at a different wavelength as compared to the corresponding wild-type luciferase and/or has enhanced thermostability as compared to the corresponding wild-type luciferase. In general, the residue corresponding to 357 in Photinus pyralis luciferase is changed from an acidic amino acid to a non-acidic amino acid and preferably an uncharged polar amino acid such as tyrosine. Mutant luciferases in accordance with the invention can produce a large (50nm) wavelength shift in emitted light and have good thermostability. The resultant colour shift can be reversed by addition of coenzyme A. These properties make the mutant particularly useful in a variety of assays.

3. 发明授权

EP0778945B1 LUCIFERASE LABELLING METHOD 失效
标题翻译：荧光素酶标记方法
公开(公告)号：EP0778945B1
公开(公告)日：2001-10-04
申请号：EP95929977.7
申请日：1995-08-30
申请人： The Secretary of State for Defence
发明人： SQUIRRELL, David, James , MURPHY, Melenie, Jane
IPC分类号： G01N33/535 , C12Q1/68 , G01N33/58 , G01N33/543 , G01N21/84
CPC分类号： C12Q1/6825 , G01N33/535
摘要： A method is provided for conjugating luciferase to a chemical entity, particularly to a specific binding agent such as an antibody, antigen or a nucleic acid, and more particularly an antibody, comprising (a) mixing the luciferase with one or more of D-luciferin, magnesium ions and adenosine triphosphate and (b) performing a covalent coupling reaction between the luciferase and the binding reagent using a covalent coupling reagent wherein the amount of D-luciferin, magnesium ions and/or adenosine triphosphate is sufficient to protect the luciferase activity against inhibition by the covalent coupling reagent. Preferably the step (a) is carried out by mixing the luciferase with its substrates in solution and preferably both magnesium and adenosine triphosphate are present as magnesium adenosine triphosphate (Mg ATP), optionally together with D-luciferin. In a second aspect of the invention there is provided a labelled chemical entity comprising a chemical entity conjugated to active luciferase as provided by the method of the present invention. Preferably the chemical entity is a specific binding agent suitable for use in a specific binding assay, preferably being an antibody, antigen or nucleic acid. When the binding agent is a nucleic acid, it is preferably an oligonucleotide, but may be a polynucleotide or a nucleoside, and may be used as a hybridisation probe or a chain extension primer, e.g. a PCR primer. Most advantageously the entity is an antibody as previous attempts to couple antibodies to luciferase have resulted in inactivity. Test kits are further provided.

4. 发明公开

EP0778945A2 LUCIFERASE LABELLING METHOD 失效
标题翻译：荧光素酶标记方法
公开(公告)号：EP0778945A2
公开(公告)日：1997-06-18
申请号：EP95929977.0
申请日：1995-08-30
申请人： THE SECRETARY OF STATE FOR DEFENCE
发明人： SQUIRRELL, David, James , MURPHY, Melenie, Jane
IPC分类号： C12Q1 , G01N33
CPC分类号： C12Q1/6825 , G01N33/535
摘要： A method is provided for conjugating luciferase to a chemical entity, particularly to a specific binding agent such as an antibody, antigen or a nucleic acid, and more particularly an antibody, comprising (a) mixing the luciferase with one or more of D-luciferin, magnesium ions and adenosine triphosphate and (b) performing a covalent coupling reaction between the luciferase and the binding reagent using a covalent coupling reagent wherein the amount of D-luciferin, magnesium ions and/or adenosine triphosphate is sufficient to protect the luciferase activity against inhibition by the covalent coupling reagent. Preferably the step (a) is carried out by mixing the luciferase with its substrates in solution and preferably both magnesium and adenosine triphosphate are present as magnesium adenosine triphosphate (Mg2+ATP), optionally together with D-luciferin. In a second aspect of the invention there is provided a labelled chemical entity comprising a chemical entity conjugated to active luciferase as provided by the method of the present invention. Preferably the chemical entity is a specific binding agent suitable for use in a specific binding assay, preferably being an antibody, antigen or nucleic acid. When the binding agent is a nucleic acid, it is preferably an oligonucleotide, but may be a polynucleotide or a nucleoside, and may be used as a hybridisation probe or a chain extension primer, e.g. a PCR primer. Most advantageously the entity is an antibody as previous attempts to couple antibodies to luciferase have resulted in inactivity. Test kits are further provided.

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