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Mutant TN5 transposase enzymes and method for their use

A transposase and mutant technology, applied in the direction of transferase, other methods of inserting foreign genetic materials, botanical equipment and methods, etc., can solve the problem of inactivity of wild-type Tnp

Inactive Publication Date: 2002-09-04
WISCONSIN ALUMNI RES FOUND
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  • Summary
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Problems solved by technology

[0007] A major obstacle to understanding how Tn5 transposes is that purified wild-type Tnp has no detectable activity in vitro

Method used

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  • Mutant TN5 transposase enzymes and method for their use
  • Mutant TN5 transposase enzymes and method for their use
  • Mutant TN5 transposase enzymes and method for their use

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Embodiment

[0031] review

[0032] The families of mutants disclosed herein can be obtained in a number of related ways. In the first approach, Applicants generated mutants that reverted transposition activity in vivo to an end-binding sequence that the mutant wild-type transposase did not recognize as a substrate. In the second approach, Applicants introduced directed mutations into the product of the first approach to determine the preferred structure of the mutant transposases of the invention.

[0033] In the first approach, the IE end-binding sequence was mutated to contain an adenine in place of thymine at position 12 ("IE12A"; SEQ ID NO: 5). The thymine-to-adenine change in IE12A disrupts one of the two methylation sites in wild-type IE. Applicants used sPCR, a method of combinatorial random site-directed mutagenesis, to obtain a modified transposase protein capable of restoring transposition activity to polynucleotides flanked by IE12A. Stemmer, W.P., "Rapid protein evolution i...

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Abstract

Disclosed herein are modified Tn5 transposase proteins that prefer the inner end of the transposon Tn5 over the outer end and that can be combined with the outer end in an end-specifically directed transposition method in vivo or in vitro. The combination of transposases that prefer the inner end to the inner end.

Description

[0001] Cross-references to related applications: [0002] This application claims priority to US Provisional Patent Application No. 60 / 146,686, filed August 2, 1999, which is hereby incorporated by reference in its entirety. [0003] Statement Regarding Federally Sponsored Research or Development [0004] This invention was made with federal funding NIH, Grant No. GM 50692. The Federal Government therefore has certain rights in this invention. Background of the invention [0005] Bacterial transposons such as Tn5 evolve intracellularly by maintaining low levels of mobility. This low level of mobility hampers researchers' detailed understanding of the molecular transposition process and its use, such as the development of new diagnostic and therapeutic drug sources, when the transposon is required for survival. Tn5 is a conserved "snipping" transposon of the IS4 family (Rezsohazy, R., Hallet, B., Delcour, J., and Mahillon, J., "The IS4 family of inserted sequences: evidence ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12N9/12C12N9/22C12N15/10C12N15/55C12N15/87C12N15/90
CPCC12N15/102C12N9/22C12N15/90
Inventor W·S·列兹尼科夫T·A·瑙曼
Owner WISCONSIN ALUMNI RES FOUND
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