Recombinant thermostable luciferase, method for production thereof, isolated nuclear acid, expressing vector, kit for bioluminescent assay, analytical test for coa detection in the sample

FIELD: gene and protein engineering for various luminescent assays.

SUBSTANCE: new luciferase mutant forms have been obtained. Said mutant forms have increased thermostability and optionally different emission wave length in contrast with respective wild type enzymes. In all disclosed muteins natural amino acid residue in position equivalent to 357-position in Photinus pyralis luciferase sequence is replaced with other residue, preferably with uncharged polar amino acid (in particular tyrosine) residue. Mutant luciferases of present invention are useful in various analytical systems as reporter agent.

EFFECT: Mutant luciferases with new properties.

22 cl, 15 dwg, 6 tbl, 12 ex

 

The present invention relates to a new protein, in particular mutant enzymes luciferase that exhibit distinctive properties compared with the corresponding wild-type enzyme to DNA that encodes a protein data, to the application of this enzyme in analytical tests and kits comprising them.

Luciferase fireflies catalyzes the oxidation of luciferin in the presence of ATP, Mg+ and molecular oxygen with light emission. This reaction has a quantum yield of approximately 0.88. The property of emission of light has led to its application in a variety of luminometric analytical tests, which determine the levels of ATP. Examples of such tests include those that are based on is described in EP-B-680515 and WO 96/02665, and many others that are commonly used in laboratories.

The luciferase obtained directly from insects, particularly beetles, such as fireflies or larvae of fireflies. Certain types of luciferase include Japanese GENJI fireflies or KEIKE, Luciola cruciata and Luciola lateralis, the East European Firefly Luciola mingrelica, the North American Firefly Photinus pyralis and the larvae of the Firefly Lampyrus noctiluca.

However, since many of the genes encoding these enzymes have been cloned and sequenced, can be obtained using recombinant DNA technology. Serial is a major recombinant DNA, encoding the enzymes used for transformation of microorganisms, such as E. coli, which then Express the desired enzyme product.

The color emitted by these enzymes light when used in tests in the laboratory exactly the same. It would be helpful if the wavelength is changed, or to make it easier to capture specific detector, or for use in systems that require numerous reporters, for example for monitoring of various events occurring in the same sample. One distinctive aspect of reporter molecules is the use of luciferase molecules, which emit light at different wavelengths. This can be achieved with the use of reporter molecules, including luciferase derived from different species of fireflies or larvae of fireflies. However, an alternative strategy is to obtain mutant luciferase using recombinant DNA technology, in order to obtain the difference in the wavelength of the signal. Examples of such mutants are provided in WO 95/18853.

In addition, the stability under heat luciferase wild and recombinant type is that they very quickly lose their activity when exposed to temperatures above about 30°With, especially above 35°C. This instability causes problems when the enzyme used is if stored at high ambient temperature, or if the analysis is carried out under the reaction conditions at high temperature, for example, in order to increase the reaction rate.

Known from EP-A-524448 and WO/95/25798 mutant luciferase having high thermal stability. In the first of them describes a mutant luciferase having a mutation at position 217 luciferase Japanese Firefly, in particular, substitution of the threonine residue at the residue of isoleucine. The latter describes a mutant luciferase having 60% homology relative to the luciferase from Photinus pyralis, Luciola mingrelica, Luciola cruciata or Luciola lateralis, but in which the amino acid residue corresponding to residue 354 Photinus pyralis or 356 Luciola species, mutated in such a way that it is distinct from glutamate and, in particular, other than glutamate, aspartate, Proline, or glycine.

In co-pending application for patent of great Britain No. 9823468.5 and on the basis of the international patent application is additionally described such mutants. In this case, the described proteins that possess luciferase activity and at least 60% homology to the wild-type luciferase, such as the enzyme from Photinus pyralis, Luciola mingrelica, Luciola cruciata or Luciola lateralis, but which include mutations at different positions in the protein, including among other things:

(a) amino acid residue corresponding to residue 214 in the luciferase Photinus pyralis and the OST is woven 216 luciferase Luciola mingrelica, Luciola cruciata or Luciola lateralis; or

(b) amino acid residue corresponding to residue 232 in the luciferase Photinus pyralis and the residue 234 luciferase Luciola mingrelica, Luciola cruciata or Luciola lateralis; or

(C) amino acid residue corresponding to residue 295 luciferase Photinus pyralis and the residue 297 luciferase Luciola mingrelica, Luciola cruciata or Luciola lateralis.

Applicants have found that using mutation engine (or introduction) of amino acids in different positions in the protein luciferase, you can achieve significant shifts in the wavelength of the emitted light and/or increased thermostability of the enzyme. In addition, can be enhanced proton flux emitted light, making the enzyme more suitable for in vivo analyses, where is eliminated characteristic of the larvae of the Firefly enzyme kinetics, or in vitro, where there are no SOA or other substances, “inducing characteristic of the larvae of the Firefly enzyme kinetics”.

The present invention relates to a recombinant protein having luciferase activity and at least 60% homology to the wild-type luciferase, where in the sequence of the enzyme amino acid residue corresponding to residue 357 in the luciferase Photinus pyralis, mutated compared with the corresponding wild-type luciferase so that luciferase is capable of emitting light at a different wavelength compared to the corresponding Ls what Ferati wild-type and/or has increased thermostability compared to the corresponding wild-type luciferase.

The sequence of the wild-type luciferase, which can form the basis of recombinant forms of the invention include Photuris pyralis, Luciola mingrelica, Luciola cruciata or Luciola lateralis, Hotaria parvula, Pyrophorus plagiophthalamus, Lampyris noctiluca, Pyrocoelia miyako, Photuris pennsylvanica or Phrixothrix (“train” fireflies " look Biochem. 38 (1999) 8271-8279).

Bioluminescent enzymes of the species that may use the substrate D-luciferin (4,5-dihydro-2-[6-hydroxy-2-benzo-thiazolyl]-4-thiazolecarboxamide acid), for emission of light, can form the basis of a mutant enzyme according to the invention.

Certain sequences luciferase wild type, which can form the basis of recombinant forms of the invention include Photuris pyralis, Luciola mingrelica, Luciola cruciata or Luciola lateralis, Hotaria parvula, Pyrophorus plagiophthalamus, Lampyris noctiluca, Pyrocoelia miyako and Photuris pennsylvanica.

In particular, luciferase are enzymes derived from enzyme Photuris pyralis, Luciola mingrelica, Luciola cruciata or Luciola lateralis. In the enzymes of Luciola mingrelica, Luciola cruciata or Luciola lateralis corresponding amino acid residue is at position 359 in the sequence.

It is shown that the sequence of all different luciferase are highly conservative, with a high degree of homology between them. This means that the corresponding region sequences of the enzymes can be easily determined in the study of the sequence is to establish the most similar regions, although, if you want, you can use commercially available software (for example, “Bestfit” from Genetic computer group, University pc. Wisconsin; see Devereux et al. (1984) Nucleic Acid Research 12: 387-395) in order to determine the appropriate areas or certain amino acids in different sequences. Alternative or additionally, you can determine the appropriate amino acids with reference to L.Ye et al., Biochim. Biophys Acta 1339 (1997) 39-52, where a given sequence of enzymes with numbering, which is also used in this application.

In relation to the possible replacement of the amino acid residue corresponding to residue 357 in the luciferase Photuris pyralis, most of the sequences of the wild type, have an acidic residue (aspartic acid or glutamic acid) in this position. With the exception of some forms of luciferase Photuris pennsylvanica, in which the corresponding residue (356) is a non-polar residue, a valine, or some form of luciferase Phrixothrix, where appropriate position is V354 in PvGR or PhRE, where it leucine L354. Thus, in General, the amino acid used as a replacement amino acid in this position is other than aspartic acid, glutamic acid, valine or leucine.

Therefore, in most cases, the remainder of the keys, the second amino acid is replaced with sour balance, including basic amino acids such as lysine or arginine, nonpolar amino acids, such as leucine, valine or isoleucine, uncharged polar amino acids such as tyrosine, asparagine, glutamine, phenylalanine, serine, tryptophan or threonine. In particular, it can be replaced with uncharged polar amino acid such as tyrosine, asparagine, serine or threonine. Especially preferred amino acid residues for substitution in this position is tyrosine, phenylalanine, or tryptophan, and most preferably tyrosine. In General, aromatic residues in this position lead to the greatest shifts and can also contribute to thermal stability.

Where the sequence of the wild type include sour amino acid residues in this position it appropriately mutate into other sour remnants.

It was found that when matirovanie enzyme thus shifts the wavelength emitted by the luciferase light, in some cases up to 50 nm to the red region of the spectrum. Thus, the mutant luciferase Photinus pyralis D357Y emits light at a wavelength of about 612 nm compared with the wild-type enzyme, which emits light at a wavelength of 562 nm.

The shift of wavelength 50 nm is significant potential for use in the analyses, pascalc the shift of this magnitude can easily be determined spectral. Different colored luciferase can be used as reporter molecules in studies of gene expression, making it possible to trace more than one gene, for example as described in WO 95/18853. Multiple analytical study can also be performed using luciferase as the label.

The fact that the light in this case is painted in an intense red colour, is particularly useful in analytical methodology. Red mutant may be useful in the analysis of the ATP solution that contains pigments or other compounds that can absorb shorter wavelengths of light. For example, a red-colored solution will not absorb red light. Examples painted in red solutions, which are often the object of such analysis includes samples of blood or the liquid culture medium with eukaryotic cells, which may contain colored red pH indicator.

Using the mixture of colorimetric agents, such as luciferase, can be a useful ability to form an intense red signal, especially where another agent in the sample forms a green signal. The photomultiplier used in the spectral analysis with the photocathode, can be set to detect either one or both of the peaks formed in one of the sample. In other words, it is possible to distinguish the photon flux from the red or green emitter in the same sample.

In addition, it was found that the shift in wavelength can influence the presence of cofactor coenzyme A(COA). As a result of this property raises the possibility that this enzyme can be used in the test for cofactor.

As described below, it was investigated the effect of cofactor coenzyme And the spectrum of the light emitted in vitro. With increasing concentration of the coenzyme And changes the spectral distribution, and the highest concentrations of SOA in the spectrum is dominated by wavelengths in the region of 590-630 nm with a pronounced peak at 610 nm.

Thus, in accordance with an additional aspect of the invention provides an analytical test to determine the presence in the sample SOA, where this test involves adding to the sample, which, as expected, is SOA, luciferase, as described above, together with other reagents required for the reaction of luciferase/luciferin, determination of the wavelength emitted by the break of light and the correlation of this with the presence or absence of COA.

Such analytical test may be useful in determining the state of growth or activity of cells, such as microorganisms or eukaryotic cells.

The concentration of SOA in the cells are also an important indicator of the biosynthesis of fatty acids and vary depending on the needs of the cells in a particular component. When the number of metabolic disorders, such as carcinogenesis and diabetes, there has been a breach of the metabolites of fatty acids and, respectively, unusual levels of SOA. Tests according to the invention can be used in the diagnosis of such conditions. For example, you can determine the concentration of SOA in the cellular sample, such as a blood sample from the patient, determining the wavelength of light emitted by the luciferase according to the invention used in the test. This result can be compared with those obtained with the breakdown of healthy cells, to determine whether changes wavelength, and thus that the SOA of prisutstvuet modified concentration. This may indicate the presence of painful conditions in patients. The cells prior to analysis appropriately are lysed using a known lytic agent.

It is believed that the amino acid residue at position 357 critically associated with the binding site of coenzyme A. When the surface of the enzyme luciferase was simulated (using software for the simulation of proteins SYBL, Tripos, Ltd.) to the resolution of 1 Angstrom (Å), were observed on the polar “pocket”. It turned out that this “pocket” formed by the remnants of N310, E and D357 and 8-10 Å. When browsing the molecules on top of this “pocket” is part of a larger “pocket”formed by the remnants of N310, E, D357, and 1232. It turned out that the remains of the N310 and E form a bridge across the cavity, which creates two smaller pockets (see figure 8).

Not limited to a particular theory, it is possible that the bridge remains can be flexible enough to be released, when the enzyme is in solution to provide a larger pocket (≈12 Å depth ≈8 Å wide)that allows you to link COA. This coincides with the energy calculations.

When E. coli cells expressing the mutants Photuris-luciferin-4-monooxygenase according to the invention, grew when changing various sources is s carbon determined the spectrum of the light emitted in vivo. Switching from rich medium (LB) up to a certain minimal medium with acetate or glucose as the sole carbon source resulted in the shift to longer wavelengths of the light emitted and the declining contribution of the shorter wavelengths. This can provide an additional means of controlling the wavelength of the light emitted for analytical purposes.

It was found that the mutation position 357 in the protein leads to an increased thermal stability.

Proteins can include additional mutations in the sequence, provided that the luciferase activity of the protein largely intact. Mutations consistently enhance the properties of the enzyme or in a particular way better fit for intended purpose. This may mean that they lead to increased thermostability and/or changes the color and/or Km for ATP enzymes. Examples of mutations that lead to color shifts described in WO 95/18853. Mutations that affect the values of Km, for example described in WO 96/22376 and the application for international patent number PCT/GB 98/01026.

Basically it was found that the effect of mutations is additive in terms of changes in the properties.

Mutant luciferase according to the invention may include other specific mutations to the e increase thermostability compared to the wild-type luciferase, for example, mutations described in WO 00/24878. In particular, at least one:

(a) amino acid residue corresponding to amino acid 354 luciferase Photuris pyralis (356 in the Luciola luciferase) mutated;

(b) amino acid residue corresponding to position 215 in the luciferase Photuris pyralis or (217 in the Luciola luciferase), is another hydrophobic amino acid; or

(c) amino acid residue corresponding to residue 214 in the luciferase Photuris pyralis or residue 216 luciferase Luciola mingrelica, Luciola cruciata or Luciola lateralis;

(d) amino acid residue corresponding to residue 232 in the luciferase Photuris pyralis or residue 234 luciferase Luciola mingrelica, Luciola cruciata or Luciola lateralis;

(e) amino acid residue corresponding to residue 295 in the luciferase Photuris pyralis or residue 297 luciferase Luciola mingrelica, Luciola cruciata or Luciola lateralis;

(f) amino acid residue corresponding to amino acid 14 luciferase Photinus pyralis or residue 16 Luciola mingrelica or 17 Luciola cruciata or Luciola lateralis;

(g) amino acid residue corresponding to amino acid 35 luciferase Photinus pyralis, or residue 37 Luciola mingrelica, or residue 38 Luciola cruciata or Luciola lateralis;

(h) amino acid residue corresponding to amino acid residue 105 luciferase Photinus pyralis or residue 106 gene Luciola mingrelica, 107 Luciola cruciata or 108 Luciola lateralis;

(i) amino acid residue corresponding to amino acid residue 234 luciferase Photinus pyralis or ostad the 236 Luciola mingrelica, Luciola cruciata or Luciola lateralis;

(j) amino acid residue corresponding to amino acid residue 420 luciferase Photinus pyralis or residue 422 Luciola mingrelica, Luciola cruciata or Luciola lateralis;

(k) amino acid residue corresponding to amino acid residue 310 luciferase Photinus pyralis or residue 312 Luciola mingrelica, Luciola cruciata or Luciola lateralis;

is different from the amino acids present in the corresponding sequence of the wild type and where the enzyme luciferase has increased thermostability compared with the enzyme having the amino acid position corresponding wild-type luciferase.

Thus, preferred examples of proteins according to the invention are mutated luciferase wild type, where more than one amino acid, for example up to 100 amino acid residues, preferably not more than 40 amino acids and more preferably up to 30 amino acids, are the other relative to the amino acid in the corresponding position in the corresponding wild-type enzyme.

Thus, in the preferred embodiment the protein according to the invention includes a luciferase Photinus pyralis, where in addition to the mutation at position 357, as described above, at least one:

(a) amino acid residue corresponding to amino acid 354 luciferase Photinus pyralis, is different from glutamate;

(b) amino acid OST the current corresponding to position 215 in the luciferase Photinus pyralis, is a hydrophobic amino acid other than alanine;

(c) amino acid residue corresponding to residue 214 in the luciferase Photinus pyralis, is other than threonine;

(d) amino acid residue corresponding to residue 232 in the luciferase Photinus pyralis, is other than isoleucine;

(e) amino acid residue corresponding to residue 295 in the luciferase Photinus pyralis, is different from phenylalanine;

(f) amino acid residue corresponding to amino acid 14 luciferase Photinus pyralis, is different from phenylalanine;

(g) amino acid residue corresponding to amino acid 35 luciferase Photinus pyralis, is different from leucine;

(h) amino acid residue corresponding to amino acid residue 105 luciferase Photinus pyralis, is different from alanine;

(i) amino acid residue corresponding to amino acid residue 234 luciferase Photinus pyralis, is different from aspartic acid;

(j) amino acid residue corresponding to amino acid residue 420 luciferase Photinus pyralis, is different from serine;

(k) amino acid residue corresponding to amino acid residue 310 luciferase Photinus pyralis, is other than histidine.

Alternative protein according to the invention includes the protein luciferase sequence farm is the Luciola mingrelica, Luciola cruciata or Luciola lateralis, and where in addition to the mutation at position 359, as described above, at least one:

(a) amino acid residue corresponding to the amino acid 356 luciferase Photinus pyralis, is different from glutamate;

(b) amino acid residue corresponding to position 215 in the luciferase Photinus pyralis, is a hydrophobic amino acid other than alanine or threonine;

(c) amino acid residue corresponding to residue 216 luciferase Luciola mingrelica, Luciola cruciata or Luciola lateralis, is different from glycine (Luciola mingrelica main sequence) or asparagine (Luciola cruciata or Luciola lateralis) main sequence;

(d) amino acid residue corresponding to residue 234 luciferase Luciola mingrelica, Luciola cruciata or Luciola lateralis, is different from serine;

(e) amino acid residue corresponding to residue 297 luciferase Luciola mingrelica, Luciola cruciata or Luciola lateralis, is different from leucine;

(f) amino acid residue corresponding to amino acid 16 Luciola mingrelica, or the amino acid 17 Luciola cruciata or Luciola lateralis, is different from phenylalanine;

(g) amino acid residue corresponding to residue 37 Luciola mingrelica or 38 in Luciola cruciata or Luciola lateralis, is other than lysine;

(h) amino acid residue corresponding to amino acid residue 106 Luciola mingrelica, or amino acid residue Luciola cruciata or 108 Luciola laterals, is other than glycine;

(i) amino acid residue corresponding to amino acid residue 236 Luciola mingrelica, Luciola cruciata or Luciola lateralis, is other than glycine;

(j) amino acid residue corresponding to residue 422 Luciola mingreiica, Luciola cruciata or Luciola lateralis, is other than threonine;

(k) amino acid residue corresponding to amino acid residue 312 Luciola mingrelica, Luciola cruciata or Luciola lateralis, is different from threonine (Luciola mingrelica main sequence) or valine (Luciola cruciata or Luciola lateralis) main sequence.

A particular in each case substituted amino acids, leading to the appearance of increased thermal stability, can be defined in the usual way, as shown below. In each case, various substitutions can lead to increased thermostability. Replacement can be directed to the site by mutagenesis of DNA that encodes a native or a suitable mutant proteins that, obviously, it is clear to the person skilled in the art. The invention in this case involves a determination of provisions related to thermostability.

In General, however, it may be desirable to plan the replacement amino acid other properties instead of the amino acids of the wild type. So, hydrophilic amino acid residues can in some cases it is preferable to replace the hydrophobic amine is acid residues and Vice versa. Similarly, acidic amino acid residues can be replaced by basic residues.

For example, the protein can include a protein having luciferase activity and at least 60% homology relative to the luciferase from Photinus pyralis, Luciola mingrelica, Luciola cruciata or Luciola lateralis, where in the sequence of the enzyme is at least one:

(a) amino acid residue corresponding to residue 214 in the luciferase Photinus pyralis and the residue 216 luciferase Luciola mingrelica, Luciola cruciata or Luciola lateralis, mutated and is different from threonine in the case of luciferase Photinus pyralis; or

(b) amino acid residue corresponding to residue 232 in the luciferase Photinus pyralis and the residue 234 luciferase Luciola mingrelica, Luciola cruciata or Luciola lateralis mutated and is different from isoleucine in the case of luciferase Photinus pyralis; or

(c) amino acid residue corresponding to residue 295 in the luciferase Photinus pyralis and the residue 297 luciferase Luciola mingrelica, Luciola cruciata or Luciola lateralis, mutated and is, for example, is different from phenylalanine in the case of luciferase Photinus pyralis;

and the enzyme luciferase has increased thermostability compared to the wild-type luciferase.

It is shown that the sequence of all different luciferase are highly conserved, with significant homology between them. This means that the corresponding region sequences of enzymes easily predelays in the study of sequences to establish the most similar regions, although, if you want, you can use commercially available software (for example, “Bestfit” from Genetic computer group, University pc. Wisconsin; see Devereux et al. (1984) Nucleic Acid Research 12: 387-395) in order to determine the appropriate areas or certain amino acids in different sequences.

Alternative or additionally, you can determine the appropriate amino acids when accessing L. Ye et al., Biochim. Biophys Acta 1339 (1997), 39-52.

In relation to the possible replacement of the amino acid residue corresponding to residue 214 in the luciferase Photinus pyralis, polar amino acid threonine respectively replaced by non-polar amino acid such as alanine, glycine, valine, leucine, isoleucine, Proline, phenylalanine, methionine, tryptophan or cysteine. Especially preferred replacement of the threonine residue that corresponds to residue 214 in Photinus pyralis, is alanine. The preferred substitution is cysteine. However, other polar residues such as asparagine at this position may also enhance the stability of the corresponding enzyme having a threonine at this position. Other amino acids present in this position in luciferase wild type, include glycine (Luciola mingrelica, Hotaria parvula), asparagine (Pyrophorus plagiophthalamus, GR, YG, and YE'OR, Luciola cruciata, Luciola lateralis, Lampyris noctiluca, Pyrocelia miyako, Photuris pennsylvanica LY, KW J19) and serine (Phrixothix). They can mostly be replaced by non-polar or other non-polar side chains, such as alanine and cysteine.

In relation to the possible replacement of the amino acid residue corresponding to residue 232 in the luciferase Photinus pyralis, non-polar amino acid isoleucine respectively replaced with another non-polar amino acid such as alanine, glycine, valine, leucine, Proline, phenylalanine, methionine, tryptophan or cysteine. Other amino acids present in this position in the sequence of the wild type, include serine and asparagine. Accordingly, these polar residues are substituted with non-polar residues, such as described above. Especially preferred replacement of residue corresponding to residue 232 in the luciferase Photinus pyralis and the rest of the luciferase 234 Luciola mingrelica, Luciola cruciata or Luciola lateralis, the group is alanine.

Replacement of the amino acid residue corresponding to residue 295 in the luciferase Photinus pyralis and the residue 297 luciferase Luciola mingrelica, Luciola cruciata or Luciola lateralis, can also affect thermostability of the protein. (This refers to the position 292 in the luciferase Phrixothix). In the main amino acid in this position is a non-polar amino acid phenylalanine or leucine. Accordingly replaced by other nonpolar amino acids. For example, Photinus pyralis nonpolar amino acid phenylalanine, respectively, replacing the t to another non-polar amino acid, such as alanine, leucine, glycine, valine, isoleucine, Proline, methionine, tryptophan or cysteine. Especially preferred replacement of the phenylalanine residue corresponding to residue 214 in the luciferase Photinus pyralis, is leucine.

It is also possible mutation of amino acid residue corresponding to the amino acid 14 luciferase Photinus pyralis or the amino acid 16 or 17 in the Luciola luciferase (13 in the luciferase Phrixothix). This amino acid residue (which is usually the phenylalanine, but also can be leucine, serine, arginine, or in some cases, tyrosine), respectively, substituted with another amino acid, in particular for another non-polar amino acid such as alanine, valine, leucine, isoleucine, Proline, methionine or tryptophan, preferably alanine.

Can also be effective mutation of amino acid residue corresponding to amino acid 35 luciferase Photinus pyralis or amino acid residue 37 in the luciferase Luciola mingrelica (38 in other types of Luciola spp.). This amino acid varies among wild-type enzymes, which may include the provision leucine (Photinus pyralis), but also lysine, histidine, glycine, alanine, glutamine or aspartic acid. Accordingly, the amino acid residue at this position is replaced by non-polar amino acid residue, or other non-polar amino acid such as alanine, valine, FeNi is alanine, isoleucine, Proline, methionine, or tryptophan. The preferred amino acid in this position is alanine, which differs from the wild-type enzyme.

Mutation at the amino acid corresponding to position 14 of the sequence Photinus pyralis, and/or a mutation at amino acid residue corresponding to amino acid 35 luciferase Photinus pyralis, preferably are not only mutations in the enzyme. Accordingly, they are accompanied by other mutations, defined above, in particular those in positions corresponding to the positions 214, 395 232 or luciferase Photinus pyralis.

Replacement of the amino acid residue corresponding to residue 105 in the luciferase Photinus pyralis and the residue 106 luciferase Luciola mingrelica, or residue 107 Luciola cruciata or 108 Luciola lateralis (102 Phrixothix), can also affect thermostability of the protein. In the main amino acid in this position is a nonpolar amino acid with alanine, or glycine, or serine at Phrixothix. Accordingly replaced by other nonpolar amino acids. For example, Photinus pyralis nonpolar amino acid alanine respectively replaced with another non-polar amino acid such as phenylalanine, leucine, glycine, valine, isoleucine, Proline, methionine, or tryptophan. Particularly preferred substitution of the alanine residue corresponding to residue 105 in the luciferase Photinus pyralis, is valine.

Replacement of the amino acid residue corresponding to residue 234 in the luciferase Photinus pyralis and the residue 236 luciferase Luciola mingrelica, Luciola cruciata or Luciola lateralis (231 Phrixothix), can also affect thermostability of the protein. In the main amino acid in this position is aspartic acid or glycine, and in some cases by glutamine or threonine. Accordingly replaced by non-polar or other non-polar amino acids, which are suitable. For example, Photinus pyralis amino acid residue, which is aspartic acid, respectively, are substituted with non-polar amino acid such as alanine, leucine, glycine, valine, isoleucine, Proline, methionine, or tryptophan. Especially preferred replacement of the phenylalanine residue corresponding to residue 234 in the luciferase Photinus pyralis, is glycine. Where this provision is non-polar amino acid residue, such as glycine (for example, in the Luciola luciferase), you can replace it with another non-polar amino acid.

Replacement of the amino acid residue corresponding to residue 420 in the luciferase Photinus pyralis and the residue 422 luciferase Luciola mingrelica, Luciola cruciata or Luciola lateralis (417 green Phrixothix and 418 in red Phrixothix), can also affect thermostability of the protein. In the main amino acid in this position is an uncharged polar amino acid serine, or Tr is the onin war, or glycine. Accordingly replaced by other uncharged polar amino acids. For example, in Photinus pyralis serine can be replaced by asparagine, glutamine, threonine, or tyrosine, and particularly a threonine.

Replacement of the amino acid residue corresponding to residue 310 in the luciferase Photinus pyralis and the residue 312 luciferase Luciola mingrelica, Luciola cruciata or Luciola lateralis, can also affect thermostability of the protein. Amino acid residue in this position varies among the famous luciferase protein as a histidine in the luciferase Photinus pyralis, Pyrocoelia miyako, Lampyris noctiluca and some forms of Photuris pennsylvanica, threonine - luciferase Luciola mingrelica, Hotaria parvula and Phrixothix (where he is the amino acid 307), valine - luciferase Luciola cruciata and Luciola lateralis, and asparagine - luciferase some Pyrophorus plagiophthalamus. Thus, in the main amino acid in this position is a hydrophilic amino acid, which can be substituted by another amino acid residue, which increases thermostability of the enzyme. Especially preferred replacement of the histidine residue corresponding to residue 310 in the luciferase Photinus pyralis, is arginine.

Other mutations may also be present in the enzyme. For example, in the preferred embodiment the protein has the amino acid position corresponding to amino acid 354 luciferase Photinus pyralis (356 Lucifer is e Luciola), replaced with glutamate, in particular, to an amino acid other than glycine, Proline or aspartic acid. Accordingly, the amino acid in this position is tryptophan, valine, leucine, isoleucine and asparagine, but most preferably lysine or arginine. This mutation is described in WO 95/25798. It was found that hydrophobic residues in this position reinforces the shift of the wavelength of the enzyme. In addition, the presence of a large hydrophobic (V or I), polar (N) or positively charged (K or R) amino acid at position 354 increases thermal stability.

In an alternative preferred embodiment of the protein has the amino acid at the position corresponding to the amino acid 217 in the Luciola luciferase (215 in Photinus pyralis), substituted for a hydrophobic amino acid, particularly isoleucine, leucine or valine, as described in EP-A-052448.

Proteins according to the invention include luciferase wild-type and recombinant. They have a 60% homology with respect to the sequences of the wild type as those of the enzyme from Photinus pyralis, Luciola mingrelica, Luciola cruciata or Luciola lateralis, in the sense that at least 60% of the amino acids found in wild-type enzymes are proteins according to the invention. Such proteins may have a higher degree of homology, in particular at least 70%, more preferably at least 80% and n is andmore preferably at least 90% relative to the wild-type enzymes, listed above. Such proteins of this type include allelic variants, proteins from other insect species, as well as enzymes, obtained by recombinant methods. They are easy to identify, because they are encoded by nucleic acids that hybridizing with sequences that encode the enzymes of the wild type under conditions of hybridization with a specific ionic strength. Such conditions are clearly understood by the experts in this field, and examples are provided in Sambrook et al. (1989) Molecular Cloning, Cold Spring Harbor Laboratory Press). In General, conditions of low ionic strength can be defined as 3×SCC from about room temperature to about 65°and the conditions of high ionic strength, as 0.1×SSC at about 65°C. SSC is the name of the buffer of 0.15 M NaCl, 0.015 G M sodium citrate. 3×SSC is three times stronger than SSC, and so on.

In particular, the homology sequence against the sequences according to the invention can be assessed using the method of multiple comparative analysis of the primary structure, described by Lipman and Pearson (Lipman D.J. & Pearson, W.R. (1985) Rapid and Sensitive Protein Similarity Searches, Science, vol.227, pp.1435-1441). You should calculate the “optimized” interest scheme on the following parameters for the algorithm of Lipmans-Pearson: ktup = 1, gap = 4 and blank length = 12. Sequence, for to is that you need to assess homology, it should be used as a “test sequence”, which means that the main sequence for comparison, such as a sequence of Photinus pyralis, or any of the other sequences reported by Ye et al. above, should be entered in the algorithm first.

Specific examples of proteins according to the invention are the sequences of the wild-type luciferase with one or more mutations as described above.

The invention further provides nucleic acids that encode luciferase as described above. Accordingly, nucleic acid based on the sequences of the wild type, which are well known in this field. Suitable mutation method for carrying out the desired mutation in the amino acid sequence it is easy to determine based on the information about the genetic code.

In the preferred embodiment of the invention the nucleic acid is a synthetic genome. Accordingly, the synthetic gene construct with a deletion of codons that are rarely found in genes with high expression level of ordinary expressing hosts, such as E. coli, and at the same time avoiding the introduction of codons that are rarely found in genes encoding luciferase fireflies. This approach ensures that the new gene is the use of the codon, which is optimal as d is E. I coli and expressing systems of insects.

For example, where possible, the codons of amino acids AGD, leu, ile, gl and pro was replaced on CGT or CGC (AGD), CTG, CTT or CTC (leu), ATC or ATT (ile), GGT or GGC (gly) and CCG CCA or CCT (pro), thus eliminating rare codons. In the case of the synthetic gene is shown below (SEQ ID No 1) and in figure 14, this has led in General to 139 silent mutations, creating 62 new frequent codon (11% overall). The first 8 nucleotides presented in the figure 14, form part of the ribosomal binding site and thus not encode. The coding sequence starts with a methionine residue, indicated by the arrow. This coding sequence and very similar sequences, such as sequences that have at least 90% homology, or preferably 95% homology, form a preferred aspect of the invention.

Another useful property that can be used to obtain synthetic ordered structure is the inclusion of new unique restriction sites. These sites make mutagenesis, in particular combinatorial cassette mutagenesis, gene simpler and more efficient. In particular, it may be desirable to incorporate unique restriction sites within the cDNA that encodes a subdomain In the enzyme. In addition, it may be preferential to create a unique website re is trickle at the opposite 3’end of the gene to create a simple slit structures and/or removal peroxisome target sequences.

In the example below, was designed nine new unique restriction sites, mainly in the Central third of the gene, and was received unique site Hind III at the opposite 3’end of the gene to create a simple C-terminal fused structures (figure 12).

Finally, the use of synthetic gene provides the opportunity for the introduction of mutations to improve thermal stability of the gene product, or otherwise modify the properties of the product, as it is desirable. In the example below, for example, received three silent mutations to introduce polypeptide " oven " amino acid Semenov TS, EC and D357F.

Nucleic acids according to the invention respectively include expressing vector, such as plasmid, under the control of regulatory elements such as promoters, enhancers, terminators, etc. Then these vectors can be used to transform host cells, such as prokaryotic or eukaryotic cells, such as plant or animal cell, and, in particular, prokaryotic cells such as E. coli, so that the cell is expressed the desired enzyme luciferase. Culturing the transformed thus cells using conditions that are well known in the field, will lead to the production of the enzyme luciferase is, which you can then select from the culture medium. Where cells are plant or animal cells, these cells can be obtained plants or animals. Then the protein can be extracted from plants or in the case of transgenic animal proteins can be isolated from milk. Vectors, transformed cells, transgenic plants and animals and methods of producing the enzyme under cultivation of these cells to form additional aspects of the invention.

The mutant luciferase Photinus pyralis D357Y received random mutagenesis, as described below. It was found that a single point mutation D357Y leads to a large color shift of the wavelength of the emitted light and also increased thermostability compared to the wild-type luciferase. In additional studies, it was found that a number of substitutions at this position leads to a good thermostability and/or large color shifts.

Specific examples of mutant enzymes Photinus pyralis, which fall in the scope of the invention include the following:

D357Y

D357F

D357W

D357K

D357N

D357I

E354I/D357Y

E354V/D357Y

E354C/D357Y

E354R/D357Y

E354S/D357Y

E354N/D357Y

E354K/D357M

E354R/D357L

E354W/D357W

E354H/D357W

E354R/D357F

E354K/D357F

E354S/D357F

E354M/D357F

E354A/D357R

E354A/D357F

E354T/D357Y

E354A/D357N

I351M/E354R/D357V

E354S/D357V

E354R/D357W

E354R/D35M

E354R/D357S

E354N/D357S

or equivalent variants of any of them, when they come from luciferase other types.

Mutations for the above mutants were introduced into the luciferase gene in the plasmid RET directed to the site by mutagenesis (PCR) or combinatorial cassette mutagenesis. Oligonucleotides were added to PCR in order to conduct appropriate mutations, are presented below.

Earlier it was reported that the effects of point mutations in positions 354 215 is additive. This invention provides the possibility of combining three or more of these mutations for high thermal stability in the mutant enzyme, which has a large color shift.

Luciferase proteins according to the invention will preferably be used in any bioluminescent test that uses the reaction of luciferase/luciferin as signal. In the literature there are many such analytical tests. Therefore, proteins can be included in the kits made for the production of such tests, not necessarily with luciferine and any other reagents necessary to establish a specific test.

Now the invention will be described by example with reference to the accompanying schematic figures, where:

The figure 1 presents the log-graph showing % ostume the camping activity with respect to time, during incubation at 45°With several mutant enzymes according to the invention;

The figure 2 presents the spectral peaks obtained by cultivation of E. coli cells expressing luciferase, in citrate buffer, D-luciferine, where the enzyme is used (a) recombinant luciferase Photinus pyralis wild-type, (b) mutant D357K, (C) mutant D357N, (d) mutant D357W, (e) mutant D357I, (f) mutant D357F, (g) mutant D357Y and (h) double mutant E354I/D357Y;

The figure 3 presents a graph showing the % residual activity with respect to time, for the three mutant enzymes, E354I, D357Y and double mutant (DM) E354I/D357Y;

The figure 4 shows the emission spectra (a) of recombinant wild-type enzyme and (b) double mutant (DM) E354I/D357Y;

The figure 5 presents a graph showing the attenuation rate of photon emission of recombinant wild-type enzyme (◆) r-wt and mutant enzyme D357K;

The figure 6 presents a chart of molecular modeling, showing potential “pocket” linking SOA in luciferase;

The figure 7 presents the spectra of in vivo bioluminescence emitted by the E. coli cells expressing the mutant luciferase .pyralis D357Y (a) growth on LB; (b) growth on minimal medium and sodium acetate; (C) growth on minimal medium and glucose;

The figure 8 presents the spectra Bialy is inessence in vivo, emitted from E. coli cells expressing the mutant luciferase .pyralis E354K/D357M (a) growth on LB, (b) growth on minimal medium and sodium acetate; (C) growth on minimal medium and glucose;

The figure 9 presents a graph showing the effect of SOA on the spectral distribution of the light emitted by the mutant luciferase .pyralis D357Y;

The figure 10 presents a graph showing the normalized data on the impact of SOA on the spectral distribution of the light emitted by the mutant luciferase .pyralis D357Y;

The figure 11 presents a graph showing the effect of SOA on the spectral distribution of the light emitted by the mutant luciferase .pyralis E354I/D357Y (figure 11a) and normalized data (figure 11b);

The figure 12 presents the modification of the restriction sites used to construct the synthetic gene luciferase;

In the figure 13 presents the design used in the synthesis of the luciferase gene;

The figure 14 shows the cDNA sequence (SEQ ID No 1) synthetic gene luciferase (including nucleotides 1-8, which form a part of the ribosomal binding site, but are not coding) and encoded amino acid sequence, which begins with a methionine residue, indicated by the arrow (SEQ ID No 2); and

The figure 15 shows thermostability of the mutants, including mu is the ants, encoded synthetic genome, at 50°C.

Example 1

Identification and characterization of mutant luciferase

Prepared two libraries Photuris-luciferin-4-monooxygenase (Photinus pyralis)obtained PCR [M.Fromant et al., Anal. Biochem. (1995) 224, 347-353]. One library, including the PCR products of full-length luc gene, cloned in expressing system RETA T7 (Novagen Inc., Madison, WI, USA). The second library, including the PCR products short section of a gene luc, covering amino acids 199-353, cloned in the vector pBSK(+) (Stratagene, La Jolla, CA, USA).

Library rate expressed in E. coli strain BL21(DE3), (In E. coli F-dcm ompT hsdS(rB-mB-) galλ (DE3)).

Library pBSK(+) expressed in cells of E. coli NV (supE44 ara14 galK2 lacY1 ∀(gpt-proA)62 prsL20 (Strr) xyl-5 mtl-1 recA13 ∀ ((mrcC-mrr) HsdS- (r-m). Both Rita and pBSK(+) gene have β-lactamase and give the cells of E. coli containing the plasmids, resistance to ampicillin.

The strain of E. coli transformed received by the library by electroporation using a pulse generator for BIORAD E. coli and grown overnight at 37°on LB agar containing ampicillin at a concentration of 50 μg/ml of Cells were transferred to a nylon membrane (Osmonics, Minnetonka, Minnesota, USA) and were sprayed with a solution of luciferin (500 µm D-luciferin, potassium salt in 100 mm sodium citrate buffer, pH 5.0). The colony was estimated using the documentation and analysis systems is Alphalmager™ 1200 (Flowgen, Lichfield, Staffordshire, UK). This gave the bioluminescence emitted within a certain period of time, receiving the image light emitted from the colonies. The brightness of the luminescence was taken as an indicator of thermal stability of luciferase.

Then colonies were tested for thermal stability. Colonies were selected based on the brightness of the light emitted and were isolated for further characterization. In some tests screening colonies of E. coli were incubated at 42°C for 2 h before the screening, so you can select thermostable mutants. Colonies selected in the first screening, transferred to nylon membranes and cultured overnight in LB medium containing ampicillin. “Patches” were sprayed with a solution of luciferin and evaluated in Alphalmager™. This secondary screening helped to positively identify clones to analyze the activity of luciferase in vitro. The E. coli clones expressing possible thermostable enzymes were analyzed by in vitro luciferase activity and thermostability.

In tests in vitro luciferase activity was determined at room temperature using an analytical system for determining the activity of luciferase Promega (Promega Corporation, Madison, WI, USA).

Luciferase reaction was started by adding 10 μl of crude cell EXTRACTA 100 ál analytical mixtures for determining the activity of luciferase Promega (1 in 2 dilution). The resulting bioluminescence was determined using a luminometer Biotrace M3.

Crude cell extracts were prepared as described in technical Bulletin Promega no. 101. Aliquots night cultures of E. coli were literally reagent for lysis of cell cultures (25 mm Tris-phosphate, pH of 7.8, 1 mm dithiothreitol (DTT), 2 mm 1,2-diaminocyclohexane-N,N,N’,N’-tetraoxane acid, 10% glycerol, 1% Triton X-100, 1.25 mg/ml of hen lysozyme) for 10 min at room temperature. Then the crude lysates were kept on ice before analysis.

Properties of enzymes additionally tested in the experiments for time dependent inactivation. The Eppendorf tubes containing 50 μl aliquots of the crude cell extract was incubated in a water bath at a given temperature. At certain time points, the tubes were removed and cooled on ice before analysis. The remaining luciferase activity was expressed as percentage of initial activity.

Build log graphs of the percentage of residual activity with respect to time of incubation and used to calculate the values of t1/2. T1/2 is the time required for the enzyme lost 50% of its initial activity after incubation at this temperature. The values of t1/2 (time to reduce the activity to 50% of the initial activity was determined in crude extracts at 37°With log graphs % of the remaining activity in relation to time (not shown).

Plasmid DNA from E. coli clones expressing mainly thermostable luciferase, as defined above, and sequenced to identify the mutations responsible for thermostability of the enzyme.

Plasmid DNA was prepared using a kit QIAGEN QIAprep Spin Miniprep (QIAGEN Ltd., Crawley, W. Sussex, UK)following the Protocol for use of microcentrifuge (QIAprep Miniprep Handbook 04/98).

DNA sequencing in all cases conducted Babraham Techno, Cambridge, UK using a DNA sequencing machine (ABI PRISM™ 377 and the reaction set to cycle sequencing with terminator ABI PRISM™ BigDye™ (Perkin Elmer Applied Biosystems), which is based on dideoxyadenosine method [F. Sanger et al., Proc. Natl. Acad. Sci. USA 74, (1977) 5463-5467].

As a result of this work identified a new mutant D357Y.

Crystal structure of luciferase [.Conti et al., Structure 4 (1996) 287-298] showed that the position 357 is located on the surface of the protein and close to the position 354, which may have an impact on the stability and spectral properties. This indicates that this region may be important in terms of thermal stability of the enzyme.

D357Y is particularly thermostable mutant being the most thermostable luciferase with a single amino acid substitution.

Example 2

Site-directed mutagenesis to obtain al the other mutants at position 357

In order to evaluate the different mutations at position 357 conducted site-directed mutagenesis using the kit directed to the site of mutagenesis, Stratagene QuikChange™ (Stratagene, La Jolla, CA, USA). In all experiments with directed on site by mutagenesis used plasmid pPW601a J54 (PJW, MoD Report, 3/96). All products of mutagenesis reactions were transformed into E. coli strain XLl-Blue [e14-(mcrA-)(mcrCB-hsdSMR-mrr)171 endA1 supE44 thi-1 gyrA96 relA1 lac recB recJ sbcC umuC::Tn5 (Kanr) uvrC [F’ proAB laclqZM15 Tn 10 (Tetr) Amy Camr]]. Oligonucleotide primers were synthesized Sigma-Genosys Ltd., Cambridge, UK, and was designed using semantic doping system [A.R.Arkin et al., Biotechnology, (1992)10, 297-300, W., Huang et al., Anal. Biochem. 218, 454-457] and used to design degenerate oligonucleotide primers with getting groups of possible mutations in more than used a separate primers for each amino acid replacement.

Was thus given to the library of mutant luciferase with substituted amino acids.

Used the following oligonucleotides and their complementary partners):

Oligonucleotide primer (5’→3’)Replacement of amino acids
cacccgagggggat[tat]aaaccgggcgcgg (SEQ ID NO 4)Y
cacccgagggggat[(gac)(tc)(c)laaaccgggcgcggtcgg (SEQ D NO 5) A,I,L,T,V,P
cacccgagggggat[(t)(gat)(gc)laaaccgggcgcggtcgg (SEQ ID NO 6)C,F,L,W,Y,X
cacccgagggggat [(AC)(Yes)(gc)]aaaccgggcgcggtcgg (SEQ ID NO 7)R,S,K,N,H,Q

Library of mutants was subjected to screening as described previously, thermal stability. The number of colonies to be screened was calculated using equation [S.Climie et al., J. Biol. Chem. 265 (1990) 18776-18779]

N=[ln(1-P)]/[Ln((n-1)/n)]

where N represents the number of colonies to be screened, n represents the number of possible codons in the target position, and R represents the probability that each codon in a mixture selected for screening at least once. The equation is based on the value of P=0,95. The resulting directed to the site of mutagenesis mutants were analyzed for luciferase activity and characterized in the research on time-dependent data of thermoinactivation.

The mutants identified in this way, as desired, was cultured in 400 ml of LB medium containing ampicillin to a≈0.5 in. The expression of luciferase is then induced by adding isopropyl-β-thiogalactoside (IPTG) to a final concentration of 1 mm. The cells are then incubated at 30°C with shaking for 3 h before deposition by centrifugation. Obtained by centrifugation of cell sediment resuspendable in 10 ml of reagent for extraction of protein B-PER™ (Pierce Chemical Company, Rochford, USA), 1 mm DTT with obtaining a crude extract, following the Protocol B-PER™ for maximum extraction of bacterial protein. The recovered mixture of protease inhibitor, 500 ál (product No. R, Sigma, Saint Louis, Missouri, USA) was added to a solution of B-PER™ for inhibition of endogenous proteases. Then the cell lysate was centrifuged at 30,000 g for 30 minutes

The supernatant of the crude extract was subjected to fractionation with ammonium sulfate. The faction, which was deposited between 30% and 55% saturation, included luciferase activity. This material resuspendable in 0.5 ml of Tris-HCl, pH 8.0, 1 mm DDT and used for thermoinactivation and spectral studies.

Introduced substitution D357L, T, V, W, R, I, S, K, N and F. these mutants have been characterized in studies of crude extracts in vitro thermoinactivation.

Partially purified extracts were diluted, 1 to 11, in the buffer for thermoinactivation: 50 mm potassium phosphate buffer, pH of 7.8, containing 10% saturated ammonium sulfate, 1 mm dithiothreitol and 0.2% BSA.

110 μl aliquots of protein solution incubated at 4°or 45°during certain periods of time, and cooled on ice before analysis. Luciferase activity was then determined as described in example 1, using a reagent for the test for determining the activity of luciferase Promega (1 in 2 dilution).

The results are presented in tables 2 and 3 on figure 1. The values of t1/2 of the definition is whether in crude extracts at 40° (Table 2) and 45°With (table 3).

Table 2
MutantT1/2
D357K2,2
D357R4,2
D357S4,6
D357N4,8
D357V5,9
D357T7,3
D357L11,3
D357I18,0
Lazzari<1,0
Table 3
MutantT1/2
D357W2,5
D357F6,5
D357Y10,4
Lazzari<1,0

All substitutions increased thermostability compared to recombinant wild type.

Example 3

Changes the wavelength of the emitted light

It was also observed that replacement of amino acids at position 357 impact on the spectrum emitted by the light enzyme in vivo. An aliquot (250 μl) of cell cultures of E. coli as described in example 2 were cultured over night at 37°C, centrifuged in microcentrifuge and supernatant was removed. Cells expressing various mutant luciferase, were incubated in citrate b is fere (pH 5.0), containing 150 ál of D-luciferin, and analyzing the light emitted by the reaction, in vivo when determining the emission spectra using an for SPECTRAmax microplate® (Molecular Devices Corp. California, USA). For mutants D357Y, F and I, have seen significant changes in the spectral peak, and the distribution of wavelengths (figure 2(a)-(g)). These results are summarized in table 4 below.

In addition, it was estimated by eye in a dark room luminescence mutants in vivo. Mutants D357 showed their different colors of luminescence spectra. In particular, D357Y, F and I, showed a significant shift towards longer wavelengths of the light emitted.

It turned out that in some cases (for example, D357F) color change of the light emission occurred not only due to the shift in λMach, but also due to differences in the contribution to the spectrum by different wavelengths of visible light.

Table 4
MutantλmaxDeviation from Lazzari (nm)
Lazzari558-
D357K556-2
D357N5580
D357W5580
D357I606+48
611+53
D357Y613+55

Recombinant wild-type enzyme (r-wt) was used to compare λmax luminous emission of some of the mutants 357 in vivo. D357Y, F and I, showed significant improvements in their maximum wavelength.

Example 4

Properties of the enzyme in the presence or absence of COA

D357Y was partially purified by precipitation with ammonium sulfate as described in example 1. This partially purified enzyme D357Y (5 ml) was mixed with 150 ál test for determining the activity of luciferase Promega. Another aliquot was mixed with an equivalent buffer for analysis, which was absent SOA (25 mm Tris-tricin, pH of 7.8, 5.0 mm MgSO4, 0.1 mm EDTA, 2 mm DTT, 470 μm D-luciferin, 530 mm ATP). Shot emission spectra of the two reactions, and they are presented in figures 9 and 10.

The spectra show a pronounced difference in the bioluminescent emission in the absence and the presence of SOA with a significant shift in λmax. The impact of SOA on the kinetics of the luciferase reaction can also see the difference in the values of RLU. (RLU - relative light units).

This difference in emission has given rise to use of the enzyme in the test for detecting the presence of COA.

Example 5

Preparation and properties of double mutant

Using the receiving directed to the site of mutagenesis, as described in example 2, was designed double mutant E354I+D357Y to consider any cumulative effects on thermal stability and color of the light emitted.

Partially purified double mutant E354I+D357Y was diluted 1 to 11 in the buffer for thermoinactivation: 50 mm potassium phosphate buffer, pH of 7.8, containing 10% saturated ammonium sulfate, 1 mm dithiothreitol and 0.2% BSA.

110 μl aliquots of protein solution incubated at 45°during certain periods of time, and cooled on ice before analysis. Then determined the luciferase activity as described previously, using a reagent for determining the activity of luciferase Promega (1 in 2 dilution).

The double mutant showed a marked improvement of thermostability compared with the single mutants E354I and D357Y separately (see figure 3). Research thermoinactivation partially purified double mutant confirmed the increased thermostability of the mutant with a value of t1/2, equal to 7.7 min inactivation at 45°C.

It was noted that the double mutant shows a much more intense red luminescence than the individual mutants E354I and D357Y, showing additivity color luminescence.

Also were shot emission spectra of recombinant wild-type and crude extract double mutant E354I+D357Y using buffer analysis is a, described in example 3.

The spectra of emission of the captured in vivo, showed λmax at 611 nm. However, the range had a significantly greater contribution to the luminescence of the red range of wavelengths, leading to a more intense red look a visual assessment of the eye. The spectra of emission of the crude extracts showed a definite change in the spectral shape and the shift of wavelength equal to 44 nm, compared with Lazzari (see figure 4).

The spectrum of emission of the double mutant in vivo was shown as tapering bandwidth maximum wavelength of the light emitted (613 nm)and reduced contribution from wavelengths of light that are in the field of 540-560 nm.

Pronounced effects of these mutations indicates the importance of this region of the enzyme for color bioluminescent light.

Example 6

Enhanced photon flux

Observed that the bioluminescence of E. coli cells expressing the mutant D357K is very bright compared to other mutants under this provision. The kinetics of the luminescence of this enzyme were analyzed using a luminometer, using which you can define the intensity of photon emission over time. Aliquots containing cell extracts of E. coli containing the recombinant enzyme wild-type or mutant D357, was added to the mixture for the test for determining the activity of luciferase, which does not VK is Ocala any reagents, contributing characteristic of the larvae of the Firefly enzyme kinetics, such as coenzyme A. Observed that the decay rate of photon emission, defined over time (15) for both enzymes was significantly lower for the mutant D357K (figure 4). In other words, the mutant enzyme has the kinetics of the reaction, which inhibited to a lesser extent for at least the first 15 s of the reaction compared to recombinant wild-type enzyme.

Example 7

Combinatorial cassette mutagenesis at positions E and D357

Stage 1

Construction of plasmids pPW601aJ54 for mutagenesis cassette

Two new unique restriction site was built in the luc gene in the plasmid pPW601a/J54 using two pairs of synthetic oligonucleotides (see below). In General, using six silent mutations introduced into the gene restriction sites Spel and Kpnl, 63 base pairs separately. The plasmids containing these new sites, called pPW601aJ54SpeI/kpni restriction sites. The presence and spatial proximity of these restriction enzymes cut sites made it possible to apply combinatorial cassette mutagenesis to study the impact of random substitutions of amino acids in positions 354 and 357 in the primary sequence hturis-luciferin-4-monooxygenase.

Nucleotides marked in the construction, form site recognition, endonuclease and in the upper case position of point mutations required to create a site.

Stage 2

Design magazines and design libraries

Synthesized a pair of synthetic oligonucleotides that, when the annealing was made double-stranded cassette, which could directly ligitamate in plasmid pPW601aJ54SpeI/kpni restriction sites, split on new restriction sites. The cartridge is built for all possible combinations of 20 existing in vivo amino acid in position 354 and 357 in the primary sequence.

Loop library 2A

5’-ctagtgctattctgattacacccNNG/CggggatNNG/Caaaccgggcgcggtcggtaaagtggta-3’

(SEQ ID NO 12)

Loop library 2B

5’-cactttaccgaccgcgcccggtttG/CNNatccccG/CNNgggtgtaatcagaatagca-3’

(SEQ ID NO 13)

2 μg of each loop library of oligonucleotides were mixed in buffer containing 50 mm Tris-HCl, pH 7.4, 25 mm NaCl, and was heated to 100°C for 3 min, This solution was slowly cooled in the heating unit to <50°annealing of complementary sequences. Subjected to annealing the oligonucleotides were then ligated into a plasmid pPW601aJ54SpeI/kpni restriction sites, which were digested Spel and Kpnl. Then aliquots of the reaction mixture when legirovanii used to transform cells of E. coli HB101 by electroporation. After electroporation transformera the nnye cells were sown on plates with LB agar, containing 50 μg/ml of ampicillin, and cultured overnight at 37°C. the next day were randomly selected from cups 869 colonies and used for inoculation in 1 ml LB containing ampicillin in 96-well plates (Beckman). The tablets covered and cells were cultured overnight at 37°when shaken.

Stage 3

Screening of randomly selected clones in vivo

The next morning, 50 ál aliquots night cultures in the stationary growth phase was transferred into two transparent plastic with a round bottom 96-well plate to micrometrology (Dynex). One tablet was covered and incubated in a heating block for 8 min (the surface temperature of the block 45°C), while the other was kept at 37°C. Then determined and recorded the luciferase activity in the cells of both tablets in vivo at room temperature when added to the wells, 50 μl of 100 mm sodium citrate buffer, pH 5.0 containing 0.5 mm D-luciferin and then when moving the tablet in capture with a camera (Alpha Imager). The light emitted by the heated and control cultures, integrated within 1 or 2 min and the image is recorded on thermal paper tape.

For the second stage of screening was selected seventy-nine cultures, showing the highest bioluminescence that was determined by jar the spine of the recorded image. This time the cultures were incubated for 16 min in a heating block before staging analysis. Of the 55 clones selected based on the results of screening for thermostability in vivo, has selected 25 for performing spectral analysis in vivo. These clones were cultured overnight in LB medium at 37°and the next morning 200 ál night cultures were centrifuged, and cell E. coli precipitation resuspendable in 150 μl of 100 mm sodium nitrate buffer, pH 5.0 containing 0.5 mm D-luciferin. Then resuspendable the cells were placed in a white plastic tablet for micrometrology and analyzed the in vivo spectrum of the bioluminescent emission emitted from each of the mutant luciferase using fluorimetry for 96-well plates Molecular Devices Spectramax. The results are summarized in table 1 below.

Stage 4

Identification of mutants

Plasmid DNA was prepared from 25 clones, selected for screening in vivo, and sequenced using specific primers for sequencing. Identified mutations leading to amino acid substitutions at positions 354 and 357 in the primary sequence. One mutant was also included an additional mutation that results in the replacement of amino acids in position 1351 (table 5).

td align="center"> Mutant 600
Table 5
MutationThe maximum wavelengths (nm)
enzyme
1E354V/D357Y614
2E354I/D357Y612
3E354C/D357Y612
4E354R/D357Y600
5E354S/D357Y612
6E354N/D357Y608
7E354K/D357M556,606
8E354R/D357L588
9E354W/D357W610
10E354H/D357W606
11E354R/D357F596
12E354K/D357F608
13E354S/D357F610
14E354M/D357F610
15E354A/D357R556
16E354A/D357F610
17E354T/D357Y612
18E354A/D357N560
19I351M/E354R/D357V606
20E354S/D357V556,608
21E354R/D357W
22E354R/D357M596
23E354R/D357S592
24E354N/D357S600
LazzariE354/D357552

where Lazzari means the recombinant wild-type.

The number of mutant luciferase were selected on the basis of the results of the analysis in vivo on thermostability. Most of these Lucifers also shows significant changes in the spectrum of light emitted in vivo in many cases with a large contribution from the longer wavelengths of light (>580 nm). The number of spectra also showed a significant narrowing of the width of the band around a single peak at 610-614 nm.

Replacement E and D357 respectively hydrophobic and aromatic amino acid, for example E354V, D357Y, lead to the largest change in the spectrum in vivo, which shows a single peak with a narrow bandwidth of about 612 nm.

Example 8

Screening of in vitro conditions on thermal stability

Were prepared by lysis does not contain cell extracts of selected clones and determined the stability of luciferase from each extract in the experience of thermal inactivation. 50 µl of each extract was placed in an Eppendorf tube and incubated in a water bath heated to 45°during 4, 9 and 16 minutes At the corresponding time point was selected and is iquote and determined the remaining luciferase activity. Table 6 shows the percentage of remaining activity with respect to time for all mutant enzymes, and recombinant enzyme wild-type.

Table 6
The number of enzymeThe percentage activity remaining after incubation at 45°
1100958775.4
21009984.767.7
3100927353.3
4100948971.4
51008572.253
61009384.871
710063.73111.7
810058.6194.9
910085.465.342.3
1010065.527.810.6
1110088.67054
12 100906952
131008360.539

tr>
The number of enzymeThe percentage activity remaining after incubation at 45°
14100806139
151001.70.1Nd
16100907663
17100917860
18100191.8Nd
19100171.4Nd
20100171.1Nd
21100716334
22100804021
231002940.6
241002840.4
251000.1ndNd
Lazzari1000.05ndNd

where “nd” means not done.

The data show that the most thermostable luciferase were those who had an aromatic amino acid at position 357 (Y, F or W) and large hydrophobic (V or I), polar (N) or positively charged (K and R) the amino acid at position 354.

Example 9

Effect of cultivation conditions on the spectrum of light emitted in vivo

Evaluated the effects of different carbon sources on the spectrum of light emitted by the cells of E. coli BL21(DE3)expressing mutant luciferase D357Y or EC+D357M (7 above).

50 ml of cell culture was incubated until the logarithmic growth phase in LB medium and then harvested by centrifugation. Cellular precipitate resuspendable in 1 ml of sterile distilled water and then 100 μl aliquot of this suspension was used for inoculation in 5 ml of fresh LB, minimal M9 medium+2 mm sodium acetate or minimal medium + 2 mm glucose in sterile tube with a capacity of 25 ml Cultures were allowed to grow at 37°when shaken, and after 90 min (D357Y) or 120 min (enzyme 7) were selected 200 μl aliquot of cells was centrifuged and resuspendable in 150 μl of 100 mm sodium citrate buffer, pH 5.0 containing 0.5 mm D-luciferin. Then resuspendable cells was transferred into a tablet for mi is rodirovanie and analyzed the spectrum of the bioluminescent emission, emitted by each of the mutant luciferase using fluorimetry for 96-well plates Molecular Devices Spectramax. The results are presented on figures 7 and 8.

The results show that switching from rich medium (LB) (figures 7a, 8A) to a certain minimal medium with either acetate (figures 7b, 8b), or glucose (figures 7C, 8C) as the sole carbon source resulted in shifts to longer wavelengths of emitted light, and lower contribution from shorter wavelengths.

Example 10

Purification and spectral characterization of recombinant wild-type and mutant luciferase

Recombinant wild-type enzyme Photinus pyralis and mutant luciferase D357Y and E354I+D357Y was purified to homogeneity in order to analyze the impact of the cofactor coenzyme And the spectrum of the bioluminescent reaction. All three luciferase was isolated in the form of the slit structures module-binding carbohydrates of 143 amino acids from anaerobic fungus Piromyces equii. It was shown that the RAS selectively associated with swollen under the action of acid cellulose and soluble carbohydrates by galactomannan and glucomannan, forming the basis for a simple one-stage scheme of the affine cleanup.

Luciferase, merged with SMW, was associated with the cellulose in the raw, not containing cell extracts, washed and then was selectively suirable with COI is whether the soluble polysaccharides. Slit proteins are purified in this way was used in tests to determine the wavelength of the light emitted in the reaction mixtures containing various amounts of coenzyme A. the Enzyme (5 μl) was added to 100 ál for analysis, 25 mm Tris-tricin, pH of 7.8, 5.0 mm MgSO4, 0.1 mm EDTA, 530 μm ATP and 470 μm D-luciferin containing various amounts of coenzyme A. In figures 9-11 shows the effects of increasing concentrations of coenzyme And the spectrum of light emitted peeled luciferase D357 and E354I + D357Y.

When analyzing the in vivo spectrum of the bioluminescent light emitted by the cells of E. coli expressing Photuris-luciferin-4-monooxygenase, merged with the end of fungal RAS, did not show any significant differences compared to cells expressing the native luciferase. Similarly, when the analysis of the in vitro spectrum of the bioluminescent light emitted by a commercial source of purified recombinant luciferase (Promega), was identical to the spectrum of light emitted from the slit protein.

The observed differences, therefore, are associated with concentrations of SOA. With increasing concentration of SOA was changing the spectral distribution, the highest concentrations of SOA in the spectrum was dominated by wavelengths in the region of 590-630 nm with a pronounced peak at 610 nm. The spectral shift is most pronounced for double what atanta, where there is a significant narrowing of the width of the strip is about the only peak at a wavelength of 610 nm (figure 11).

Example 12

Obtaining synthetic luciferase Photinus pyralis, mutated such that it has 214C/354K/357F

Was designed and assembled synthetic luc gene of the pair of oligonucleotides using synthetic strategy described above. The sequence of the gene was designed to create a luciferase with amino acids S, K and 357F.

Synthesized twenty-nine pairs of overlapping synthetic oligonucleotides involving Sigma-Genosyc Ltd PAGE was purified and ligated into three ordered structure of approximately 550 BP (IDRIS 1, 2 & 3, figure 3). Each ordered structure then separately ligated into the vector S(+) and the resulting constructs were used to transform cells of E. coli XL1-Blue. Plasmid DNA was prepared from clones, including the assembled insert and sequenced to confirm the attachment ORS. The presence of n-1 oligonucleotides (by-products of oligosynthesis) in the ordered structures is complicated by the build process. DNA sequencing revealed only right ordered structure IDRIS 2, and used PCR to amendments ordered structure IDRIS 3, which included a deletion of one base pair at the 5’-end design. Ordered structure fully the LFS was achieved by legirovaniem mixture of plasmids, including IDRIS 1 with IDRIS 2 and 3.

Then legirovannoi DNA was used to transform cells of E. coli XL1-Blue and selected clones expressing active enzyme, using the analysis in vivo. Took a few clones and sequenced to confirm the presence and affection of synthetic luc gene having the sequence represented in figure 14. Full LFS was named IDRIS (FA).

The synthetic gene was integrated into the vector pBSK(+) between sites of Wamn I and Sal I in polylinker. In this position of the gene is not in a frame to be read alphabetical and has considerable remoteness from the lac promoter. But producirovanie sufficient amount of luciferase for preliminary characterization of the enzyme. Prepared raw not containing cell extracts of E. coli XL1-Blue, expressing IDRIS (FA), of the night cultures using the method of Promega lysis.

Then tested thermal stability of the enzyme extract at 50°C for 20 min and compared with thermostable mutant E354I+D357Y. Optimized with the new codon triple mutant was significantly more thermostable compared to the mutant E354I+D357Y (figure 15).

1. Recombinant thermostable luciferase, which is characterized by amino acid sequence that corresponds to amino acid sequence of the wild-type luciferase replacing natural is th residue in position, corresponding to position 357 in the sequence of the luciferase Photinus pyralis, another residue, an introduction which provides the improvement of thermostability of the modified enzyme and, optionally, changing the wavelength of the emitted light compared to the wild-type enzyme, or multiple amino acid substitutions, one of which is specified replacement position corresponding to position 357 in the sequence of the luciferase Photinus pyralis.

2. Recombinant luciferase according to claim 1, characterized in that the sequence of the indicated luciferase wild type corresponds to the luciferase from Photinus pyralis, Luciola mingrelica, Luciola cruciata or Luciola lateralis, Hotaria parvula, Pyrophorus plagiophthalamus, Lampyris noctiluca, Pyrocoelia miyako or Photuris pennsylvanica.

3. Recombinant luciferase according to claim 2, characterized in that the sequence of the wild-type luciferase is a sequence of enzyme derived from Photinus pyralis, Luciola mingrelica, Luciola cruciata or Luciola lateralis.

4. Recombinant luciferase according to claim 2, characterized in that the sequence of the indicated luciferase wild type corresponds to the luciferase from Photinus pyralis, Luciola mingrelica, Luciola cruciata or Luciola lateralis, Hotaria parvula, Pyrophorus plagiophthalamus, Lampyris noctiluca or Pyrocoelia miyako and amino acid residue corresponding to residue 357 in the luciferase Photinus pyralis, is different from aspartic acid or glutamic acid.

5. Recombine the Naya luciferase according to claim 2, characterized in that the sequence of the indicated luciferase wild type corresponds to the luciferase from Photuris pennsylvanica and amino acid residue corresponding to residue 357 in the luciferase Photinus pyralis, is other than valine.

6. Recombinant luciferase according to claim 1, characterized in that the amino acid residue corresponding to residue 357 in the luciferase Photinus pyralis, is different from aspartic acid, glutamic acid or valine.

7. Recombinant luciferase according to claim 6, characterized in that the amino acid residue corresponding to residue 357 in the luciferase Photinus pyralis, is an uncharged polar amino acid.

8. Recombinant luciferase according to claim 7, characterized in that the amino acid residue corresponding to residue 357 in the luciferase Photinus pyralis, is tyrosine, phenylalanine or tryptophan.

9. Recombinant luciferase of claim 8, wherein the amino acid residue corresponding to residue 357 in the luciferase Photinus pyralis, is tyrosine.

10. Recombinant luciferase according to any one of the preceding paragraphs, wherein the protein has at least 80%homology relative to the luciferase from Photinus pyralis, Luciola mingrelica, Luciola cruciata or Luciola lateralis.

11. Recombinant luciferase according to claim 1, characterized in that in addition to the substitution at position 357, contains at least one of the trail is proposed substitution in comparison with the corresponding wild-type luciferase:

(a) substitution of amino acid residue corresponding to amino acid 354 luciferase Photinus pyralis (356 in the Luciola luciferase);

(b) replacing the amino acid residue corresponding to position 215 in the luciferase Photinus pyralis (217 in the Luciola luciferase), another hydrophobic amino acid, or

(c) substitution of amino acid residue corresponding to residue 214 in the luciferase Photinus pyralis or residue 216 luciferase Luciola mingrelica, Luciola cruciata or Luciola lateralis;

(d) replacing the amino acid residue corresponding to residue 232 in the luciferase Photinus pyralis or residue 234 luciferase Luciola mingrelica, Luciola cruciata or Luciola lateralis;

(e) replacing the amino acid residue corresponding to residue 295 in the luciferase Photinus pyralis or residue 297 luciferase Luciola mingrelica, Luciola cruciata or Luciola lateralis;

(f) replacing the amino acid residue corresponding to the amino acid 14 luciferase Photinus pyralis or residue 16 luciferase Luciola mingrelica or residue 17 luciferase Luciola cruciata or Luciola lateralis;

g) replacing the amino acid residue corresponding to amino acid 35 luciferase Photinus pyralis or residue 37 luciferase Luciola mingrelica or residue 38 luciferase Luciola cruciata or Luciola lateralis;

(h) replacing the amino acid residue corresponding to amino acid residue 105 luciferase Photinus pyralis or residue 106 luciferase Luciola mingrelica, residue 107 luciferase Luciola cruciata or residue 108 Luciola lateralis

(i) replacing the amino acid residue corresponding to amino acid residue 234 luciferase Photinus pyralis or residue 236 luciferase Luciola mingrelica, Luciola cruciata or Luciola lateralis;

(j) replacing the amino acid residue corresponding to amino acid residue 420 luciferase Photinus pyralis or residue 422 luciferase Luciola mingrelica, Luciola cruciata or Luciola lateralis;

(k) replacing the amino acid residue corresponding to amino acid residue 310 luciferase Photinus pyralis or residue 312 luciferase Luciola mingrelica, Luciola cruciata or Luciola lateralis.

12. Recombinant luciferase according to claim 11, characterized in that the substituted amino acid residue corresponding to amino acid 354 luciferase Photinus pyralis (356 in the Luciola luciferase).

13. Recombinant luciferase according to item 12, wherein the amino acid residue corresponding to residue 214 in the luciferase Photinus pyralis or residue 216 luciferase Luciola mingrelica, Luciola cruciata or Luciola lateralis replaced by another hydrophobic amino acid.

14. An isolated nucleic acid encoding a recombinant luciferase and characterized by nucleotide sequence that essentially coincides with the nucleotide sequence in accordance with the genetic code determines the amino acid sequence of recombinant luciferase according to any one of claims 1 to 13.

15. Nucleic acid according to 14, characterized those who, it contains a synthetic gene.

16. Nucleic acid according to 14, characterized in that the application of the codon optimized for a particular expressing the owner and/or introduced unique restriction sites.

17. Nucleic acid for 14 or 15, characterized in that it contains nucleotides 9-1661 SEQ ID NO: 1 or a sequence that has with her, at least 90%homology.

18. Expressing a vector containing a nucleic acid according to any one of p-17 under the control of the control elements ensuring the expression of the indicated nucleic acids.

19. A method of obtaining a recombinant luciferase according to any one of claims 1 to 13, providing for the transformation of the host cell with the vector according p, cultivation of specified cells and separation from her luciferase.

20. The method according to claim 19, characterized in that a host cell is a recombinant cell E. coli.

21. Set for use in bioluminescent analysis, containing the luciferase and luciferin, characterized in that it contains recombinant thermostable luciferase according to any one of claims 1 to 13.

22. Analytical test to determine the presence in the sample SOA, providing for adding to a sample suspected to contain the SOA, the recombinant protein according to any one of claims 1 to 13, together with other reagents that are necessary for p is Ogadenia interaction luciferase with luciferine, measurement of the wavelength emitted by the sample light and the judgment results on the presence or absence of COA.

23. Test item 21, characterized in that it is applicable in the diagnosis of diabetes.



 

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