CN1367840A - Mutant TN5 transposase enzymes and method for their use - Google Patents

Abstract

This article discloses a modified Tn5 transposase protein that prefers the inner terminus of transposon Tn5 to the outer terminus, and can be used in combination with transposases that prefer the inner terminus to the outer terminus in in vivo or in vitro terminal-specific guided transposition methods.

Description

Mutant TN5 transposases and methods of use thereof

Cross-references to related applications:

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.

Statement Regarding Federally Sponsored Research or Development

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

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 for a conserved transposase motif ", Mol Microbiol. 9: 1283-1295 (1993)), which encodes a 53 kD transposase protein (Tnp) responsible for its movement. The amino acid and nucleic acid sequences of wild-type Tn5 transposase are known. Ahmed, A. and Podemski, L. "The Corrected Sequence of T5" Gene 154(1), 129-130 (1995), which is hereby incorporated by reference in its entirety. Attached herein is the nucleic acid sequence encoding wild-type Tn5 transposase, namely SEQ ID NO: 1. Also attached is the polypeptide sequence encoded by SEQ ID NO: 1 corresponding to the wild-type Tn5 transposase, i.e. SEQ ID NO: 2.

The Tnp protein facilitates the movement of this entire element by first binding to one of two 19bp specific binding sequences (called outer ends) (OE; SEQ ID NO: 3), which then form a nucleoprotein structure (called a synapse) , blunt cleavage at each end, ligation to target DNA, and subsequent strand transfer (Reznikoff, WS, Bhasin, A., Davies, DR, Goryshin, IY, Mahnke, LA, Naumann, T., Rayment, I., Steiniger- White, M, and Twining, SS, "Tn5: A Molecular Window to Transposition", Biochem. Biophys. Res. Commun. 266:729-34 (1999)). The Tn5 transposase can also facilitate the movement of single inserted sequences by combining the OE and inner end (IE; SEQ ID NO: 4) sequences. IE was also 19 bp in length, identical to OE in 12 of 19 positions (Fig. 1). In vivo, the Tn5 transposase showed a clear preference for E. coli OE. By the presence of 2 dam methylation sites (GATC palindromic sequence), i.e. the addition of 4 methyl groups at each inner end sequence (IE ^ME ; also shown in SEQ ID NO: 4, no methylation shown), Inhibition of transposase recognition and binding to IE in Escherichia coli (Yin, JCPKrebs, MP, and Reznikoff, WS, "Effect of dam methylation on Tn5 transposition", J.Mol.Biol., 199:35 -45 (1988), which is hereby incorporated by reference in its entirety). This methylation reduces transposition by reducing protein-DAN initial recognition (Jilk, RA, York, D., and Reznikoff, WS, "Composition of the outer terminus of the transposase Tn5", J. Bacteriol. 178: 1671-1679 (1996)).

A major obstacle to understanding how Tn5 transposes is that purified wild-type Tnp has no detectable activity in vitro. Recently, a double mutant hyperactive form of the transposase ("Tnp EK/LP") was developed that facilitates all steps of the in vitro transposition reaction. The Tnp EK/LP protein differs from wild-type Tn5 Tnp at positions 54 (Glu to Lys) and 372 (Leu to Pro), and a non-essential but beneficial change at position 56 prevents the so-called arrester protein produce. This modified hyperactive Tnp protein retains a clear preference for the OE-terminus (or OE-like) of the wild-type Tn5 transposase. Tnp EK/LP clarifies many aspects of Tn5 transposition that were previously underexplored in vivo.

In vitro, polynucleotide transposition is a powerful tool for introducing random or targeted mutations into the genome. Useful in vitro transposition systems based on Tn5 transposition are disclosed in US Patent No. 5,925,545 and International Publication No. WO 00/17343, both of which are incorporated herein by reference in their entirety.

Tnp proteins with the ability to recognize both IE and OE and a preference for binding IE are needed to allow us to direct the transposition of nucleic acids and facilitate more complex (compared to using Tnp with a single specificity for OE) transposition and gene Engineering strategies (described in the aforementioned patents and applications). Tnp with increased preference for IE ^ME is also desired, since DNA methylation in common dam+ bacterial hosts inhibits binding of existing Tn5 transposases and reduces the ability of existing transposases to facilitate movement of IE-defined transposons.

Summary of the invention

The present invention can be summarized as disclosed herein as a modified transposase protein that favors the promotion of flanking wild-type Tn5 transposase inner ends (IE) over outer ends (OE) relative to wild-type Tn5 Tnp Transposition of the target sequence, whether or not the IE sequence is methylated.

In a related aspect, the invention can also be generalized as disclosed herein relative to wild-type Tn5 Tnp, a modified transposase that prefers IE relative to OE and is hyperactive in terms of transposition frequency .

On the other hand, the present invention can also be generalized as disclosed herein relative to wild-type Tn5 Tnp, a modified transposase that prefers IE over OE and is able to perform well even when the IE sequence is methylated. High levels catalyze transposition. In contrast, wild-type Tn5 transposase cannot efficiently recognize methylated IE sequences.

In another related aspect, the invention can be generalized as transposases of the invention comprising mutations relative to the wild-type Tn5 transposase that either (1) alter the binding of the transposase to DNA ends or (2) increase Transposition or (3) has both functions. Such mutations may be end-sequence-specific (as shown in the example for altering DNA binding) or non-specific (as shown in the example for increasing transposition).

In another aspect, the present invention can be summarized as that the transposase of the present invention (1) has a stronger preference for IE than OE and (2) differs from wild-type Tnp in at least one of the following amino acids: amino acid 58, amino acid 334 and amino acid 372 bits.

On the other hand, the present invention can be summarized as the difference between the transposase of the present invention and wild-type Tnp in that it contains at least one of the following mutations: amino acid 58 glutamic acid is mutated to valine, amino acid 334 glutamic acid is mutated to Lysine and amino acid 372 leucine were mutated to glutamic acid.

Another aspect of the invention can be summarized that by reducing the preference for OE, the transposases of the invention also exhibit a stronger preference for IE. Mutations at amino acid 8 of the wild-type transposase reduce the transposase's preference for OE and thereby increase its apparent preference for IE. This modification is accomplished by mutating arginine to cysteine.

The transposase proteins of the invention can facilitate the transposition of more IE-flanked target sequences than wild-type Tn5 transposase in vitro or in vivo. Suitable methods for assaying transposase activity in vitro are disclosed herein and in US Patent No. 5,925,545, which is hereby incorporated by reference in its entirety. Methods suitable for assaying transposase activity in vivo are disclosed herein.

The difference between the modified Tn5 Tnp of the present invention and the wild-type Tn5 Tnp lies in a change in at least one amino acid position, which is selected from: (1) a change in amino acid 58, which reduces or eliminates Tnp and methylated DNA residues Negative interactions between bases and (2) a change at amino acid 334, which alters DNA binding. In addition to the changes described herein, the modified Tnp of the present invention also contains a change at position 56 (eg, from Met to Ala), which prevents the production of so-called repressor proteins that interfere with transposition. In addition, mutant Tn5 transposase proteins may also contain mutations other than those described above. Other mutations relative to wild-type Tn5 Tnp are disclosed below. The role of each mutation is disclosed herein below, and it can be seen that the applicants of the present invention have identified amino acid residues of the protein that have a direct impact on function, and that the same position Other modifications on may have equivalent, stronger or weaker effects. The amino acid sequence of the wild-type Tn5 transposase is listed in the sequence listing. Exemplary changes relative to wild-type transposases are listed herein.

The present invention can also be generalized that the enzyme disclosed in the present invention contributes to a simple in vitro system and method, when any transposable element DNA is flanked by IE ends opposite to each other, this system and method can transfer the donor This transposable element of DNA is introduced into the target DNA. Some other requirements for donor DNA or target DNA are also envisioned. It is believed that there are very few, if any, Tn5s (preferring insertion sites), so this system can be used to introduce desired sequences randomly into target DNA. Therefore, it is believed that using the modified transposase described herein and simple donor DNA, the system and method can be broadly applied to introduce changes in any target DNA regardless of its nucleotide sequence. It can therefore be used to solve many problems of interest to those skilled in the field of molecular biology.

Finally, the changes described herein are disclosed as changes at the protein level, but it is within the ability of those skilled in the art to modify polynucleotides encoding Tn5 transposase proteins to encode modified proteins of the invention. Those skilled in the art also appreciate the degeneracy of the genetic code and that multiple codes can direct the production of an amino acid residue.

The present invention will be more fully understood upon consideration of the following detailed description and accompanying drawings.

Brief description of the drawings

Figure 1 shows the structure of Tn5 transposase and the nucleotide sequences of Tn5 outer end (OE), inner end (IE), methylated inner end (IE ^ME ) and modified IE (IE12A).

Figure 2 is a schematic representation of papillization assays observing the molecular basis of transposition in vivo.

Figure 3A shows the positions of mutations observed in 4 consecutive cycles (A, B, C, D) of mutagenesis/recombination. Figure 3B shows a graph of the in vivo transposition profile for each of the mutations of Figure 3A.

Figure 4 shows the relative preference of mutant transposases obtained in successive cycles of mutagenesis/recombination of OE and IE with E. coli dam- strain.

Figure 5A shows plasmids suitable for in vitro transposition methods. Figure 5B shows transposition products obtained by in vitro transposition catalyzed by the mutant transposase of the present invention. Figure 5C is a further characterization of the lane 2 product of Figure 5B.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Random mutagenesis studies of the Tnp gene have shown that mutations in the protein that increase transposition rates can be isolated (Krebs and Reznikoff, 1988; DeLong, A., and Syvanen, M., "Transaction of the Tn5 Transposase Mutation ", P.N.A.S.U.S.A. 88:6072-6 (1991); Wiegand, T.W. and Reznikoff, W.S., "Characterization of two highly transposable Tn5 mutations", J.Bacteriol.174:1229-1239 (1992); Weinreich, M.D.Gasch, A., & Reznikoff, W.S., "Evidence for unproductive multimerization leading to cis-preference for the Tn5 transposase," Genes. Dev. 8:2363-2374 (1994); Zhou, M and Reznikoff, W.S., "Changing Tn5 Mutants with DNA Binding Specificity" J. Mol. Biol. 271:362-73, (1997) which are hereby incorporated by reference in their entirety). This makes transposases unique compared to most of the enzymatic proteins that exhibit maximal activity in vivo. This is likely because the high rate of transposition is detrimental to the survival of the transposon, so the 'optimum' level exhibited by the transposase is much lower than the maximum level it can achieve.

Applicants have isolated a series of related mutant transposase proteins that differ from the wild-type Tn5 transposase and that display a preference for inner ends (IEs) and sometimes methylation in the transposition system The inner end (IE ^ME ) of α is more preferred than the outer end (OE), which is not methylated due to the lack of methylation sites. A mutant transposase end preference can be characterized by either (1) the in vivo transposition frequency observed when it is used in a system with OE or IE ^ME termini flanking the target polynucleotide, or (2) when It is the in vivo transposition frequency observed for a paired system of target nucleotides flanked by OE and IE ^ME ends. Although Applicants have exemplified a number of transposase proteins that differ from wild-type Tn5 by 1, 4, 5 and 7 mutations, the effect of each particular mutation can be reasonably predicted from their analysis.

Example

review

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.

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 in vitro by DNA shuttling", Nature 370:389-391 (1994) and Stemmer, W.P., "DNA shuttling by random fragmentation and reassembly: in vitro recombination for molecular evolution", Proc. sPCR was developed and described by Natl. Acad. Sci. U.S.A. 91: 10747-10751 (1994), both of which are hereby incorporated by reference in their entirety. Briefly, DNA is manipulated in vitro in the sPCR method to introduce point mutations and allow them to recombine randomly in a population of mutated sequences. The mutated gene can be cloned into a plasmid (optionally selecting for increased in vivo activity). Clones with the desired phenotype are then used as substrates for subsequent rounds of mutagenesis/recombination and selected for further improved phenotypes.

Screening (alternative selection) (analyzing an appropriate number of colonies (~10 ⁴ ) per round) can be coupled with sPCR methods (Crameri, A., Whitehorn, EA, and Stemmer, WP, "Improved green Fluorescent proteins", Nat. Biotechnol. 14:315-319 (1996) and Zhang, JH, Dawes, G., and Stemmer, WP, "Fucosidases evolved directly from galactosidases by DNA shuttling and screening" , Proc.Natl.Acad.Sci.USA94, 4505-4509 (1997), both of which are incorporated herein as a reference.Use the papillation (papillation) test described in the following examples as the transposase mutant (capable of Screening method for reverting transposase activity to polynucleotides flanked by IE12A). The papillary assay is a modification of the assay described by Krebs, MP, and Reznikoff, WS, "Produces lacZ translational fusions resulting in transposition Use of Mutant Tn5 Derivatives", Gene 63:277-85 (1998), which is incorporated herein by reference in its entirety. In the papillary assay, in-frame productive transposition into an actively expressed gene results in the formation of β-Gal fusion protein. These cells turn blue (due to the presence of X-gal) and grow at an increased rate in the colony (lactose utilization). The rate of these papillae formation can be compared to the transposition promoted by the mutant protein rate compared.

Applicants determined that these mutant transposases (identified using the first approach) catalyze the transduction of polynucleotides flanked by either wild-type OE or wild-type IE (i.e. unmethylated nucleic acid) in dam- strains in vivo. seat capacity. Among the mutants identified using this method, Applicants identified a near-wild-type mutant transposase ("Tnp sC7") that retained reactivity with polynucleotides flanking OE ("Tnp sC7"), but had very High reactivity with polynucleotides flanking IE, and when tested in dam+ strains (i.e. nucleotides methylated, hereinafter referred to as "IE ^ME "), with polynucleotides flanking IE More responsive. Tnp sC7 contains seven mutations relative to the wild-type transposase.

In the second approach, a related mutant transposase ("Tnp sC7v2.0") containing 4 of the 7 mutations in Tnp sC7 was subsequently assayed and still exhibited a high IE ^ME :OE reactivity ratio . Both Tnp sC7 and Tnp sC7v2.0 contain a mutation (R8C) that inhibits OE-associated activity, two mutations that specifically increase IE ^ME -associated activity (E58V, E344K), and a mutation that increases IE ^ME or OE flanked by Mutation for polynucleotide transposition (L372Q).

Mutant Tnp

Figure 2 shows a modified papillary assay for screening for productive transposition. In the first plasmid used ("pRZ9904(IE12A/IE12A)"), a pair of inverted IE12A ends flanked a polynucleotide containing the lacZ gene, but which lacked the promoter and translation initiation site. The second plasmid ("pRZ9905") used in this assay encodes a transposase capable of mobilizing lacZ-containing polynucleotides. It is not difficult for those skilled in the art to construct plasmids with these properties. Materials and methods are listed in detail below.

Five random mutants that suppress terminal sequence mutations and generate papillae after the first round of mutagenesis/recombination cycles. An equal amount of plasmid encoding one of these transposases was then used as the initial substrate for a second round of mutagenesis/recombination. After the second round, the mutated transposase gene was cloned into vector DNA and screened a second time for transposition reactivity with IE12A-defined polynucleotides by papillization. From this second round, a total of 6 active mutants were isolated. A third round of mutagenesis/recombination was then performed and screened for reactivity with the mutant end sequences. Hundreds of colonies in this round were positive for papillary. Of these, 7 were significantly more active than the others, and they were isolated and used as templates for the fourth round. In the fourth round of screening, hundreds of transposed colonies were visually observed during the screening process. None of them had the same activity as the most active mutant (Tnp sC6) isolated from the third round of mutagenesis/recombination.

The most active isolates from each round were sequenced and tested for transposition reactivity to IE12A-defined transposons using a quantitative papillary assay (see Figures 3A and 3B). Figure 3A shows the mutations relative to wild-type Tnp: Tnp sA5 (best papillae in the first round), sB2 (best papillae in the second round), sC6 (best papillae in the third round ) and sD5 (best papillae for round four). Figure 3B shows the transposition activity of the four isolates in an in vivo papillary assay. Tnp WT was also tested but failed to promote a detectable transposition. Mutation Q81H was the only mutation that distinguished the most active mutant sC6 (isolated in the third round) from the significantly less active mutant sB2 (isolated in the second round). Another mutation in the third round, Tnp sC7, was similar to the fourth round isolate, sD5, except that it also had two other mutations (D217A and E344K). The reactivity of Tnp sC7 with the IE12A defined transposon was similar to that of the fourth round isolate sD5 (data not shown).

Mutant Tnp is able to promote transposition of OE and IE-defined transposons

The mutant Tnp described above are of primary interest because they can restore transposition activity to the transposon flanked by the IE12A terminus (which is inactive in wild-type Tnp). Although these transposase mutants had increased function at these transposons, the transposability did not reach the level of activity required in vitro. In fact, the in vivo reactivity of Tnp sC6 with the IE12A terminus was only reverted to a level similar to that of Tnp WT and OE-defined transposon responses (data not shown).

Interestingly, however, the majority of isolated mutants were highly reactive to transposons defined by at least one native end sequence (IE or OE). By Goryshin, I.Y.Kil, Y.V., and Reznikoff, W.S., "The length of DNA and the torsional constraint on IS50 transposition", Proc Natl Acad Sci U SA91: 10834-10838 (1994) (herein incorporated by reference in its entirety) In mating experiments, the preference for these transpositions was determined with dam- (DNA unmethylated) strains.

Figure 4 shows the in vivo transposition reactivity of 25 mutant Tnp with IE and OE defined transposons in a mating assay of the dam- strain, normalized to the reactivity of wild-type Tnp (6.5 × 10 ^-5 Normalized to 1). In this assay in the dam environment, Tnp WT exhibited roughly equivalent levels of activity regardless of whether the substrate polynucleotide was flanked by IE or OE. On the other hand, many mutants showed higher reactivity to IE than to OE. This is not surprising since these mutants were screened with IE12A, which differs from IE by only 1 nucleotide. In contrast, OE differs from IE12A by 6 nucleotides. One mutant in particular, Tnp sC7, exhibited a very interesting phenotype. It showed a markedly high reactivity with IE transposons, while showing little change in the frequency of movement of OE transposons. The ability to discriminate between IE and OE is important because it is beneficial for multipart transposition using IE and OE ends separately, which can direct reactions one way or the other by providing transposases with a preference for IE or OE. A preference about 5 times higher for IE than OE might be appropriate, and certainly a preference for IE 10 times higher would be more appropriate. A 20-fold higher preference is best. Figure 4 shows that such a mutant transposase can be obtained by a person skilled in the art using the methods described herein. Specifically, mutants sB1, sC6, sC7, sD1 and sD3 are examples of such mutants.

But more remarkably, the transposition activity of Tnp sC7 is not only uninhibited by methylated IE (which reduces Tnp WT levels by about 102), but also prefers IE ^ME transposons flanked by IE, as mated in Table 1 As shown in the results of the experiment, it was shown that the transposition frequency of OE by both Tnp sC7 and Tnp WT was reduced in the dam+ strain. Since Tnp binding to OE is not affected by dam methylation, this difference only reflects a difference in transposition reactivity between dam+ and dam strains. In addition to this decrease, the Tnp sC7-promoted IE-defined transposition rate was even higher than in the dam+ strain due to the presence of methylation at both ends. In contrast, methylation of the terminal sequences inhibits recognition by wild-type transposases. The following attention was directed to Tnp sC7 based on its ability to discriminate between IE and OE and its insensitivity to IE methylation.

Table 1 In vivo transposition rate of Tnp WT and Tnp sC

JCM101(dam-) RZ212(dam+)

IE frequency OE frequency IE/OE IE ^ME frequency OE frequency IE ^ME /OETnp WT 6.5×10 ^-5 6.5× ^{10 -5} 1.0 1.0×10 ^-8 3.1×10 ^-6 3.2×10 ^-3 Tnp sC7 1.8×10 ^-3 3.7× ^10-5 50 2.6× ^10-3 3.3× ^10-6 794

The role of each mutation of Tnp sc7 in transposition activity

To understand how each mutation affects the activity of the protein and to maximize the activity of the protein and its ability to discriminate between IE ^ME and OE, Applicants used the information obtained from sC7 to strategically generate more mutant transposases, As mentioned above they contain 7 mutations relative to wild type transposase. Since comprehensive testing of all possible combinations of these 7 mutations is too cumbersome (128 possible combinations), these 7 mutations were divided into 2 categories for genetic engineering. In each mutation of the first class, a unique mutation from sC7 was engineered to revert to the wild type at that position. This group of mutant transposases, called "minus one," included all possible mutants containing 6 of the 7 mutations. In the second class of individual mutants, the wild-type transposase was engineered to contain exactly one of the seven mutations from sC7. This group of mutant transposases, called "plus one," includes all possible mutants that contain only 1 of the 7 mutations. All these mutant proteins were analyzed for in vivo transposition reactivity with IE ^ME and OE by mating assays. Table 2 lists the results of this analysis.

Table 2 Results of the 'minus one/plus one' analysis

'minus one IE ^ME normalize' minus one OE normalize 'add one IE ^ME normalize 'plus one OE normalize

"Frequency One" Frequency "Frequency One" Frequency Wild type 1.0×10 ^-8 1.0 3.1×10 ^-6 1.0sC7 2.6×10 ^-3 1.0 3.3×10 ^-5 1.0RC8C 3.1×10 ^{- 3} 1.2 6.0×10 ^-6 1.8 ---- ---- 4.0×10 ^-7 0.1E58V 1.8×10 ^-6 6.9×10 ^-4 1.1×10 ^-6 0.3 4.0×10 ^-4 4×10 ⁴ 1.4× ^10-6 0.5A157T 3.1× ^10-3 1.2 4× ^10-6 1.2 ---- ---- 6.6× ^10-7 0.2T171S 2.8× ^10-3 1.1 2.6× ^10-6 0.8 5.6× ^10-9 0.6 1.8×10 ^-6 0.6D217A 3.7×10 ^-3 1.3 4.5×10 ^-6 1.4 ---- ---- 3.4×10 ^-6 1.1E344K 5.5×10 ^-4 0.2 1.7×10 ^-5 5.2 4.3×10 ^{- 8} 4.3 6.8× ^10-7 0.2L372Q 2.6× ^10-4 0.1 2.8× ^10-7 0.1 8.9× ^10-8 8.9 1.1× ^10-5 3.5

a. 'Minus one' contains all mutations present in Tnp sC7 except the indicated sites. For example R8C 'minus one' contains a wild-type arginine at position 8.

b. 'Plus one' is Tnp WT, except that the noted amino acids are mutated to residues in Tnp sC7. For example R8C 'plus one' contains cysteine at amino acid position 8.

Sequence-specific mutations. Mutation E58V had the deepest effect on the activity of Tnp sC7 among all mutations. This mutation in the wild-type background (E58V' plus one') increased IE ^ME -associated transposition 40,000-fold, whereas removal of the mutation from Tnp sC7 (E58V' minus one') decreased overall activity by more than 1,000 times. This mutation has relatively minimal effect on OE-associated activity.

Mutation E344K exhibited this similar, though much weaker, sequence-specific effect on activity. When it was removed from Tnp sC7 (E344K 'minus one'), the IE ^ME -associated activity was reduced 5-fold while the OE-associated activity was stimulated about 5-fold. This result is mirrored in the 'plus one' data, as E344K stimulates IE ^ME -associated activity 4-fold and reduces OE-associated activity by about 5-fold in the wild type background.

Non-sequence-specific-mutation. Mutation L372Q strongly stimulates Tnp sC7 reactivity with IE ^ME . When it was removed from sC7, the associated activities of both IE ^ME and OE were reduced to the same extent. When added to a wild-type transposase, its 'plus one' phenotype stimulates the activity of both substrates.

other mutations. Of the remaining 4 mutations, the R8C mutation is the most interesting. When this mutation was added to the wild-type transposase, OE-associated transposition was reduced nearly 10-fold. When this mutation was removed from sC7 OE, the associated activity increased about 2-fold. In methylated hosts, its removal from sC7 had little effect on IE ^ME -associated activity, which by itself reduced IE ^ME -associated activity to below detectable levels. When the remaining three (A157T, T171S, D217A) mutations were removed from sC7, there was little effect on IE ^ME or OE related activity. To determine whether any combination of these mutations resulted in an increase in overall IE ^ME- associated activity at the expense of its specificity for IE ^ME over OE, Applicants engineered paired and triple reversion mutants of sC7 at these positions. Constructs containing all three of these backmutations had the second highest IE ^ME- related activity and the highest ability to discriminate between IE ^ME and OE (Table 3). This construct Tnp containing four mutations (R8C, E58V, E344K, L372Q) was renamed Tnp sC7 v2.0.

Table 3 contains the indicated in vivo frequencies of mutated Tnp sC7 that reverted to wild type. Numbers in parentheses indicate sites of conversion to wild type.

IE ^ME OE IE ^ME /OE

Frequency Normalized Frequency Normalized Tnp sC7 2.6×10 ^-3 1.0 3.3×10 ^-6 1.0 794Tnp sC7(157,171) 6.5×10 ^-3 2.5 7.0×10 ^-6 2.1 933Tnp sC7(157,217) 8.4× 10 ^-3 3.3 2.25×10 ^-5 6.8 373Tnp sC7(171,217) 5.8×10 -3 2.3 1.18×10 ^-5 3.6 502Tnp sC7(157,171,217)α 7.4× ^{10 -3 2.8} 7.1×10 ^-6 2.1 1042

α Renamed to sC7v2.0

Abbreviation: OE = outer end

  IE = inner end

IE ^ME = inner end of dam methylation

Tnp sC7 is less than the sum of its individual mutations. Tnp E58V (E58V 'plus one') mutant was ^4x104 more active than Tnp WT alone. The compound mutant with E58V removed (E58V 'minus one') was 1.8 x ¹⁰² more active than Tnp WT. It can then be predicted that the activity of Tnp sC7 will increase:

(4×10 ⁴ )(1.8×10 ² )=7.2×10 ⁶ However in reality the stimulation of the compound mutation is much lower (2×10 ⁵ ). In other words, adding E58V to the other 6 mutations resulted in a 1.1×10 ³ -fold increase, whereas adding E58V to the wild-type background did not see a 4×10 ⁴ -fold stimulation. This may be because both E58V and E334K stimulate the same step (initial DNA binding) and when mixed are less than additive.

Tnp sC7v2.0 efficiently transposes IE ^ME -defined transposons in vitro

The ability of Tnp sC7v2.0 to promote transposition of IE ^ME- defined transposons in vitro was tested under the same conditions (with Tnp EK/LP) developed for OE-defined transposon movement (Goryshin, IY, and Reznikoff, WS, "In Vitro Transposition of Tn5", J. Biol. Chem. 273:7367-7374 (1998), which is hereby incorporated by reference in its entirety). The substrate plasmid pGT4, a high copy number pUC19-based vector with inverted IM ^ME end sequences flanked by a kanamycin resistance gene, was purified as a supercoiled monomer (see Materials and Methods). This plasmid was constructed to allow digestion with the Pvull restriction endonuclease to release the transposon from the donor backbone DNA (see Figure 5A). Tnp is produced and purified by cloning the nucleotide sequence into an expression vector, expressing the protein in host cells and isolating the protein from extracts of the host cells (all using standard methods known to those skilled in the art). sC7v2.0.

Incubation with Tnp sC7v2.0 for 3 hours resulted in 66% conversion of supercoiled plasmids to transposition products and intermediates. In Figure 5B, lane 1 is the unreacted substrate pGT4. Lane 2 shows the transposition activity of IE ^ME transposon promoted by Tnp sC7v2.0, and lane 3 is the result of pGT4 digested with PvuII restriction endonuclease.

Although transposition reactions performed in this manner can produce many different DNA products, the reactions are explained by the analysis of certain diagnostic fragments that have been identified by Goryshin and Reznikoff (1998) (supra). Figure 5C is a reproduction of lane 2 of Figure 5B. Lane 1 is the excised transposon. It is an intermediate of the paired-end fragmented but not strand-transferred plasmid. Band 2 is the donor backbone DNA released by double-end excision of the transposon. As shown in lane 3 of Figure 5B, these bands, which are PvuII digestion products, have the same molecular weight. Lane 3 represents the substrate plasmid that underwent cleavage at one end of the transposon. It migrated to the same position as the linearized plasmid (data not shown). Bands 4 and 5 are two different types of strand transfer products. Lane 4 is the result of intramolecular insertion of a transposon into the unreacted plasmid. This results in a relaxed circular DNA that is longer than the original substrate plasmid by more than the length of the inserted transposon. The band indicated by 5 is the result of intramolecular reverse movement. These transposition products are the size of transposons but circular. These circularized transposition products can contain different numbers of nodes and thus migrate to different positions on the gel.

Structural/Functional Analysis

The hyperactive Tn5 transposase mutant Tnp sC7v2.0 (7.4×10 ⁵ -fold increased transposition of flanking IE ^ME transposons) contained mutations at amino acids 8, 58, 344 and 372. Analysis of Tnp sC7 revealed that excess activity conferred by E58V and E344K depends on the end sequence of the transposon. The existence of the E58V phenotype was not surprising, as a previous random mutagenesis screen with OE-flanked transposons isolated proteins with sequence-specific activity either at amino acid 47 or at amino acid 54 (Zhou et al. and Reznikoff, 1977; Zhou, M., Bhasin, A., and Reznikoff, WS, "Molecular genetic analysis of transposase-terminal DNA sequence recognition: three adjacent Cooperativity in specific interactions" J. Mol. Biol. 276:913-925 (1997)). However, the sequence-specific activity of the mutation at amino acid 344 suggests that this residue also interacts with transposon end DNA, which is indeed unexpected.

Recently, a protein-DNA co-crystal representing TnpEK/LP in complex with pre-excised (non-donor backbone) OE DNA was solved. In this complex, amino acid 58 was shown to specifically interact with OE at position 10. This mutates the mutated residue adjacent to position 12, the nucleotide that was originally selected for expression. Positions 10, 11 and 12 are all different between IE and OE, and are adjacent to one of the two major groove regions containing modifications by dam methylases (position 11 on the upper strand and position 12 on the lower strand).

The structure also shows that amino acid 344 interacts with DNA at position 7 of OE. When position 7 is not one of the seven that distinguish IE from OE, it is located between position 4 and the region of nucleotides 10, 11 and 12 (distinguishing the two). Therefore, the difference in the activity of Tnp E344K on IE and OE may be due to the influence of the front and rear sequences of these two regions. We therefore propose that both mutations, E58V and E344K, can interact with end-sequence DNA and alter the function of the transposase at the level of initial sequence recognition.

The presence of a leucine to glutamine mutation (L372Q) in all sequenced mutants (sA5, sB2, sC6, sC7 and sD5) was both familiar and surprising. It is familiar because a hyperactive mutation at this position has been previously isolated. This was surprising because a mutation was previously isolated that resulted in a leucine to proline (L372P) substitution at this position (Weinreich et al., 1994). This L372P mutation results in 2 consecutive proline residues in an alpha helix. It destabilizes amino acids 372-390 in co-crystals. It has been proposed that this mutation alters the conformation of this catalytic domain in relation to the C-terminal dimerization domain. This change improves activity by increasing the distance between the N and C termini. The close proximity of these ends in Tnp WT is thought to reduce the transposition rate of Tnp WT (Reznikoff et al., 1999). Wild-type leucine residues visible in early partial Tnp structures (Davies, D.R., Braam, L.M., Rezikoff, W.S., and Rayment, I., "Three-dimensional analysis of Tn5 transposase-associated proteins determined to 2.9A resolution Structure ", J BiolChem.274:11904-11913 (1999)) is buried in the hydrophobic pocket, may make this hydrophobic pocket unstable due to the substitution of glutamine. Whereas with PCR random mutagenesis method (making only one nucleotide change in this codon), and possibly at residues (methionine, valine, arginine, proline, or glutamine) Only 5 different amino acid substitutions resulted) 2 different hyperactive mutations were isolated, so it is possible that more direct mutagenesis methods such as codon randomization could reveal other interesting mutants.

The mutation R8C, which reduces OE-associated transposition almost 10-fold in the wild-type background (R8C 'plus one') and increases approximately 2-fold when removed from the sC7 background (R8C 'minus one'), is less easily interpreted. In the co-crystal structure, the arginine residues are not in the region where the DNA contacts. However, this structure represents only a resting image of the complex after cleavage, and the region likely made contact with DNA in the original synapse.

IE Methylation

The inner terminus of the Tn5 transposon contains 2 GATC signal sequences, which add 4 methyl groups to the main groove at each terminus. In this study we were able to isolate a mutation, E58V, which not only overcomes this binding inhibition but also exhibits a preference for transposons in which methyl groups are present (presumably because of increased binding affinity). Additionally, the co-crystal structure of Tnp EK/LP in complex with pre-cleaved DNA revealed that glutamic acid 58 directly interacts with position 10 of OE in the main groove. This region is near the methyl group on the adenine of the IE ^ME nontransferred strand. The fact that this single amino acid change can lead to dramatic changes in the phenotype leads us to propose that it is this methyl group that is responsible for the inhibition of transposase binding to IE ^ME . The inhibition of Tnp WT binding to IE ^ME is likely due to the interaction between this methyl group and the negatively charged side chain of glutamic acid58. Replacing this residue with valine not only eliminated this undesirable interaction but also increased binding affinity due to the hydrophobic pocket between the side chain of the valine residue and the methyl group.

General Materials and Methods

Media and Reagents

As described by Zhou M., & Reznikoff, W.S., (supra), supplemented with ampicillin, chloramphenicol, 5-bromo-4-chloro-3-indole-β-D-galactoside and phenyl- β-D-Galactoside Glucose-Based Miller Medium (Miller, J., Experiments in Molecular Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. (1992)) (Trp-XG-PG plate) was papillary test. Following transformation during site-directed mutagenesis, cells were cultured in SOC medium following the transmutation protocol (Promega). All other bacterial cultures were performed in Luria Broth (Sambrook, J., Fritsch, E.F. & Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.) . When needed, antibiotics (Sigma) were added at the following concentrations: ampicillin 100 μg/ml; chloramphenicol 20 μg/ml; nalidixic acid 20 μg/ml; gentamicin 5 μg/ml; tetracycline 15 μg/ml. Taq DNA Polymerase, T4 Polymerase, T4 Ligase, dNTPs and all components in the Alternative Mutagenesis Kit were purchased from Promega. Restriction enzymes were purchased from Promega or New England Biolabs. Oligonucleotides used in site-directed mutagenesis, sPCR and sequencing were purchased from Research Genetic. The radioactive nucleotides used in the sequencing were from Amersham.

Plasmid construction

Plasmid pGT4 was constructed as a high copy number plasmid containing the kanamycin resistance gene flanked by two inner termini. Designed so that the transposon can be liberated from its pUC vector backbone by digestion with PvuII.

Fourteen pRZ9905(sC7) derivatives and pRZ9905(sC7 version 2.0) were constructed by exchanging restriction fragments between pRZ9905 and pRZ9905.

bacterial strain

Plasmid cloning and directed evolution procedures were performed in JM109 (Promega). Site-directed mutagenesis procedures were performed with strains ES1301 (Promega) and JM109. In mating assays, transposase plasmids were transformed into strain RZ212 [Δ(lac-proA, B), ara, str, recA56, srl, thi/pOX38-Gen] and then coupled into 14R525 [F-nalr] .

directed evolution process

Directed evolution of the Tn5 transposase was performed using sPCR essentially as described by Stemmer (1994). Forty microliters (~4 μg) of a pRZ9905 plasmid miniprep was partially digested in 50 μl of a solution containing 100 mM Tris-HCl (ph=7.0), 5 nM MgCl and 90 ng DNase I. After incubation at room temperature for 7 minutes, EDTA was added to 10 mM to stop the reaction. After addition of loading dye (Sambrook et al., 1989), digested DNA was electrophoresed on 2% NuSieve gels (FMC BioProducts) next to pGEM DNA markers (Promega). A gel slice containing a DNA fragment of 200-600 bp in size and another gel slice containing a DNA fragment of 600-1000 bp in size were excised from the gel. DNA from these two pieces was purified separately by phenol chloroform extraction (Sambrook, et al., 1998). After ethanol precipitation, the DNA pellet was dried and resuspended directly in 50 μl assembly reaction mix. The assembly mix contains 0.2mM dNTPs, 2.0mM MgCl, 50mM KCl, 10mM Tris-HCl (pH 9.0 at 25°C) and 0.1% Triton X-100. After adding 0.5 units of Taq DNA polymerase, the DNA was reassembled according to the following thermocycling protocol: 94°C for 30 seconds; 50 cycles of 94°C for 20 seconds; 65°C for 1 minute; and 72°C for 2 minutes; cool to 4°C. For each sample (200-600bp and 600-1000bp), the transposase gene was amplified by performing a standard PCR amplification reaction using 5 μl of the assembly reaction product as a DNA template. The transposase-encoding fragment was digested with AflII and BglII and ligated into purified AflII/BglII digested pRZ9905 vector DNA.

The ligation product was transformed into electrocompetent JM109 cells containing plasmid pRZ9904. After culture, cells were plated on Trp-XG-PG plates containing chloramphenicol and ampicillin for selection. The plates were incubated at 32°C for 14 days. At this point, pRZ9905 plasmid DNA was isolated from all colonies exhibiting at least one papillae and retransformed into the papillary assay to verify their papillary plus phenotype. A total of 5 pRZ9905 derivatives (out of 20,000 colonies initially screened) proved to be Papillary+. Equal amounts of all five of these plasmids were then used as substrates for a second round of mutagenesis and selection. This process was repeated for a total of 4 rounds of selection (-20,000 colonies/round).

quantitative papillary test

The IE12A in vivo transposition activity of Tnp WT, Tnp sA5, Tnp sB2, Tnp sC6 and Tnp sD5 was compared using a quantitative papillary assay. Competent cells of strain JM109 carrying the plasmid pRZ9904(IE12A/IE12A) were transformed with the appropriate transposase-encoding pRZ9905. After outgrowth, transformed cells were plated on Tnp-XG-PG plates containing chloramphenicol and ampicillin. Plates were incubated at 32°C until colonies appeared (~18 hours). Individual colonies were then picked with a sterile needle and spotted on fresh plates in a 4x4 grid pattern to evenly distribute all colonies. One protein was spotted per plate containing 16 colonies. Plates were incubated at 32°C and transposition was quantified by observing the emergence of papillae at 24 hour intervals. Data are presented as the mean number of papillae per colony.

mating test

Mating experiments were performed as previously described (Yin et al., 1988; Goryshin et al., 1994). Bacterial cells carrying the transposon containing plasmid pFMA52-187 (with two OEs or two IEs) and the F factor pox-Gen were transformed with the appropriate transposase-encoding plasmid pRZ9905. The donor used for library screening was strain JCM101 [ΔlacZX74, raps, dam-3]. All other matings were performed with E. coli RZ212 [Δ(lac-proA, B), ara, str, recA56, srl, thi]. Each combination of transposase and end sequence was analyzed in triplicate. The value is expressed as the average value of these three data.

in vitro transposition assay

The substrate plasmid PGT4 was isolated from DH5α cells using the Qiafilter plasmid mega kit (Qiagen). Supercoiled monomeric plasmids were isolated from 1% agarose gels using the Qiaquick gel purification kit (Qiagen). The reaction was carried out at 37°C under the conditions determined by Reznikoff and Goryshin (1998). The concentration of pGT4 was 35.5 nM. Tnp sC7v2.0 was added to a concentration of 280 nM.

The foregoing is not intended to limit the scope of the present invention. It should be understood that the present invention includes all various changes and modifications within the scope of the claims of the present invention.

Sequence Listing <110> W.S. Reznikoff (Reznikoff, William S)

T.A. Naumann (Naumann, Todd A) <120> Transposase and method of use thereof <130>960296.96471<140><141><150>US 60/146686<151>1999-08-02<160>5<170 >PatentIn Ver.2.1 version<210>1<211>1431<212>DNA<213>transposon Tn5<220><221>CDS<222>(1)..(1428)<400>1atga ata act tct GCT CTT CAT CAT GCC GCC GAC TGG GCT AAA TCT GTG 48MET ILE THR Seru His Ala Ala Ala Ala Lys Ser Val1 10 15TTC TCG GCG GCG GCG GCC CGC CGCC CGCCCCTGCC TTG GCC TTG GCC TTG GCC TTG GCC TTG GCC TTG GCC TTG GCC TTG GCC TTG GCC TTG GCC TTG GCC TTG GCC TTG GCC TTG GCC TTG GCC TT 96 Ala Leu Gly Asp Pro Arg Arg Thr Ala Arg Leu Val

20 25 30AAC GCC GCC CAA TTG GCA AAA TAA TAA TCT GGT AAA TCA ACC ATC 144ASN Val Ala Gln Leu Ala Lys Tyr Serle Thr Ile

35 40 45TCA TCA GAA GAA GCC ATG CAG GAA GGC GCT TAC CGA TTT ATC 192SER GLU GLU MET GLN GLN GLN GLN GLN GLN GLN GLN GLN GLN GLN GLN GLU

50                  55                  60cgc aat ccc aac gtt tct gcc gag gcg atc aga aag gct ggc gcc atg   240Arg Asn Pro Asn Val Ser Ala Glu Ala Ile Arg Lys Ala Gly Ala Met65                  70                  75                  80caa aca gtc aag ttg gct cag gag ttt ccc gaa ctg ctg gcc att gag 288Gln Thr Val Lys Leu Ala Gln Glu Phe Pro Glu Leu Leu Ala Ile Glu

85 90 95GAC ACC ACC TCT TTG AGT TAT CGC CAC CAG GCC GCC GAA GAG CTT GGC 336ASP THR Ser Tyr ARG His Gln Val Ala Glu Leu GLY

100 105 110AAG CTG GC TCT ATT CAG GAT AAA TCC CGC GGC GGG TGG GTT CAC TCC 384LYS Leu GLY Serle Gln Asr Ser His Ser

115 120 125GTT CTC TTG CTC GCC ACC ACC ACA TTC CGC ACC GGA TTA CTG Cat 432VAL Leu Leu Glu Ala PHR VR Val Gly Leu leu his

130                 135                 140cag gag tgg tgg atg cgc ccg gat gac cct gcc gat gcg gat gaa aag   480Gln Glu Trp Trp Met Arg Pro Asp Asp Pro Ala Asp Ala Asp Glu Lys145                 150                 155                 160gag agt ggc aaa tgg ctg gca gcg gcc gca act agc cgg tta cgc atg 528Glu Ser Gly Lys Trp Leu Ala Ala Ala Ala Thr Ser Arg Leu Arg Met

165 170 175GGC AGC ATG AGC AGC AAC GTG ATT GCG GCG GAC CGC GAA GCC GCC GCC GCC GCC GCC GCC GCC GCC GCC GCC GCC GCC

180 185 185ATT CAT GT CAG GAC AAA CTG GCG Cat AAC GAG CGC TTC GTG 624ILE HIS Ala Tyr Leu Gln Asn Glu ARG PHE VAL VAL

195 200 205GTG CGC TC AAG CAC CAC CCA CGC AAG GAC GAC GAG TCT GGG TAT CTG 672VAL ARS HIS PRO ARG LYS ASP Val Gly Leu Tyr Leu Tyr Leu Tyr Leu

210                 215                 220tac gac cat ctg aag aac caa ccg gag ttg ggt ggc tat cag atc agc   720Tyr Asp His Leu Lys Asn Gln Pro Glu Leu Gly Gly Tyr Gln Ile Ser225                 230                 235                 240att ccg caa aag ggc gtg gtg gat aaa cgc ggt aaa cgt aaa aat cga 768Ile Pro Gln Lys Gly Val Val Asp Lys Arg Gly Lys Arg Lys Asn Arg

245 255CCA GCC CGC AAG GCG AGC TTG AGC CGC AGT GGC ACG CGC ACG CTA 816Pro Ala Ala Ser Leu Arg Serg Ile Thr Leu

260 265 270AAA CAG GGG AAT ATC ACG CTC AAC GCG GCC GCC GAG GAG AAC 864lys GLN GLY Asn Ile Thr Leu Ala Val Leu Glu Ile Asn

275 280 285CCG CCC AAG GGT GAG ACC CCG TTG AAA TGG TTG CTG CTG ACC GAA 912PRO PRO LYS GLY GLU Leu Leu Leu Leu Thr Ser Glu Thir Seru Glru's glue

290                 295                 300ccg gtc gag tcg cta gcc caa gcc ttg cgc gtc atc gac att tat acc   960Pro Val Glu Ser Leu Ala Gln Ala Leu Arg Val Ile Asp Ile Tyr Thr305                 310                 315                 320cat cgc tgg cgg atc gag gag ttc cat aag gca tgg aaa acc gga gca 1008His Arg Trp Arg Ile Glu Glu Phe His Lys Ala Trp Lys Thr Gly Ala

325 330 335gga GCC GAG AGG CAA CGC ATG GAG GAG GAG GAG GAT AAT CTG GAG CGG Ala Glu ARG GLN ARG MET GLU GLU GLU Glu Glu Glu Glu MG MET

340 345 350GTC TCG ATC CTC TCG TTT GCG GCG GTC AGG CTG TTA CTC AGA GAA 1104VAL Serle Leu Serite Ala Val ARG Leu Gln Leu ARG GLU

355 360 365AGC TTC ACG CTG CCG CAA GCA CTC AGG GCG CAA GGG CTG CTA AAG GAA 1152SER PHE Thr Leu Gln Ala GLN GLN GLN GLN Leu Lys Glu Lyn Gelican

370                 375                 380gcg gaa cac gta gaa agc cag tcc gca gaa acg gtg ctg acc ccg gat   1200Ala Glu His Val Glu Ser Gln Ser Ala Glu Thr Val Leu Thr Pro Asp385                 390                 395                 400gaa tgt cag cta ctg ggc tat ctg gac aag gga aaa cgc aag cgc aaa 1248Glu Cys Gln Leu Leu Gly Tyr Leu Asp Lys Gly Lys Arg Lys Arg Lys

405 410 415GAG AA GCA GGT AGC TTG CAG TGG GCT TAC ATG GCG AGA CTG 1296Glu Lys Ala GLY Seru Gln Tr Met Ala Ala Ala Ala ARG Leu Leu ARA's Leu Leu ARA's Leu ARA -I that Ala Mg Leu ARA ALEU that thesia

420 425 430GGC GGT TTT AGC AGC AGC AAG CGA ACC GGA AGC TGC TGC GCC 1344GLY PHE MET ASRLS ARG THR GLY Ile Ala Serp Gly Ala Ala

435 440 445CTC TGG GAA GGT TGG GAA GCC CTG CAA AGT AAA CTG GGC TTT CTT 1392leu TRP GLU GLY TRU ALA Leu Gln Leu ASP GLY PHE Leu Leu

450 455 460GCC GC AAG GAT CTG ATG GCG GCG CAG GGG AAG AAG AAG AAG AAGA 1431ALA LYS ASP Leu Met Ala Gln GLN > 2MET ILE THR Serg Ala Ala Ala Ala Ala Ala Lyser Val

20 25 30Asn Val Ala Ala Gln Leu Ala Lys Tyr Ser Gly Lys Ser Ile Thr Ile

35 40 45Ser Ser Glu Gly Ser Glu Ala Met Gln Glu Gly Ala Tyr Arg Phe Ile

50 55 60ARG Asn Pro Asn Val Sera Glu Ala Ile ARG LYS ALA GLY ALA MET65 70 75 80GLN ThR Val LYS Leu Ala Gln Glu Glu Leu Leu Ala Ile Glu Glu

85 90 95Asp Thr Thr Ser Leu Ser Tyr Arg His Gln Val Ala Glu Glu Leu Gly

100 105 110Lys Leu Gly Ser Ile Gln Asp Lys Ser Arg Gly Trp Trp Val His Ser

115 120 125Val Leu Leu Leu Glu Ala Thr Thr Phe Arg Thr Val Gly Leu Leu His

130                 135                 140Gln Glu Trp Trp Met Arg Pro Asp Asp Pro Ala Asp Ala Asp Glu Lys145                 150                 155                 160Glu Ser Gly Lys Trp Leu Ala Ala Ala Ala Thr Ser Arg Leu Arg Met

165 170 175Gly Ser Met Met Ser Asn Val Ile Ala Val Cys Asp Arg Glu Ala Asp

180 185 190Ile His Ala Tyr Leu Gln Asp Lys Leu Ala His Asn Glu Arg Phe Val

195 200 205Val Arg Ser Lys His Pro Arg Lys Asp Val Glu Ser Gly Leu Tyr Leu

210 215 220Tyr ASP HIS Leu Lysn Gln Pro Glu Leu Gly GLY TYR GLN ILE Ser2225 230 235 240ile Pro Gln Lysp Lys ARG GLY LYS ARG LYS ARG LYS ARG LYS ARG LYS ARG LYS ARG LYS ARG LYS ARG LYS ARG LYS ARG LYS ARG LYS ARG LYS ARG LYS ARG LYS ARG LYS ARG LYS ARG LYS ARG LYS ARG LYS ARG LYS ARG LYS ARG LYS ARG LSN ARS ARS ARSN AS ARSN's LG Lg Lg Lg Lg Lg Lg Lg Lg Lg Lug Gelican

245 250 255Pro Ala Arg Lys Ala Ser Leu Ser Leu Arg Ser Gly Arg Ile Thr Leu

260 265 270Lys Gln Gly Asn Ile Thr Leu Asn Ala Val Leu Ala Glu Glu Ile Asn

275 280 285Pro Pro Lys Gly Glu Thr Pro Leu Lys Trp Leu Leu Leu Thr Ser Glu

290 295 300Pro Val Glu Serou Ala Gln Ala Leu ARG Val Ile Tyr Tyr ThR30 310 320HIS ARG TRG Ile Glu PHE HIS LYS ALA TRP LYS THR GLY ALA

325 330 335Gly Ala Glu Arg Gln Arg Met Glu Glu Pro Asp Asn Leu Glu Arg Met

340 345 350Val Ser Ile Leu Ser Phe Val Ala Val Arg Leu Leu Gln Leu Arg Glu

355 360 365 Ser Phe Thr Leu Pro Gln Ala Leu Arg Ala Gln Gly Leu Leu Lys Glu

370 375 380ALA GLU HIS Val Gln Sergr Val Leu Thr Pro ASP385 395 400GLU CYS Glu Leu Gly Tyr Leu ASP LYS ARG LYS ARG LYS ARG LYS

405 410 415Glu Lys Ala Gly Ser Leu Gln Trp Ala Tyr Met Ala Ile Ala Arg Leu

420 425 430Gly Gly Phe Met Asp Ser Lys Arg Thr Gly Ile Ala Ser Trp Gly Ala

435 440 445Leu Trp Glu Gly Trp Glu Ala Leu Gln Ser Lys Leu Asp Gly Phe Leu

450                 455                 460Ala Ala Lys Asp Leu Met Ala Gln Gly Ile Lys Ile465                 470                 475<210>3<211>19<212>DNA<213>转座子Tn5<400>3ctgactctta tacacaagt                                            19<210>4<211>19< 212> DNA <213> Transposition TN5 <400> 4CTGTCTTG AtcagartCT 19 <210> 5 <211> 19 <212> DNA <213> Transplant TN5 <400> 5CTGTCTCAGAGAGATCT 19

Claims (17)

1. A mutant Tn5 transposase protein modified relative to the wild-type Tn5 transposase protein, characterized in that the difference between the mutant transposase and the wild-type protein is selected from the following positions: amino acids 58 and amino acid 372, wherein the mutant Tn5 transposase protein prefers the inner end of Tn5 over the outer end of Tn5. 2. The mutant Tn5 transposase according to claim 1, wherein said transposase contains valine at amino acid 58. 3. The mutant Tn5 transposase according to claim 1, wherein said transposase contains a mutation at amino acid 372. 4. The mutant Tn5 transposase according to claim 1, wherein said transposase contains glutamine at amino acid 372. 5. The mutant Tn5 transposase according to claim 1, wherein the difference between the mutant transposase and the wild-type protein lies in amino acid 58 and 372. 6. The mutant Tn5 transposase according to claim 1, wherein said transposase contains valine at amino acid 58 and glutamine at amino acid 372. 7. mutant type Tn5 transposase as claimed in claim 1, is characterized in that, the difference of described mutant type transposase and wild-type Tn5 transposase is to be selected from the difference of following position: amino acid 8 and Amino acid 344. 8 . The mutant Tn5 transposase according to claim 7 , wherein the amino acid 8 position of the transposase is cysteine. 9. The mutant Tn5 transposase according to claim 7, wherein the amino acid 344 of the transposase is lysine. 10. The mutant Tn5 transposase according to claim 7, wherein the difference between the mutant Tn5 transposase and the wild-type Tn5 transposase lies in amino acid 8 and amino acid 344. 11. The mutant Tn5 transposase according to claim 1, wherein said transposase contains cysteine at amino acid 8 and lysine at amino acid 344. 12. The mutant Tn5 transposase as claimed in claim 1, wherein the transposase contains cysteine at amino acid 8, valine at amino acid 58, and amino acid 344. Lysine and glutamine at amino acid 372. 13. A polynucleotide encoding the transposase of claim 1. 14. A polynucleotide encoding the transposase of claim 12. 15. A system for in vitro transposition of transposable DNA sequences, characterized in that said system comprises: The mutant Tn5 transposase modified relative to the wild-type Tn5 transposase according to claim 1; A donor DNA molecule comprising a transposable DNA sequence flanked at the 5' and 3' ends by wild-type IE sequences; and A target DNA molecule into which a transposable element can be transposed. 16. The system of claim 15, wherein the IE sequence is methylated. 17. A method for in vitro transposition, characterized in that said method comprises the steps of: Mix the target DNA molecule and the mutant Tn5 transposase described in 1 with a donor DNA molecule containing the transposable transposable molecule of interest in a suitable reaction buffer at subphysiological temperature A DNA sequence, wherein the DNA sequence of interest is flanked by a wild-type Tn5IE sequence at its 5' and 3' ends, reacted until the modified transposase binds to the IE sequence; and Raising the temperature to physiological temperature for a period of time is sufficient for the enzyme to catalyze transposition in vitro.

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