Modified biotin-binding protein (version), nucleic acid coding it, vector and carrier for biotin binding

FIELD: chemistry.

SUBSTANCE: claimed inventions deal with a modified protein, nucleic acid, coding such protein, a vector, containing nucleic acid, and a carrier for biotin binding, which such protein is immobilised on. The characterised modified biotin-binding protein is obtained by the introduction of a mutation from one to several amino acid residues into a sequence, represented in SEQ ID NO:2, or an amino acid sequence, identical to the said sequence by 98% or more, and the presence of the biotin-binding activity, where at least one residue, selected from the group, consisting of residues from 1) to 4), presented below, is substituted with the residue of acidic amino acid or residue of neutral amino acid; 1) residue of arginine in position 104 SEQ ID NO: 2; 2) residue of lysine in position 141 SEQ ID NO: 2; 3) residue of lysine in position 26 SEQ ID NO: 2 and 4) residue of lysine in position 73 SEQ ID NO: 2.

EFFECT: claimed inventions make it possible to obtain the biotin-binding protein and can be applied for biotin binding.

14 cl, 6 dwg, 11 tbl, 3 ex

 

The technical field to which the invention relates

According to this application claims priority of Japanese patent application No. 2008-208766, registered on August 13, 2008

The present invention relates to a modified biotinidase squirrel.

Prior art

Avidin is a protein derived from the protein of the egg, and the streptavidin is a protein derived fromStreptomyces avidinii. Avidin and streptavidin, each has a very high affinity (KD=10-16up to 10-14) to Biotin (D-[(+)-CIS-hexahydro-2-oxo-1H-thieno-(3,4)-imidazole-4-valerate]), and this affinity is one of the strong interactions between two biological molecules. Their molecular masses of approximately 60 kDa. Currently the interaction of avidin/streptavidin-Biotin is widely used in the fields of biochemistry, molecular biology and medicine (Green, (1975), Adv. Protein Chem., 29: 85-133; Green, (1990), Methods Enzymol., 184: 51-67). Avidin and streptavidin, each forms a tetramer, and one subunit of the tetramer binds one molecule of Biotin.

A problem in using the avidin is its nonspecific binding. Avidin may not specifically be linked with the cells, but also with DNA, proteins, and biological material such as a membrane. For example, when determining substances with �using bind avidin-Biotin avidin't specifically binds with substances other than the target substance is used, which increases the background. Reasons for high non-specific binding of the avidin include its high isoelectric point and chain of sugars contained in the amount of approximately 10% of the molecular weight. Avidin is a strongly basic protein with a high isoelectric point of 10 or more, and overall positively charged. Accordingly, it is believed that avidin easily associated with biological substances, which in many cases are negatively charged.

In addition, it is believed that the chain of sugars on the surface of the avidin bind to biological substances (Marttila et al., (2000) FEBS Lett, 467, 31-36). In order to reduce non-specific binding of the avidin was investigated, for example, chemically modified neutravidin, whose chains of sugars removed the avidin using glycosidase (Bayer, et al., (1995) Appl Biochem Biotechnol, 53(1), 1-9), and the biosynthesis of the avidin purchase without modification chains of sugars as a result of substitution of the aspartic residue at position 17 (target glycosylation in avidyne) on the residue isoleucine (Marttila et al., (2000) FEBS Lett, 467, 31-36). In addition, there is research to reduce the isoelectric point of the avidin by transformation of the residue of lysine or arginine residue in avidyne in neutral amino acid or an acidic AMI�akisato using genetic engineering (Marttila, et al., (1998) FEBS Lett, 441, 313-317).

Although these modifications can reduce nonspecific binding, for example, DNA and cells to avidin, reduction of nonspecific binding with human sera to be used in clinical testing, has not been significantly investigated. In addition, the biosynthesis mutant Videnov requires gene-expression systems insect. Accordingly, modification of the sequence of the avidin require a long time of cultivation and high cost, therefore, still not logged in to use in practice.

In accordance with the present study regarding the affinity between Biotin and a Biotin binding protein such as avidin or streptavidin, binding with fluorescent Biotin increases with significant modifications to the structure of streptavidin (Aslan et al., (2005) Proc Natl Acad Sci U. S. A. 102, 8507-8512). Unfortunately, biotinlabeled the ability of this protein is significantly reduced.

The inventors of the present invention purified protein exhibiting antibacterial activity againstMagnaporthe griseafrom edible mushroomPleurotus cornucopiae. As found, the protein possessed biotinidase activity and was named tlavideo (tamaminin 1). In the patent WO 02/072817 as disclosed amino acid sequence of the protein travidia 1 and nucleotide seq�the sequence of the gene, encodes a protein (SEQ ID NO: 1 and 2 in the patent WO 02/072817). Was also identified homologue travidia 1 (tamaminin 2) fromPleurotus cornucopiaeand, as shown, he had a strong biotinidase activity. As found, the protein possessed biotinidase activity and was named tlavideo (tamaminin 1). In the patent WO 02/072817 as disclosed amino acid sequence of the protein travidia 2, and the nucleotide sequence of the gene encoding the protein (SEQ ID NO: 3 and 4 in the patent WO02/072817), and was successfully received their recombinant protein. Tumaviciene 1 and 2 can be expressed inEscherichia coli. In particular, tamaminin 2, which can be easily obtained by purification using iminobiotin column and has a higher resistance to heat compared with the streptavidin is a great bioinvasion protein. However, although nonspecific binding travidia 2 with nucleic acids and/or proteins is lower than the current of the avidin, it's comparable to the streptavidin.

List of links

Patent document

Patent document 1: WO02/072817 A1

Patent document 2: WO2008/081938 A1

Non-patent document

Non-patent document 1: Marttila, et al., (2000) FEBS Lett, 467, 31-36

Non-patent document 2: Bayer, et al., (1995) Appl Biochem Biotechnol, 53(1), 1-9

Non-patent document 3: Marttila, et al., (1998) FEBS Lett, 441, 313-317

Non-patent document�UNT 4: Alon, et al., (1990) Biochem Biophys Res Commun, 170, 1236-1241

Non-patent document 5: Aslan, et al., (2005) Proc Natl Acad Sci U. S. A. 102, 8507-8512

Non-patent document 6: Weber, et al., (1989) Science, 243: 85-88

Non-patent document 7: Livnah, et al., (1993) Proc. Natl. Acad. Sci. U. S. A., 90, 5076-5080

Non-patent document 8: Qureshi, et al., (2001) J. Biol. Chem., 276(49), pp. 46422-46428

A brief summary of the invention

The technical problem

The aim of the present invention is the provision of a modified bytesmessage protein that exhibits superior characteristics such as low non-specific binding and/or additionally increased affinity of Biotin binding while preserving the specific characteristics travidia, i.e. high biotinidase ability.

The solution of a technical problem

The present invention successfully improved performance travidia 2 (in the present description hereinafter may be referred to as TM2) by modifying the amino acid sequence (SEQ ID NO: 2) natural travidia 2.

Preferred embodiments of the present invention

The present invention preferably includes the following implementation options:

Method 1

Modified biotinidase protein comprising the amino acid sequence represented by SEQ ID NO: 2, amino acid sequence, region�giving from one to several mutations of amino acids in the sequence, represented by SEQ ID NO: 2, or amino acid sequence identical to the sequence represented by SEQ ID NO: 2, 80% or more, and having biotinidase activity, where

one or more residues selected from

1) the arginine residue at position 104 of SEQ ID NO: 2;

2) the lysine residue at position 141 of SEQ ID NO: 2;

3) the lysine residue in position 26 of SEQ ID NO: 2; and

4) the lysine residue in position 73 of SEQ ID NO: 2

replaced by the balance of acidic amino acid or a residue of a neutral amino acid;

Method 2

Modified biotinidase protein according to method 1, where amino acid residue selected from 1) to 4) of the replaced amino acid residue having a hydrophobicity index of 2 or less;

Method 3

Modified biotinidase protein according to method 1, where 1) the arginine residue at position 104 of SEQ ID NO: 2 and/or 2) a lysine residue at position 141 of SEQ ID NO: 2 is replaced with an acidic amino acid residue or a neutral amino acid residue;

Method 4

Modified biotinidase protein according to the method 3, where 1) the arginine residue at position 104 of SEQ ID NO: 2 and/or 2) a lysine residue at position 141 of SEQ ID NO: 2 is replaced with an acidic amino acid residue;

Method 5

Modified biotinidase protein according to the method 3 or 4, where 1) the arginine residue at position 104 of SEQ ID NO: 2 and/or 2) a lysine residue at position 11 of SEQ ID NO: 2 is replaced with a glutamic acid residue;

Method 6

Modified biotinidase protein according to any of methods 1 through 5, where the aspartic acid residue in position 40 of SEQ ID NO: 2 is replaced with an asparagine residue;

Method 7

Modified biotinidase protein according to any one of methods 1 to 6, which is selected from the group consisting of:

modified bytesmessage protein (R104E-K141E), in which the arginine residue at position 104 of SEQ ID NO: 2 is replaced with a glutamic acid residue, and the lysine residue at position 141 is replaced by glutamic acid residue;

modified bytesmessage protein (D40N-R104E) in which the aspartic acid residue in position 40 of SEQ ID NO: 2 is replaced with an asparagine residue and the arginine residue at position 104 is replaced by a glutamic acid residue;

modified bytesmessage protein (D40N-K141E) in which the aspartic acid residue in position 40 of SEQ ID NO: 2 is replaced with an asparagine residue, and the lysine residue at position 141 is replaced by glutamic acid residue; and

modified bytesmessage protein (D40N-R104E-K141E) in which the aspartic acid residue in position 40 of SEQ ID NO: 2 is replaced with an asparagine residue, the arginine residue at position 104 is replaced by a glutamic acid residue and a lysine residue at position 141 is replaced by glutamic acid residue;

Method 8

Modified bio�invasively protein according to any one of methods 1 to 7, which satisfies at least one requirement selected from the following requirements a) to l):

a) aspartic residue at position 14 of SEQ ID NO: 2 is not modified or is replaced by glutamine or aspartic acid;

(b) the serine residue at position 18 of SEQ ID NO: 2 is not modified or is replaced with threonine or tyrosine;

(c) the tyrosine residue at position 34 of SEQ ID NO: 2 is not modified or is replaced by serine, threonine or phenylalanine;

(d) a serine residue at position 36 of SEQ ID NO: 2 is not modified or is replaced with threonine or tyrosine;

(e) an aspartic acid residue in position 40 of SEQ ID NO: 2 is not modified or is replaced by asparagine;

(f) the tryptophan residue at position 69 of SEQ ID NO: 2 is not modified;

(g) a serine residue at position 76 of SEQ ID NO: 2 is not modified or is replaced with threonine or tyrosine;

(h) a threonine residue at position 78 of SEQ ID NO: 2 is not modified or is replaced by serine or tyrosine;

(i) the tryptophan residue at position 80 of SEQ ID NO: 2 is not modified;

(j) the tryptophan residue at position 96 of SEQ ID NO: 2 is not modified;

(k) a tryptophan residue at position 108 of SEQ ID NO: 2 is not modified; and

l) residue aspartic acid at position 116 of SEQ ID NO: 2 is not modified or is replaced with glutamic acid or asparagine;

Method 9

Modified biotinidase protein according to the method of 1, comprising the amino acid�ing sequence, identical to 90% or more of the sequence represented by SEQ ID NO: 2;

Method 10

Modified biotinidase protein according to any one of methods 1 to 9, which satisfies at least one property selected from the following properties (i) to (iv):

(i) having an isoelectric point lower than the point of a protein consisting of the amino acid sequence represented by SEQ ID NO: 2;

(ii) exhibiting low non-specific binding to nucleic acids and/or proteins in comparison to a protein consisting of the amino acid sequence represented by SEQ ID NO: 2;

(iii) exhibiting lower fibrocartilaginous activity compared to a protein consisting of the amino acid sequence represented by SEQ ID NO: 2; and

(iv) exhibiting higher biotinidase activity compared to a protein consisting of the amino acid sequence represented by SEQ ID NO: 2;

Method 11

Modified biotinidase protein comprising the amino acid sequence represented by SEQ ID NO: 2, amino acid sequence having mutations from one to several amino acids in the sequence represented by SEQ ID NO: 2, or amino acid sequence identical to 80% or more sequence predstavljen�th SEQ ID NO: 2, and having biotinidase activity, where the aspartic acid residue in position 40 of SEQ ID NO: 2 is replaced with an asparagine residue;

Method 12

Modified biotinidase protein according to the method 11, where biotinidase activity is higher than the protein consisting of the amino acid sequence represented by SEQ ID NO: 2;

Method 13

Nucleic acid that encodes the protein according to any one of methods 1 to 12;

Method 14

A vector containing a nucleic acid according to the method 13; and

Method 15

The carrier, which immobilized protein according to any of methods 1 through 12.

The preferred implementation options for performing the present invention will be described below.

Tamaminin

Tumaviciene represent a new bytesmessage proteins that are found in the edible fungusPleurotus cornucopiae(patent WO 02/072817). This document illustrates:

- tamaminin 1 and tamaminin 2 characterized 65,5% homology of amino acids and firmly bonded with Biotin;

- tamaminin 2 highly expressed in the soluble fractions inEscherichia coli; and

- that when tamaminin 2 expressed inEscherichia coli, A 4.5-hour culture provides purified recombinant protein of high purity in an amount of from about 1 mg per 50 ml of culture; it is very�June high value even higher than the values for the avidin and streptavidin, which are known as biotinidase proteins.

Used in the present description, the term "tamaminin 2" refers to tamaminin 2 or a variant thereof. The present invention provides a modified TM2 by modifying specific amino acid residues TM2 or its variants, which demonstrates non-specific binding to nucleic acids and/or proteins. In the present description "tamaminin 2" and "TM2" refer to wild-type TM2 or its variants, unless expressly stated otherwise. However, these terms can be used as a generic name that includes the modified TM2 of the present invention, TM2 wild-type, variant type and the modified TM2 type, depending on the context. In addition, since TM2 shows biotinidase activity, it may be denoted in the present description as "bytesmessage squirrels."

Specifically, TM2 (wild type) usually can be a protein comprising the amino acid sequence of SEQ ID NO: 2 or a protein encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1. Alternative, TM2 may be a variant of a protein comprising the amino acid sequence of SEQ ID NO: 2 or variant of a protein encoded by a nucleic acid comprising the nucle�LiDE sequence of SEQ ID NO: 1, where options have biotinidase activity equivalent to the activity travidia 2. Options TM2 may be a protein comprising the amino acid sequence with deletion, substitution, insertion and/or addition of one or more amino acids in the amino acid sequence SEQ ID NO: 2 in which biotinidase activity equivalent to the activity of TM2. Substitution can be a conservative substitution, which means the replacement of a certain amino acid residue by another residue having similar physical and chemical characteristics. Non-limiting examples of conservative substitutions include replacement of one amino acid residue containing an aliphatic group (for example, Ile, Val, Leu or Ala), and other the replacement of one polar residue for others, like swapping between Lys and Arg, or Glu and Asp, or Gln and Asn.

Variants or mutants result in deletions, substitutions, insertions and/or additions of amino acids can be obtained from the DNA encoding the natural protein, by applying the well-known method, for example, site-specific mutagenesis (see, for example, an article in Nucleic Acid Research, Vol. 10, No. 20, p. 6487-6500, 1982, included in this description by reference in full). Used in the present description the expression "one or more amino acids" refers to the possible number of amino acids that �might be removed, substituted, inserted or added by site-specific mutagenesis. It should also be noted that used in the present description the expression "one or more amino acids" can sometimes identify one or more amino acids.

Site-specific mutagenesis can be carried out as follows using synthetic oligonucleotide primers that are complementary, single-stranded DNA phage, subjected to mutations, except for specific bugs mating, which is consistent with the desired mutation. More specifically, the above synthetic oligonucleotides are used as primers for the synthesis chain, complementary to the phage, and the host is transformed received desperatley DNA. Culture of the transformed cells were plated in agar and containing a single phage cells form plaques. Then, theoretically, 50% of new colonies contain the phage having a mutation in a single chain, and the remaining 50% have the original sequence. Received plaque hybridized with a synthetic probe labeled by treatment with kinase, at a temperature, which allows you to gibridizatsiya with those colonies that are characterized by the complete coincidence of the DNA possessing the above desired mutation, but it cannot gibridizatsiya with those colonies that have the original �EP. Then plaques that hybridized with the probe, harvested and cultured for DNA.

It is noted that the methods of introducing deletion, substitution, insertion and/or addition of one or more amino acids in the amino acid sequence of a biologically active peptide while retaining its activity include not only the above site-specific mutagenesis, but also a method that includes processing a gene mutagen and a method which enables selective cleavage of the gene, then the deletion, substitution, insertion or addition of the selected nucleotide and finally, the cross-linking of the digested fragments. More specifically, used in the present invention TM2 is a protein which consists of amino acid sequence with deletion, substitution, insertion, or addition of one to ten amino acids in the amino acid sequence of SEQ ID NO: 2, and which has biotinidase activity.

Variant or mutant TM2 may also be a protein which includes an amino acid sequence identical, at least 80%, preferably identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98%. or identical, at least 99%, and more preferably identical, at least 99.3% of the amino acid sequence SEQ ID NO: 2, and which has a similar TM2 biotinidase activity.

The percent identity between two amino acid sequences can be determined by visual inspection and mathematical calculation. Alternatively, the percent identity of two protein sequences can be determined by comparing information on the sequences using the computer program GAP, which is based on the algorithm of Needleman, S. B. and Wunsch, C. D. (J. Mol. Biol., 48: 443-453, 1970), and which is available to the University of Wisconsin Genetics Computer Group (UWGCG). The preferred default parameters for the GAP program include: (1) the scoring matrix blosum 62, as described by Henikoff, S. and Henikoff, J. G. (Proc. Natl. Acad. Sci. USA 89: 10915-10919, 1992); (2) 12 points for the space; (3) 4 points for the length of the gap; and (4) no penalty for end gaps.

To compare sequences can also be used other programmes used by specialists in this field of technology. The percent identity can be determined by comparing the information about the sequences using the program BLAST, opinoi, for example, Altschul et al. (Nucl. Acids. Res., 25, p. 3389-3402, 1997). This program can be accessed on the Internet at the website of the national center b�tehnologicheskoi information (NCBI) or from a data Bank of DNA of Japan (DDBJ). Various conditions (parameters) to search for identity using the BLAST program details are given on these websites and some of the plants can vary in a suitable manner, although the search is usually carried out using the default values. Alternatively, the percent identity between two amino acid sequences can be determined using a program such as a computer program of processing of genetic information GENETYX Ver. 7 (Genetyx) or FASTA algorithm. In this alternative, the search may be performed using the default values.

The percent identity between two sequences of nucleic acids can be determined by visual inspection and mathematical calculation, or more preferably by comparing the information about the sequences using a computer program. Ordinary preferred computer program is the Wisconsin package, version 10.0 GAP program from Genetics Computer Group (GCG; Madison, State of Wisconsin) (Devereux et al., Nucl. Acids Res. 12: 387, 1984). When using this software GAP can be compared not only sequences of two nucleic acids, but also of two amino acid sequences and comparison of nucleic acid sequence and amino acid sequence. In the present description preferably�the default parameters for the GAP program include: (1) using the program GCG unary comparison matrix (containing a value of 1 for identical and 0 for non-identical) for nucleotides, and the weighted comparison matrix of amino acids by Gribskov and Burgess, Nucl. Acids Res. 14: 6745, 1986, as described by Schwartz and Dayhoff, eds., Atlas of Polypeptide Sequence and Structure, National Biomedical Research Foundation, pp. 353-358, 1979, or other applicable comparison matrix; (2) a penalty of 30 for each amino acid gap and an additional penalty of 1 for each symbol in each gap or penalty of 50 for each gap in the nucleotide sequence and an additional penalty of 3 for each symbol in each gap; (3) no penalty for end gaps; and (4) no maximum penalty for long gaps. Other programs compare sequences that are used by professionals in the art and which can be used in the present invention include the BLAST program, version 2.2.7, which can be downloaded at the website of the US National Library of Medicine (http://www.ncbi.nlm.nih.gov/blast/bl2seq/bls.html), or the algorithm UW-BLAST 2.0. Setup standard default settings for UW-BLAST 2.0 are described at the following website: http://blast.wustl.edu. In addition, the BLAST algorithm uses a scoring matrix for amino acids BLOSUM 62 and selection parameters that can be applied as follows: (A) inclusion of a filter to the masked segment of the requested sequence having low compositional complexity (in the determination using the program SEG from Wootton and Federhen (Computers and Chemistry, 1993); see also Wooton and Federhen, “Analysis of compositionally biased regions in sequence databases,” Methods Enzymol., 266: 544-71, 1996) or for the masked segments including internal repeats of short periodicity (in determining the program XNU from Claverie and States (Computers and Chemistry, 1993)); and (B) the expected probability of matches expected to be found only by chance in accordance with the statistical model thresholds or E-sets (Karlin and Altschul, 1990), no statistically significant differences for messages matching database sequences (if a statistically significant difference caused by a certain coincidence is higher than the threshold E is set, the match is not reported); preferred numerical value of the threshold E-many is either 0,5 or in ascending order of preference 0,25, 0,1, 0,05, 0,01, 0,001, 0,0001, 1e-5, 1e-10, 1e-15, 1e-20, 1e-25, 1e-30, 1e-40, 1e-50, 1e-75 or 1e-100.

Variant or mutant TM2 may also be a protein which is encoded by a nucleic acid comprising nucleotide sequences which hybridise with chain, complementary to the nucleotide sequence of SEQ ID NO: 2 under stringent conditions, and which has the same biotinidase activity, and TM2.

Used in the present description the expression "under stringent conditions" refers to hybridization under conditions of moderate or high stringency. Specifically, conditions of moderate stringency can be easily determined by those skilled in the art based on, for example, the length of the DNA. Basic conditions formulated in the book of Sambrook et al. Molecular Cloning: A Laboratory Manual, 3rd ed. Chapter 6, Cold Spring Harbor Laboratory Press, 2001 and include the use of: solution for pre-washing 5×SSC, 0,5% SDS, 1.0 mm EDTA (pH 8,0); hybridization conditions of about 50% formamide, 2×SSC to 6×SSC, preferably 5-6×SSC and 0.5% SDS at about 42°C (or other similar hybridization conditions, such as the stark solution in about 50% formamide at about 42°C), and washing conditions at approximately 50-68°C, 0.1 to 6×SSC and 0.1% SDS. Preferably, conditions of moderate stringency conditions include hybridization (and washing conditions) of about 50°C, 6×SSC and 0.5% SDS. Conditions of high stringency can also be readily determined by the skilled in the art based on, for example, the length of the DNA.

Typically, such conditions include hybridization at higher temperatures and/or at lower salt concentrations than under conditions of moderate stringency (for example, hybridization in the presence of about 0.5% SDS at about 65°C with 6×SSC to 0.2×SSC, preferably 6×SSC, more preferably 2×SSC, even more preferably of 0.2×SSC or 0.1×SSC), and/or flushing, and they can be defined as hybridization conditions of the type described above and includes rinsing at about 65-68°C in 0.2 in 0.1×SSC and 0.1% SDS. In a buffer solution for use in �hybridizati and washing SSC (1×SSC consists of 0.15 M NaCl and 15 mm sodium citrate) can be replaced by SSPE (1×SSPE consists of 0.15 M NaCl, 10 mm NaH2PO4and 1.25 mm EDTA; pH of 7.4), and rinsed for about from 15 minutes to one hour after the completion of hybridization.

If desired, can be used a commercial kit for hybridization, where the probe does not use a radioactive substance. A specific example is hybridization, in which the system is used ECL direct promisiunea and definitions (product of Amersham). Stringent hybridization may be carried out at 42°C for 4 hours after adding to the hybridisation buffer set blocking agent and NaCl in amounts corresponding to 5% (wt./about.) and 0.5 M; the washing can be carried out twice in 0.4% SDS and 0.5×SSC for 20 minutes each at 55°C, then once in 2×SSC for 5 minutes at room temperature.

Biotinidase active variants or mutants TM2 can be measured using any of the known methods. For example, it can be determined using a method based on fluorescent Biotin as described in article Kada et al. (Biochim. Biophys. Acta., 1427: 33-43 (1999)). This method is a test system that uses such a nature of the fluorescent Biotin that if it is associated with bioinvasion site in biotissues protein, the intensity of its fluorescence decays. Alternatively, biotinidase�th activity variants or mutant proteins can also be estimated using the sensor, capable of measuring the binding of the protein with Biotin, such as a biosensor operating on the principle of surface plasmon resonance.

In a modified travidia of the present invention amino acid residues, which are preferably not subjected to modification, will be described below.

Modified tamaminin in which nonspecific binding is reduced according to the present invention

The modified TM2 of the present invention includes the amino acid sequence represented by SEQ ID NO: 2, amino acid sequence having one to several amino acid mutations in the sequence represented by SEQ ID NO: 2, or amino acid sequence identical to the sequence represented by SEQ ID NO: 2, 80% or more, and having biotinidase activity, and is characterized in that the modified TM2 is a protein (TM2 wild-type and variant type TM2), in which one or more residues selected from the group consisting of:

1) the arginine residue at position 104 of SEQ ID NO: 2;

2) the lysine residue at position 141 of SEQ ID NO: 2;

3) the lysine residue in position 26 of SEQ ID NO: 2; and

4) the lysine residue in position 73 of SEQ ID NO: 2

replaced by the balance of acidic amino acid or a residue of a neutral amino acid.

Isoelectric point (pI) of the wild type TM2, designed for DOS�ve its primary structure, approximately 7,4, while the real measured value is from about 8.2 to about 8.6. Thus, wild-type TM2 is a protein with a pI ranging from neutral to slightly acidic. The degree of nonspecific binding TM2 much smaller than that of the avidin, and is approximately equal to that of streptavidin which is a neutral protein.

The inventors of the present invention have conducted studies to further reduce nonspecific binding TM2. These include experiments using TM2 in which the principal balance of the modified amino acid residue acidic or neutral amino acids, they were held on the basis of the assumption that even in proteins with pI ranging from neutral to slightly acidic, such as TM2, nonspecific binding can be further reduced by reducing the pI.

It is known that the affinity of binding of streptavidin or the avidin with Biotin is reduced in some cases by replacing one or more amino acids. Accordingly, in experiments performed modification of amino acid residues, at the same time, the audit, described below, showed that not only reduced isoelectric point, but also not attenuated excellent characteristics, i.e., high biotinidase ability TM2.

As a result of intensive ISS�of adavani the inventors of the present invention have found, modified TM2 protein with mutations of one or more residues selected from the group consisting of:

1) of the arginine residue (R104) at position 104 of SEQ ID NO: 2;

2) lysine residue (K141) at position 141 of SEQ ID NO: 2;

3) lysine residue (K26) at position 26 of SEQ ID NO: 2; and

4) lysine residue (K73) at position 73 of SEQ ID NO: 2,

satisfies the requirements specified above as the modified TM2 type of the present invention, having a low pI. Thus, the invention was made.

The amino acid after mutation represents the balance of acidic amino acids (aspartic acid or glutamic acid) or the balance of neutral amino acids (asparagine, serine, glutamine, threonine, glycine, tyrosine, tryptophan, cysteine, methionine, Proline, phenylalanine, alanine, valine, leucine or isoleucine).

In addition, because non-polar amino acid may lead to nonspecific binding due to hydrophobic interaction, the balance of the acidic or neutral amino acid preferably has a hydrophobicity index of 2 or less. The hydrophobicity index determines the degree of hydrophobicity of each amino acid residue, as described, for example, article Kyte and Doolittle, J. Mol. Biol., 157, 105-132 (1982), and is well known to specialists in this field of technology. "The remnants of acidic amino acid residues or a neutral amino acid�from, having a hydrophobicity index of 2 or less are aspartic acid and glutamic acid, which are the remnants of an acidic amino acid, or asparagine, serine, glutamine, threonine, glycine, tyrosine, tryptophan, methionine, Proline and alanine, which are neutral amino acid residues.

More preferably 1) the arginine residue at position 104 of SEQ ID NO: 2 and/or 2) a lysine residue at position 141 of SEQ ID NO: 2 is replaced with an acidic amino acid residue or a neutral amino acid residue. More preferably 1) the arginine residue at position 104 of SEQ ID NO: 2 and/or 2) a lysine residue at position 141 of SEQ ID NO: 2 is replaced with an acidic amino acid residue.

K26 is preferably replaced by A (alanine), K73 preferably replaced by Q (glutamine), R104 is preferably replaced with E (glutamic acid) or D (aspartic acid), and K141 preferably replaced with E (glutamic acid) or D (aspartic acid); and R104 is more preferably replaced by E, and K141 more preferably replaced by E.

The modified TM2 of the present invention, having a low pI, has an isoelectric point that is significantly lower than that of the wild type TM2. Specifically, in paragraphs 1-8) of example 1 revealed that the isoelectric point of each of the modified TM2 having the mutation, reduced by 1 or more compared with isoelectric points of contact�th TM2 wild-type.

The results show that the modified TM2 protein of the present invention is characterized by lower non-specific binding with nucleic acids and/or protein compared to the protein binding TM2 wild type or mutant type. Specifically, in paragraphs 1-11) of example 1 showed a reduction in nonspecific binding with DNA. Moreover, in paragraphs 1-9) of example 1 showed a reduction in nonspecific adsorption of whey protein. Non-specific binding and adsorption are both reduced to approximately 60% of the binding of wild-type TM2 as a result of mutations K26, K73 or K141. In R104-K141 (both R104 and K141 motroway) non-specific binding and adsorption both fall to levels close to 20% of the binding TM2. On the contrary, although it was reported on the reduction of nonspecific binding with DNA or cells with the avidin mutant in basic amino acids (Marttila et al., (2000) FEBS, 467, pp. 31-36), for such a large decrease nonspecific binding to serum proteins has not been reported.

The study of the binding of the modified TM2 of the present invention, having a low pI, fibronectin revealed a significant reduction of binding of fibronectin. Fibronectin is a cell adhesion molecule that is present in the extracellular matrix, and causes background noise especially in the determination of protein in plasma or serum. Such clicks�zoom, lower binding to fibronectin is preferred. As shown in paragraphs 1-10) of example 1, the level of binding of fibronectin decreased in all mutants compared to TM2. In particular, the levels of binding of fibronectin with K141 and R104-K141 both significantly reduced to 10 to 20% of the level of binding of the wild type TM2. It was not known any relationship between the magnitude of pI and fibronectine ability, and there really has not been any clear correlation between the value of pI and the degree of reduction of binding of fibronectin. In this regard, the lower levels of binding of fibronectin with mutants K141 and R104-K141 is unpredictable very large.

In one preferred modified TM2 of the present invention, which has reduced binding to fibronectin, lysine at position 141 in the amino acid sequence modified TM2 in the acidic amino acid or neutral amino acid, more preferably modified to an acidic amino acid or neutral amino acid with a hydrophobicity index of 2 or less, more preferably modified to E (glutamic acid) or D (aspartic acid) and most preferably in E.

Alternatively, in another modified TM2, which has reduced binding to fibronectin, R104 and K141, each modified to an acidic amino acid and�and neutral amino acid more preferably, each modified to an acidic amino acid or neutral amino acid with a hydrophobicity index of 2 or less, more preferably modified to E (glutamic acid) or D (aspartic acid) and most preferably in E.

Modified tamaminin having improved biotinidase ability

TM2 is very firmly binds to Biotin with a rate constant of Association (ka) at 9.19×105(M-1h-1), the rate constant of dissociation (kd) 6,83×10-6(h-1) and the dissociation constant (KD) is 7.43×10-12(M). ka, kd and KD streptavidin, another bytesmessage protein measured in a similar manner, were, respectively 2,28×106(M-1h-1), 2,52×10-6(h-1and of 1.11×10-12(M). This means that the strength of binding TM2 with Biotin in the same order as the strength of binding streptavidin, but slightly lower than that of streptavidin (patent WO 2008/081938 A1). In order biotinidase protein was associated with Biotin more quickly or more, preferably higher biotinidase ability.

The inventors of the present invention successfully obtained high-affinity modified TM2 having further improved biotinidase ability, by modifying the amino acid adhering to the shaft�in wild-type TM2, high source biotinidase ability. In high-affinity TM2 of the present invention, at least the aspartic acid residue at position 40 (D40) in the amino acid sequence SEQ ID NO: 2 representing TM2, modified. Amino acid after the modification is preferably N (asparagine).

Modification D40 can be carried out in conjunction with a modification of R104, K141, K26, and/or K73 for the reduction described above nonspecific binding (method 6), or may be performed alone (method 11). As shown in examples 2 and 3, the modified TM2 in which modified D40, has biotinidase activity, significantly higher than the activity of the wild type TM2.

Modified tamaminin having reduced non-specific binding and improved biotinidase ability

Modified tamaminin obtained with combined mutations of the amino acids described above, a "modified travidia with reduced non-specific binding" and "modified travidia with increased biotinidase ability", was characterized by reduced non-specific binding and increased thus biotinidase ability. The thus obtained modified tamaminin contains a mutation in at least one�th amino acid residues from K26, K73, R104 and K141 in the amino acid sequence TM2, and further comprises a mutation of amino acid residues D40, replaced by a mutation in N (method 6).

The amino acid after mutation K26, K73, R104 and/or K141 is an acidic or neutral amino acid, preferably an acidic or neutral amino acid with a hydrophobicity index of 2 or less (Kyte and Doolittle, J. Mol. Biol., 157, 105-132 (1982)). More preferably K26 mutated to A (alanine), K73 mutated to Q (glutamine), R104 mutated to E (glutamic acid) or D (aspartic acid) and K141 mutated to E (glutamic acid) or D (aspartic acid); and more preferably R104 mutated to E, and K141 mutated in E.

In a modified travidia, which is modified as R104 and D40 were observed increased affinity and reduction of nonspecific protein binding (in paragraphs 3-7 and 3-9) of example 3).

Modified tamaminin in which modified everything R104, K141 D40 and was characterized by a decrease in the isoelectric point, increased affinity, decreased nonspecific protein binding, decreased fibronectine ability and decrease non-specific binding to nucleic acids (in points 3-6), 3-7) 3-9), 3-10) and 3-11) of example 3). Moreover, unexpectedly, significantly improved the stability of the protein structure to heat (in paragraphs 3-8) approx�RA 3).

Accordingly, it is preferable to modify both amino acid residues D40 and R104 and more preferably to modify all residues D40, R104 and K141.

As described above, the modified TM2 of the present invention preferably, but not limited to those selected from the group consisting of:

modified bytesmessage protein (D40N-R104E) in which the aspartic acid residue in position 40 of SEQ ID NO: 2 is replaced with an asparagine residue and the arginine residue at position 104 is replaced by a glutamic acid residue;

modified bytesmessage protein (D40N-K141E) in which the aspartic acid residue in position 40 of SEQ ID NO: 2 is replaced with an asparagine residue, and the lysine residue at position 141 is replaced by glutamic acid residue; and

modified bytesmessage protein (D40N-R104E-K141E) in which the aspartic acid residue in position 40 of SEQ ID NO: 2 is replaced with an asparagine residue, the arginine residue at position 104 is replaced by a glutamic acid residue and a lysine residue at position 141 is replaced by a glutamic acid residue.

In a preferred embodiment of the modified TM2 protein of the present invention includes, for example, any of the amino acid sequences of SEQ ID NO: 8, 10, 16, 20, 22, 24 and 25.

More preferred are D40N-R104E, D40N-K141E and D40N-R104E-Key most preferred is� D40N-R104E-K141E.

The modified TM2 protein of the present invention satisfies at least one property selected from the following properties (i) to (iv):

(i) has an isoelectric point lower than the point of a protein consisting of the amino acid sequence represented by SEQ ID NO: 2;

(ii) exhibits lower nonspecific binding to nucleic acids and/or proteins in comparison to a protein consisting of the amino acid sequence represented by SEQ ID NO: 2;

(iii) has lower fibrocartilaginous activity compared to a protein consisting of the amino acid sequence represented by SEQ ID NO: 2; and

iv) exhibits a greater biotinidase activity compared to a protein consisting of the amino acid sequence represented by SEQ ID NO: 2.

In respect of the properties (i), since the isoelectric point of the wild type TM2 is approximately from 8.5 to 8.8, the isoelectric point of the modified TM2 of the present invention is preferably 8.0 or less and more preferably of 7.7 or less.

In respect of the properties (iii), whereas fibrocartilaginous property of the wild type TM2 defined as less from 1.3 to 1.4, fibronectins activity of the modified TM2 of the present invention is preferably 1.0 or less ,25 or less, or 0.15 or less.

Amino acid residue that is desirable not to modify the modified TM2 of the present invention

Modification of amino acid residues in the modified TM2 of the present invention should not affect biotinidase activity. In this regard, biotinidase pocket streptavidin, one bytesmessage protein, has to some extent been identified. The amino acid homology between streptavidin and TM2 is only about 50%. The inventors of the present invention compared the amino acid sequence of TM2 and streptavidin, in order to obtain information about biotinidase TM2 pocket. As a result, it was found that among the amino acids constituting biotinidase pocket streptavidin, residues directly interacting with Biotin, i.e., N23, S27, Y43, S45, N49, W79, S88, T90, W92, W108, W120 and D128 (Weber, et al., (1989) Science 243: 85-88, Livnah, et al., (1993) Proc. Natl. Acad. Sci. U. S. A. 90: 5076-5080) correspond N14, S18, Y34, S36, D40, W69, S76, T78, W80, W96, W108 and D116 TM2, respectively, and that biotinidase pocket highly conservative.

The only difference was that N (asparagine) at position 49 streptavidin in TM2 is a D (aspartic acid) at position 40, and biotinidase ability TM2 D40N, in which aspartic acid is modified to asparagine, as in streptavidin increased as described above. These Reza�taty guess what biotinidase pockets TM2 and streptavidin have very similar structures and that these amino acid residues are significantly involved in the binding with Biotin.

In particular, since four of the tryptophan residue (W69, W80, W96 and W108), are believed to be very important parts of the structure bytesmessage pocket, it is preferable that they have not been modified. At the same time, other amino acids in TM2, believed to be involved in the binding with Biotin is also preferred that the amino acid residues (N14, S18, Y34, S36, S76, T78 and D116), assumed to be directly interacting with Biotin, were not modified. Alternatively, in the case when these modified residues, preferably amino acids have been modified in the amino acids having similar properties and structures, to keep biotinidase ability. For example, asparagine (N14), it is desirable to modify in glutamine (Q) or aspartic acid (D) and preferably aspartic acid; aspartic acid (D40), it is desirable to modify the asparagine (N); serine (S18, S36, and S76) to modify the threonine (T) or tyrosine (Y), and preferably threonine; tyrosine (Y34) it is desirable to modify the serine (S), threonine (T) or phenylalanine (F), and preferably phenylalanine; threonine (T78) preferably Modific�in the presence of serine (S) or tyrosine (Y), and preferably serine; and aspartic acid (D116) it is desirable to modify to glutamic acid (E) or asparagine (N) and preferably asparagine.

A method of modifying amino acids

The modified TM2 of the present invention can be obtained by modifying amino acids or amino acid TM2 using the known method, which causes a mutation in the amino acid sequence without any restrictions. Preferably the modification is achieved by obtaining the nucleotide sequence of a nucleic acid encoding the modified protein of the present invention.

For example, in order to modify the amino acid in a specific position of the amino acid sequence, can be applied a method using PCR (Higuchi, et al., (1988), Ho, et al., (1989)). This means that they are carrying out PCR using a primer containing the erroneously paired codon to mutate the target with obtaining a DNA that encodes a target mutant, and allow the DNA to Express the target mutant.

Variants or mutants caused by deletions, substitutions, insertions and/or additives, can be obtained from natural DNA encoding the protein, by applying the well-known technique, for example, site-specific mutagenesis (see, for example, the journal Nucleic Acid Research, Vol. 10, No. 20, p. 6487-6500, 1982, included in this description in the quality�e reference in its entirety). Site-specific mutagenesis can be carried out as follows using synthetic oligonucleotide primers that are complementary, single-stranded DNA phage, subjected to mutations, except for specific bugs mating, which is consistent with the desired mutation. More specifically, the above synthetic oligonucleotides are used as primers for the synthesis chain, complementary to the phage, and a host cell transformed received desperatley DNA. Culture of transformed cells plated onto agar, and from single cells containing phage to form plaques. Then, theoretically, 50% of new colonies contain the phage having a mutation in a single chain, and the remaining 50% have the original sequence. Received plaque hybridized with a synthetic probe labeled by treatment with kinase, at a temperature, which allows you to gibridizatsiya with those colonies that exhibit a complete pairing with the DNA having the above desired mutation, but it cannot gibridizatsiya with those colonies that have the original chain. Then plaques that hybridized with the probe, harvested and cultured for DNA.

Nucleic acid that encodes the modified protein tamaminin 2 (TM2)

The present invention provides a nucleic acid that encodes �modifitsirovannyi TM2 of the present invention. A nucleic acid includes, for example, the nucleotide sequence obtained by modifying the nucleotide sequence (SEQ ID NO: 1) TM2 in the nucleotide sequence encoding the modified TM2 protein having a modified amino acid or modified amino acids. The nucleotide sequence of the subject modification is not limited as long until the nucleotide sequence after modification encodes a modified amino acid or modified amino acid. Their examples include a nucleic acid having a nucleotide sequence, modified for the modification of the present invention in a nucleic acid consisting of the nucleotide sequence of SEQ ID NO: 1, or nucleic acid, which hybridise with a complementary chain of nucleic acid, consisting of nucleotide sequence SEQ ID NO: 1, under stringent conditions, and encodes a protein having biotinidase activity (denoted in the present description hereinafter referred to as "TM2 gene").

A nucleic acid of the present invention preferably encodes the amino acid sequence: SEQ ID NO.: 8, 10, 16, 20, 22, 24 or 25, and more preferably encodes the amino acid sequence of SEQ ID NO: 22 or 24. A nucleic acid of the present image�etenia preferably comprises nucleic acid sequences: SEQ ID NO: 7, 9, 15, 19, 21 or 23, and more preferably comprises nucleic acid sequence SEQ ID NO: 21 or 23.

A vector containing a nucleic acid of the present invention

The present invention provides a vector containing a nucleic acid encoding the modified TM2 protein. The vector is an expression vector for expression of the modified TM2 protein.

Nucleic acid that encodes the modified TM2 protein of the present invention, is a nucleic acid, as described in the section "Nucleic acid that encodes the modified protein tamaminin 2", and is not particularly limited.

The vector may be of the recognition site of restriction enzymes or sequencing used in the Gateway system (Invitrogen), such as aatB1, aatB2 or aatB3, on one end or on both ends of the nucleic acid encoding the modified TM2 protein. Moreover, the promoter and terminator, which operate in the desired cells of the host can be localized, respectively, above and below, the nucleic acid encoding the modified TM2 protein.

The type of site recognition by restriction enzymes is not particularly limited, but the expression vector preferably has only one type of site recognition. Number of recognition sites is not particularly limited, but is Odie� or more and preferably 10 or more.

Moreover, a nucleic acid sequence encoding a linker amino acid sequence (which is not particularly limited and may be usually used by professionals in the art, for example, a sequence containing a large number glycinol and Surinov) consisting of at least one amino acid, preferably at least five amino acids, more preferably at least ten amino acids, more preferably at least 25 amino acids and at most 50 amino acids, may be located between the site of recognition by the restriction enzyme and nucleic acid modified TM2, or sequence between aatB and nucleic acid modified TM2. In addition, for example, can be located in the sequence encoding the site of the recognition enterokinase or protease, such as factor Xa, although it is not particularly limited.

For example, if the gene encoding the antibody such as scFv or Fab, is inserted into this expression vector in reducing conditions that are not suitable for expression of the hybrid protein, such as intracytoplasmic protein, a nucleic acid sequence encoding a leader peptide such as a signal peptide or signal the�AI, may be located between the promoter and the unit including the sequence for inserting nucleic acid encoding the modified tamaminin.

The vector of the present invention preferably is an expression vector. In addition to the gene expression unit of the expression vector may include a unit to allow replication in appropriate cells of the host, e.g. origin of replication or a marker of drug resistance for selection of the desired host cells. A host is not particularly limited, but preferably is an Escherichia coli. In addition, there may be included a suitable control system for the expression, such as a system of repression of lactose in Escherichia coli.

The medium for the immobilization of a modified travidia

The present invention provides a carrier for immobilization of the modified TM2 protein of the present invention.

The material constituting the carrier may be any known material, and examples include, but are not limited to, cellulose, Teflon, nitrocellulose, agarose, dextran, chitosan, polystyrene, polyacrylamide, polyester, polycarbonate, polyamide, polypropylene, nylon, polydivinylbenzene, latex, silica, glass, fiberglass, gold, platinum, silver, copper, iron, stainless steel, ferrite, silicon wafer, polyethylene, polyethyleneimine, a polymer of lactic acid, resins, polysaccharides, proteins (e.g., albumin), carbon, and combinations thereof. The preferred media has a certain level of strength and a stable composition and is characterized by low non-specific binding.

A solid carrier can be any shape, including but not limited to, beads, magnetic beads, thin films, microtubules, filters, plates, microplates, carbon nanotubes and touch micrometric. Flat solid media, such as thin films and tablets may be provided with holes, channels, bottom filter grids or the like, as known in the art.

In an embodiment of the present invention, the balls can have a diameter spheres in the range from about 25 nm to about 1 mm. In the preferred embodiment, the implementation of the balls can have a diameter in the range from approximately 50 nm to approximately 10 μm. Beads size can be selected depending on the intended purpose. Since some bacterial spores have a size of approximately 1 μm, preferred beads to capture these spores have a diameter greater than 1 micron.

Immobilization of protein on the carrier is not particularly limited, and may be implemented � by using a known method of binding protein with a carrier. Specifically, a suitable method can be selected by the specialists in the art depending on the media type and the like.

Advantages of the invention

The present invention proposes a modified tamaminin that exhibits improved properties, such as the reduction of nonspecific binding and/or further improvement of Biotin binding while retaining the characteristics of travidia, i.e. high biotinidase abilities. The use of a modified travidia to determine, for example, in immunotest method or hybridization of the nucleic acid for measurement of an analyte using a binding avidin-Biotin may reduce the background, to increase the sensitivity and maintain the property of binding to Biotin under stringent conditions (e.g. at high temperature in the presence of a denaturing agent and enzyme).

Brief description of figures

[Fig.1] In Fig.1 shows non-specific binding of modified proteins TM2 of the present invention with a low pI with serum proteins, immobilized on magnetic beads (**p<0.01 relative to TM2).

[Fig.2] In Fig.2 shows non-specific binding of modified proteins TM2 of the present invention with a low pI with fibronectin (*p<0.01 relative to TM2).

[Fig.3] Fig.3 pre�shown nonspecific binding of modified proteins TM2 of the present invention with a low pI with DNA.

[Fig.4] In Fig.4 shows nonspecific adsorption of serum protein on magnetic beads with immobilized TM2 protein of the present invention, characterized by low non-specific binding of high affinity, (*p<0.1 in relation to TM2).

[Fig.5] Fig.5 shows non-specific binding of proteins TM2 of the present invention, characterized by low non-specific binding of high affinity, with fibronectin (*p<0.01 relative to TM2).

[Fig.6] Fig.6 shows non-specific binding of proteins TM2 of the present invention, characterized by low non-specific binding of high affinity with DNA, where the upper, middle and lower columns presents the results for wild-type TM2, TM2 R104EK141E and TM2 D40NR104EK141E, respectively.

Examples

The present invention will be described specifically with reference to the following examples, but the examples are not intended to limit the technical scope of the present invention. Specialists in the art can easily add modifications/changes to the present invention on the basis presented in this paper describe. And such modifications/changes are included in the technical scope of the present invention.

Example 1: Creation and analysis of TM2 with low pI

1-1)Create a TM2 with low pI

For �CSOs to reduce the isoelectric point TM2, the principal amino acids in TM2 was replaced by a neutral amino acid or acidic amino acid to create the following seven mutants.

(1) the TM2 Mutant in which the lysine in position 26 is replaced by alanine (in the present description hereinafter referred to as "TM2 K26A"; its nucleotide sequence is presented in SEQ ID NO: 3 and amino acid sequence in SEQ ID NO: 4);

(2) the TM2 Mutant in which lysine at position 73 replaced by glutamine (in the present description hereinafter referred to as "TM2 K73Q"; its nucleotide sequence is presented in SEQ ID NO: 5 and amino acid sequence in SEQ ID NO: 6);

(3) the TM2 Mutant in which arginine at position 104 is replaced by glutamic acid (in the present description hereinafter referred to as "TM2 R104E; its nucleotide sequence is presented in SEQ ID NO: 7 and amino acid sequence in SEQ ID NO: 8);

(4) the TM2 Mutant in which lysine at position 141 is replaced by glutamic acid (in the present description hereinafter referred to as "TM2 K141E; its nucleotide sequence is presented in SEQ ID NO: 9 and amino acid sequence in SEQ ID NO: 10);

(5) the TM2 Mutant in which lysine at position 33 is replaced by threonine, and lysine at position 37 is replaced by alanine (in the present description hereinafter referred to as "TM2 K33TK37A"; its nucleotide sequence is presented in SEQ ID NO: 11 and amino acid followers�nost in SEQ ID NO: 12);

(6) the TM2 Mutant in which lysine at position 33 is replaced by threonine, the lysine at position 37 is replaced by alanine, and arginine at position 104 is replaced by glutamic acid (in the present description hereinafter referred to as "TM2 K33TK37AR104E"; its nucleotide sequence is presented in SEQ ID NO: 13 and amino acid sequence in SEQ ID NO: 14); and

(7) the TM2 Mutant in which arginine at position 104 is replaced with glutamic acid, and lysine at position 141 is replaced by glutamic acid (in the present description hereinafter referred to as "TM2 R104EK141E"; its nucleotide sequence is presented in SEQ ID NO: 15 and amino acid sequence in SEQ ID NO: 16).

(8) the TM2 Mutant in which the lysine at position 19 is replaced by threonine (in the present description hereinafter referred to as "TM2 K19T", its nucleotide sequence is presented in SEQ ID NO: 17 and amino acid sequence in SEQ ID NO: 18).

E in R104E and T and A in K33TK37A determined by comparing the amino acid sequences of TM2 and streptavidin with reference to the relevant provisions in the sequence of streptavidin. E in K141E determined with reference to an amino acid in the corresponding position in the sequence travidia 1.

At first, in order to create a TM2 with a low pI, primers were created for the introduction of each mutation: primer Tm2NtermPci consisting of 5'-site of the TM2 gene and the sequence of the PcI encoding the cleavage site of the restriction enzymes (ACATGT) located above the 5'-site created primer Tm2CtermBam consisting of 3'-site of the TM2 gene and a BamHI sequence encoding the cleavage site of the restriction enzymes (GGATCC) below 3'-site, and a series of semantic primers containing the erroneously paired codons for each mutant and antisense primers were as follows (SEQ ID NO: 26 through 37):

Table 1: primers for creating TM2 with low pI

Table 1
NameSequence (5'-3')Length
Tm2NtermPci
AAAACA TGTCAG ACG TTC AAT CTT C25 members
Tm2CtermBam
TTTGGA TCCTTA CTT CAA CCT CGG TGC G28 members
Tm2 K26A PciIFW
TTT TTT ACA TGT CAG ACG TTC AAT CTT CAC TCA CCG GAA CCT GGT
ACA ATG AAC TCA ACT CCA AGA TGG AAT TGA CTG CAA ACG CAG ACG
GTA CTC TCA CTG GAA AGT
108 members�s
Tm2 K73Q F
TCC TGG GAG AAC AGT CAA ATT CAT TCC GCT ACG33 members
Tm2 K73Q R
TCC TGG GAG AAC AGT CAA ATT CAT TCC GCT ACG33 members
Tm2 K33,37TA F
ACT CTC ACT GGAACGTAC CTC TCCGCAGTT GGG GAT GTC39 members
Tm2 K33,37TA R
GAC ATC CCC AACTGCGGA GAG GTACGT TCC AGT GAG AGT39 members
Tm2 R104E F
TCG AGC ACT GCGGAAGGG GAC TGG GTA27 members
Tm2 R104E R
CCA TAC GTC CCCTTCCGC AGT GCT CGA27 members
Tm2 K141E Bam
TTTGGA TCCTTACTCCAA CCT TGC CG GCG 30 members
Tm2 K19T F
GAA CTC AAC TCCACGATG GAA TTG ACT27 members
Tm2 K19T R
AGT CAA TTC CATCGTGGA GTT GAG TTC27 members

From top to SEQ ID NO: 26 to 37

The recognition sites of the restriction enzymes are underlined, and the sites of mutations are shown by dashed lines.

1-2) PCR

To create TM2 gene with low pI, performed a two-step PCR. In the first stage PCR using the plasmid vector pTrc99A containing as a matrix TM2 gene, 5'-site was amplified using primer Tm2NtermPci and antisense primer Tm2 K26A R, Tm2 K73Q R, Tm2 K33TK37A R, Tm2 R104E R or Tm2 K19T R containing the erroneously paired each mutant codon, and 3'-site was amplified using primer Tm2CtermBam and semantic primer Tm2 K26A F, Tm2 K73Q F, Tm2 K33TK37A F, Tm2 R104E F or Tm2 K19T F containing the erroneously paired codon of each mutant.

In the case of TM2 K141E mutation was introduced in a single PCR reaction with primers Tm2NtermPci and Tm2 K141E Bam.

PCR was carried out under the reaction conditions: 50 µl reaction solution containing the matrix DNA (500 ng), the buffer 10×yrobest (Takara, 5 µl), 2.5 mm dNTP (4 μl), primers (25 pmol each), and 5 U/µl DNA polymerase Pyrobest (Takara, 0.5 ál) and started with 3 min at 96°C followed by ten cycles of 1 min at 96°C, 1 min at 55°C and 2 min at 72°C and finished with 6 min at 72°C in the system with programmable temperature control PC-700 (ASTEK). As a result, the PCR products that were approximately 120 BP TM2 K33TK37A and approximately 330 BP TM2 K R104E, received at 5'-site and approximately 310 BP TM2 K33TK37A, approximately 100 BP TM2 K R104E and approximately 60 BP TM2 K19T received in the 3'-site. In the case of TM2 K141E received a PCR product of 430 BP

These PCR products were subjected to electrophoresis on agarose using agarose with low melting point (SeaPlaqueGTG) in TAE buffer. Each DNA fragment was cut out with the gel and there was added the same as that of the gel with 200 mm NaCl followed by treatment at 70°C for 10 min to melt the gel. This sample was subjected to phenol extraction, extraction with phenol/chloroform and chloroform extraction, each once, and the DNA fragments of the 5'site and 3'-site was collected by precipitation with ethanol. Using these fragments as template, was carried out the second stage PCR for the creation of genes, except TM2 K141E, using primers Tm2NtermPci and Tm2CtermBam. The reaction conditions were the same as those in the first stage PCR. The result is each PCR product of 430 BP

1-3) clone king�R

The TM2 gene fragments with low pI, obtained by PCR, cloned into the vector pCR4 Blunt TOPO (Invitrogen). The ligation reaction was carried out in accordance with the instruction attached to the kit of the vector. DNA was introduced inEscherichia coliTB1 by electroporation, and plasmid DNA was extracted in accordance with a conventional method (Sambrook, et al., 1989, Molecular Cloning, A laboratory manual, 2ndedition). Nucleotide sequences of PCR products of clones, in which was reaffirmed the insert was determined from both ends using M13 primer (Takara) using a fluorescence sequencer ABI PRISM Model 310 Genetic Analyzer, Perkin Elmer) to confirm the modification of the nucleotide of the target.

The plasmid in which the gene (its nucleotide sequence was confirmed) introduced in CR4 Blupnt TOPO, double-hydrolyzable PciI and BamHI, and the DNA fragment was collected using a cleaning gel in accordance with the above-described method. The fragment was ligated into the expression vector pTrc99A forEscherichia colithat hydrolysable NcoI and BamHI in advance using ligation kit (Takara). The ligation product was transformed intoEscherichia coliTB1, and extraction of plasmid DNA and analysis by using the restriction enzymes was carried out according to a conventional method to confirm the presence of inserted gene to obtain vectors expressing TM2 protein with low pI, TM2 K26A/pTrc99A, TM2 K73Q/pTrc99A, TM2 K33TK37A, TM2 104E/pTrc99A, TM2 K141E/pTrc99A and TM2 K19T/pTrc99A. In addition, the gene encoding TM2 R104EK141E, was created by introducing mutations by PCR using vector TM2 R104E/pTrc99A as template and using primers Tm2NtermPci and Tm2 K141E Bam. The gene encoding TM2 K33TK37AR104E, was created by introducing mutations by PCR using vector TM2 K33TK37A/pTrc99A as template and using primers Tm2NtermPci and Tm2 K141E Bam and was cloned using the same method as described above.

1-4) the Expression of TM2 with a low pI in Escherichia coli

Escherichia coliTB1 transformed TM2/pTrc99A with low pI, inoculable in LB culture medium (6 ml) containing an antibiotic, ampicillin (final concentration: 100 μg/ml), and cultured with shaking at 37°C until the absorbance of 0.5 at 600 nm, OD600then added 1 mm IPTG, and then cultured with shaking at 37°C over night.Escherichia colicollected from 1 ml of culture solution by centrifugation and suspended in 20 mm phosphate buffer (pH 7, 400 μl) followed by destruction of bacterial cells by treatment with ultrasound. The solution after destruction centrifuged (15,000 rpm) to obtain a soluble fraction in the supernatant.

The soluble fraction was analyzed by immunoblotting: soluble fraction and the same volume of sample buffer 2×SDS (250 mm Tris-HCl, pH6,8, 20% 2-mercaptoethanol, 20% SDS, 20% glycerol) were mixed and heated at 95°C for 10 minutes followed by separation on SDS-PAGE for analysis by immunoblotting using antibodies against rabbit TM2 (PCT/JP2006/326260) as the first antibody and anticalcium IgG antibodies labeled with alkaline phosphatase (BIO-RAD), as the second antibody. The results of the analysis immunoblotting showed that he had detected a band of approximately 15.5 kDa eachEscherichia colitransformed TM2/pTrc99A with low pI, but the band was not detected inEscherichia colitransformed with the vector pTrc99A containing no gene TM2 with a low pI. The sizes of these bands were consistent with a molecular mass of 15.5 kDa, monomer, predicted amino acid sequence TM2.

The formation of tetramers of mutants in TM2 Undenatured state was confirmed in accordance with the method of Bayer, et al., (1996, Electrophoresis, 17(8), 1319-24). This means that mixed the SDS sample buffer containing no reducing agent, such as DTT or mercaptoethanol, and soluble fraction of the mutant TM2, with subsequent analysis by SDS-PAGE without temperature treatment. As a result of the band having the same size as the strip of wild-type TM2, determined each mutant, which demonstrated the formation of tetramers. The level of expression of soluble TM2 protein with low pI was 20 mg per 1 l culture�professional solution for each TM2 K26A, TM2 K73Q, TM2 R104E, TM2 K141E and TM2 R104EK141E. This is equivalent to the expression level of the wild type TM2.

On the contrary, in contrast to these mutants, the expression levels in TM2K33TK37A, TM2 K33TK37AR104E and TM2 K19T, each had as low as 2 mg.

1-5) measuring the activity of fluorescent Biotin

Biotinidase ability TM2 with low pI, expressed inEscherichia coliwas confirmed in accordance with the method of Biochim. Biophys. Acta, 1427, 44-48 (1999): prepared solutions (each 150 ml) of buffer for analysis (50 mm NaH2PO4, 100 mm NaCl, 1 mm EDTA (pH 7,5) containing graduale growing number of solution of the extract, Proektirovanie from 25 ml of culture solution of each TM2 with low pI using 1.5 ml of 20 mm phosphate buffer (pH 7). Each of these solutions and 10 pmol/µl solution of fluorescent Biotin (Biotin-4-fluorescein: Molecular Probe, 50 ál (500 pmol)) were mixed and introduced into the reaction at room temperature for 10 minutes followed by measuring the fluorescence intensity at Ex=460 nm and Em=525 nm using the Infinite M200 (TECAN).

The results showed that the fluorescence intensity decreased proportionally to the increase in the number of extract TM2 with a low pI. As a result, it is demonstrated that full-sized TM2 mutant in which the basic amino acid in TM2 replaced by a neutral or acidic amino acid, b is associated with�ethnobotany connection.

1-6) Cleaning TM2 with low pI

TM2 with a low pI was purified in accordance with the method of Hofmann, et al., (1980) using a column filled with 2-iminobiotin-separate (Sigma). Each culture solutionEscherichia coli(25 ml) with induced expression of TM2 with a low pI was mixed with 50 mm CAPS (pH 11, 1.5 ml) containing 50 mm NaCl to suspend cells, with subsequent destruction by ultrasound. The supernatant was applied onto a column Packed with 2-iminobiotin-agarose (500 ál). The column was fully washed with 50 mm CAPS (pH 11) containing 500 mm NaCl, followed by elution with 50 mm NH4OAC (pH 4). The amount of each purified TM2 protein with low pI was approximately at the same level as the amount, expressed inEscherichia coliwith demonstrated a purity of 95% or more.

1-7) Measurement biotinidase ability

The test of Biotin binding TM2 with a low pI was performed using Biacore (registered trademark) 3000 (Biacore, biosensor, which uses the principle of surface plasmon resonance). Biotinylating serum albumin bull (BSA) was immobilizovana on the touch matrix CM5 (Biacore) using amine crosslinking with EZ-Link (registered trademark) NHS-LC-Biotin (Of 22.4 Å) or with EZ-Link (registered trademark) NHS-LCLC-Biotin (30.5 Mm Å) (both are products PIERCE). HBS-EP (Biacore) was used as running buffer, the vehicle every TM2 with low pI (40 µl) was injected at 25°C and the flow rate of 20 µl/min for 2 minutes.

From the resulting sensorgram calculated the rate constant of Association (ka), the rate constant of dissociation (kd) and dissociation constant (KD) using computer analysis programs, BIAevaluation version 4.1. Table 2 presents the results. The values presented in the table in brackets indicate that they are below the limit of determination Biacore 3000 (ka<5×10-6). Each TM2 with low pI detects specific binding with Biotin and thus demonstrates that the mutation had no significant impact on biotissues ability.

Table 2: Analysis of the interaction between TM2 with low pI and Biotin

Table 2
The sample namekaKdKD
BSA-LCLC-Biotin (30.5 mm Å)
TM2 R104EOf 5.8×105(5,6×10-7)(9,9×10-13)
TM2 K141EOf 5.8×105(Of 1.1×10-6)(2,0×10-12)
TM2 K33TK37AOf 2.5×105 (7,2×10-7)(2.3 x 10-12)
TM2 K33TK37AR104EA 6.3×105(Of 1.4×10-6)(2,7×10-12)
TM2Of 9.2×1056,8×10-6Of 7.4×10-12
BSA-LC-Biotin (of 22.4 Å)
TH2 K26AA 5.0×105(6,5×10-7)(Of 1.7×10-12)
TM2 K73QOf 5.9×105(Of 1.7×10-6)(2,8×10-12)
TM2 R104EK14TEOf 1.2×106(5,8×10-10)(5,0×10-16)
TM2Of 9.9×105(1,2×10-6)(Of 1.3×10-12)

1-8) Electrophoresis with isoelectric focusing

The isoelectric point of each TM2 with low pI were measured by electrophoresis with isoelectric focusing using the XCell SureLock Mini-Cell (Invitrogen). Conducted�and with each instruction TM2 with low pI (500 ng) and the IEF sample buffer (pH 3 to 10 (2×), Invitrogen) were mixed and added to the polyacrylamide gel (pH 3 to 10, IEF Gel, Invitrogen) at a pH in the range from 3 to 10, followed by electrophoresis at a regulated voltage of 100 V for 1 hour, 200 V for 1 h and 500 V for 45 minutes in this order.

After electrophoresis, the gel was shaken in the buffer for blotting (of 0.7% acetic acid) for 10 minutes and then transferred to a PVDF membrane at 10 V for 1 hour using XCell ll Blot Module (Invitrogen). The PVDF membrane was injected into the reaction with a rabbit antibody against TM2 (patent PCT/JP2006/326260) serving as a first antibody and with anticalcium IgG antibody labeled with alkaline phosphatase (BIO-RAD) serving as the second antibody, with subsequent determination of the bands set II <VECTOR Black> (VECTRO) with a substrate of alkaline phosphatase.

The results revealed that the isoelectric point of each TM2 mutant was lower than the isoelectric point of the wild type TM2 1 or more. Table 3 presents the values pI, actually measured by electrophoresis with isoelectric focusing and the value of pI, calculated using Genetyx.

Table 3: isoelectric point TM2 with low pI

Table 3
The sample nameCalculated pITM2 K19T6,36,6-7,5
TH2 K26A6,37,3-7,6
TH2 K73Q6,37,7
TM2 R104E5,97,0-7,5
TH2 K141E5,96,6-7,2
TM2 K33TK37A5,86,2
TM2 R104EK141E5,16,2
TM2 K33TK37AR104E5,15,9-6,0
TM27,48,5-8,8

1-9) Nonspecific binding to human serum

In this embodiment, the implementation of nonspecific binding to human serum was investigated as unspecific adsorption of TM2 with low pI on magnetic beads with immobilized serum proteins. Specifically, in order to investigate the nonspecific binding TM2 with low pI, measured the amount of TM2 with low pI, adsorbirovanny�e on magnetic beads, to which are covalently attached to serum proteins. TM2 K26A, TM2 K73Q, TM2 R104E, TM2 K141E and TM2 R104EK141E that expressibility in a high degree inEscherichia coliused as a TM2 with a low pI, explored.

Protein human serum and magnetic beads linked with each other as follows. Magnetic beads having surfaces coated with carboxyl groups (Dynabeads M-270 carboxylic acid Acid, Dynal, 210 μl), washed with 0.01 n sodium hydroxide (210 μl) for 10 minutes and then ultrapure water (210 μl) for 10 minutes three times. To the washed magnetic beads were added hydrochloride 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, PIERCE) dissolved in cooled ultrapure water to a final concentration of 0.2 M, and the mixture was shaken at room temperature for 30 minutes. Then, magnetic beads were washed with a cooled ultrapure water (210 ml) and then with 50 mm MES buffer (pH 5.0, 210 µl).

Magnetic beads were added to 1 mg/ml protein human serum (CHEMICON, 210 µl), cialisovernight against 50 mm MES buffer (pH 5.0). The mixture was shaken at room temperature for 30 minutes for the covalent binding of the protein human serum using magnetic beads. Magnetic beads were collected with a magnet to remove the supernatant. Unreacted active groups on the beads were removed with 50 mm Tris buffer (pH 7.0, 210 µl), and then mA�magnetic beads were blocked with PBS buffer (420 μl), containing 0.5% BSA and 0.1% tween 20. Magnetic beads suspended in PBS buffer (210 μl) to obtain the magnetic beads with immobilized protein human serum.

Magnetic balls (7 μl) and 0.56 μg/ml of each TM2 with low pI were mixed to interaction at room temperature for 1 hour. Magnetic beads were collected by magnet and supernatant was removed. After washing in 20 mm potassium phosphate buffer (500 μl) containing 500 mm sodium chloride, magnetic beads were mixed with 20 µl of the sample buffer 2×SDS (250 mm Tris-HCl, pH 6,8, 20% 2-mercaptoethanol, 20% SDS, 20% glycerol), followed by heating at 95°C for 20 minutes for dissociation TM2 with low pI from magnetic beads.

This sample was subjected to SDS-PAGE and then analyzed immunoblotting to determine the number TM2 with a low pI. Rabbit antibody against travidia 2 and goat antibody against rabbit IgG labeled with alkaline phosphatase, was used as the first and second antibodies, respectively. The strip was determined by set II <VECTOR Black> (VECTRO) with a substrate of alkaline phosphatase and quantitatively determined using Las-3000 (FUJIFILM). The results are presented in Fig.1.

As shown in Fig.1, adsorption TM2 with low pI to whey protein was reduced compared with wild-type TM2 10% in TM2 R104E, 30% in TM2 K26A, 40% K73Q and K141E and 70% in TM2 R104EK141E. Thus, nonspecific binding�tion K26A, K73Q, K141E and K104EK141E was significantly lower compared to non-specific binding TM2.

1-10) Nonspecific binding to fibronectin

Binding to fibronectin of each TM2 with low pI compared with the use of the microplate, which was immobilized fibronectin. The concentration of fibronectin was adjusted to 50 μg/ml with the fixing solution exclusively for New tablet ELISA, and a solution of fibronectin (50 ml) was added to each tablet New ELISA (Sumitomo Bakelite), followed by shaking at 37°C for 4 hours for immobilization. Then the plate was washed three times with PBS containing 0.1% Triton X-100 (300 μl), and then dried in a natural way.

The TM2 mutants, K26A, K73Q, R104E, K141E and R104EK141E (50 µl each) was added to each corresponding well of the tablet was left at room temperature for 1 hour. The tablet was washed 3 times with 300 ál/well PBST (PBS buffer containing 0.1% tween 20). Then biotinylated HRP, diluted 5000 times in PBST containing 0.5% BSA, was added at 50 μl/well and allowed to react with the mutant at room temperature for 1 hour. After washing with 300 μl PBST/well, three times, to each well was added 50 μl/well of a solution of 1-step Ultra TMB-ELISA for the development of color. After stopping the color development under the influence of 2M sulfuric acid, 50 µl/well and measuring the absorbance at 450 nm using microplans�Togo reader Infinite 200. The results showed that the binding of fibronectin was significantly lower in the mutant TM2 with low pI compared to TM2.

The results are presented in Fig.2. As shown in Fig.2, the effect of reducing binding to fibronectin was elevated in R104EK141E, K141E, R104E, K26A and K73Q, in that order.

1-11) Nonspecific DNA binding

Analyzed nonspecific binding TM2 with low pI with DNA.

DNA salmon sperm, diluted stepwise from 10 mcg to 1 mcg 2×SSC buffer, denaturiruet alkali and were absorbed on Hybond N+membrane (Amersham Biosceinces) using Bio-Dot SF (BIO-RAD). After blocking the membrane with a solution of Denhardt × 5 (0,1% BSA, 0.1% ficoll, 0.1% polyvinylpyrrolidone) the membrane was immersed in a solution of 25 μg/ml TM2 with a low pI or wild-type TM2 at room temperature for 90 minutes. Then the membrane was washed with buffer TTBs extension (TBS buffer containing 0.05% tween 20) at room temperature for 5 minutes three times. The membrane was blocked with TBS buffer containing 3% skim milk and 0.1% tween 20, for 1 hour. The membrane was introduced into a reaction with biotinylated horseradish peroxidase (Vector), diluted 5000 times with TBS buffer containing 3% skim milk and 0.1% tween 20 at room temperature for 1 hour. The membrane was again washed with TBS buffer containing 0.1% tween 20, and then shaken in a solution that was a mixture of reagent 1 and reagent 2 from ECL (masham) in equal volumes, with subsequent determination by the reaction of fluorescence using Las-3000 (FUJIFILM).

The results are presented in Fig.3. As shown in Fig.3, TM2 only weakly associated with 10 μg DNA, but the binding of each mutant TM2 with a low pI was below the limit of determination. This shows that non-specific binding TM2 with low pI with DNA significantly reduced.

Based on the results of example 1 properties of the modified TM2 with low pI of the present invention are summarized in table 4.

Table 4: summary properties of the modified TM2 with low pI

tr>
Table 4
The binding of fluorescent BiotinpINonspecific binding of serum protein (%)The binding of fibronectin (A450)Nonspecific DNA binding
K19T+From 6.6 to 7.5
K26A+From 7.3 to 7.6680,85-
K73Q+7,7550,98-
R104E+From 7.0 to 7.5900,70-
K141E+From 6.6 to 7.2580,21-
K33T-K37A+6,2
K33T-K37A-R104E+From 5.9 to 6.0
R104E-K141E+6,2250,11-
TM2WT+From 8.5 to 8.8100To 1.34±

Example 2 With�the building and analysis of TM2 of high affinity

2-1) the Creation of TM2 of high affinity

To confirm the increase of affinity to Biotin in TM2 was introduced mutation of amino acid.

Several well-known streptavidin, for example, streptavidin v2 (the number of the Deposit: Q53533, Bayer, et al., (1995) Biochim Biophys Acta 1263: 60-66) and streptavidin v1 (the number of the Deposit: Q53532) from Streptomyces violaceus, and streptavidinStreptomyces avidinii(the number of the Deposit: P22629, Argarana, et al., (1986) Nucl Acids Res 14: 1871-1882) fromStreptomyces avidinii. As described in the patent WO02/072817, homology of amino acid sequences of these streptavidin with protein TM2 each is 50%, 48% and 48%, i.e. approximately 50%.

The inventors of the present invention suggested that the structure, similar to the structure of streptavidin necessary to maintain or increase biotinidase ability TM2 protein. Accordingly, the amino acid sequence of streptavidin and TM2 were compared in respect to tryptophan residues, which are believed to be is a very important part for the binding between streptavidin and Biotin, and residues that become involved in the formation of hydrogen bonds (Qureshi, et al., (2001), J. Biol. Chem. 276(49), pp. 46422-46428).

As a result, the inventors found that among residues that become involved in the formation of hydrogen bonds with Biotin, asparagine at position 49 posledovatelnostei different from the corresponding amino acids (aspartic acid at position 40) in the sequence of TM2. The inventors investigated whether or not modification of the D40 in TM2 in asparagine, the amino acid streptavidin type to increase the affinity.

First, to create a TM2 with a high affinity, primers were created for the introduction of the above mutations: primer Tm2NtermPci consisting of 5'-site of the TM2 gene and a sequence encoding the cleavage site of the restriction enzyme PciI (ACATGT) located above the 5'-site, and primer Tm2CtermBam consisting of 3'-site of the TM2 gene and a sequence encoding the cleavage site of the restriction enzyme BamHI (GGATCC), below the 3'-site that were the same as the primers described above. Series of sense primers containing the erroneously paired codons for each mutant and antisense primers were as follows (SEQ ID NO: 38 and 39):

Table 5: primers for creating TM2 of high affinity

Table 5
NameSequence (5'-3')Length
TM2 SA D40N F
TAC CTC TCC AAA GTT GGGAATGTC TAC GTG CCC TAC CCA39 members
TM2 SA D40N R
TGG GTA GGG CAC GTA GACATTCCC AAC TTT GGA GAG GTA39 members

SEQ ID NO: 38 and 39

The recognition sites of the restriction enzymes are underlined, and the sites of mutations are shown by dashed lines.

2-2) PCR and cloning

As described above, was designed TM2 mutant in which aspartic acid at position 40 was replaced by asparagine (in the present description hereinafter referred to as “TM D40N”; the nucleotide sequence of SEQ ID NO: 19 and amino acid sequence of SEQ ID NO: 20).

To construct the gene encoding the mutant TM2, repeated PCR in two stages. In the first stage PCR plasmid vector pTrc99A containing the TM2 gene, was used as template, the 5'-end was amplified using primer tm2NtermPci and antisense primer TM2 SA D40N R containing the erroneously paired codon mutant, whereas the 3'-end was amplified using a semantic primer TM2 SAD40N F containing the erroneously paired codon, and primer TM2CtermBam.

PCR was performed under the following reaction conditions: 50 µl reaction solution containing the matrix DNA (500 ng), 10×Pyrobest buffer (Takara, 5 μl), 2.5 mm dNTP (4 μl), primers (25 pmol each) and 5 U/µl DNA polymerase Pyrobest (Takara, 0.5 µl), the reaction was started with 3 min at 96°C followed by 10 cycles of 1 min at 96°�, 1 min at 55°C and 2 min at 72°C and finished by 6 min at 72°C in a system with a programmable temperature control PC-700 (ASTEK). As a result at the 5'-end were obtained PCR products of the expected size. These PCR products were subjected to electrophoresis in agarose using agarose with low melting point (SeaPlaqueGTG) in TAE buffer for purification of DNA fragments, as described above.

Using these fragments as matrices spent second stage PCR with primers Tm2NtermPci and Tm2CtermBam. The reaction conditions were the same as those in the first stage PCR. Received high-affinity fragment of the TM2 gene (430 BP was cloned into the vector pCR4 Blunt TOPO (Invitrogen) using the process described above. The result was obtained by expressing the protein TM2 D40N vector TM2 D40N/pTrc99A.

2-3) Expression of high-affinity TM2 in Escherichia coli

Escherichia coliTB1 transformed with the vector pTrc99A containing the mutant TM2 obtained in 2-2), inoculable in LB culture medium (6 ml) containing an antibiotic, ampicillin (final concentration: 100 μg/ml), and cultured with shaking at 37°C until the absorbance at 600 nm, OD600equal to 0.5.

Thereafter, the culture solution was added 1 mm IPTG, followed by shaking culture at 37°C over night.Escherichia colicollected from 1 ml of culture solution by centrifugation and suspended in 20 mm PHOS�atom buffer (pH 7, 400 μl) followed by destruction of bacterial cells by sonication. The solution is the destruction centrifuged (15,000 rpm) to collect the soluble fraction in the supernatant. Soluble fraction was subjected to analysis by immunoblotting: soluble fraction and an equal volume of 2×SDS buffer for the sample was mixed and heated at 95°C for 10 minutes followed by separation by SDS-PAGE to analyze immunoblotting using antibodies against rabbit TM2 (PCT/JP2006/326260) as the primary antibody and alkaline phosphatase labeled antibodies against rabbit IgG (BIO-RAD) as the secondary antibody.

The result was revealed strip of about 15.5 kDa inEscherichia colitransformed with the vector pTrc99A containing TM2 D40N, but was not identified inEscherichia colitransformed with the vector pTrc99A containing no mutant TM2. Band size was in accordance with the molecular mass of 15.5 kDa monomer, predicted based on the amino acid sequence of TM2. Then according to the method of Bayer et al. (1996, Electrophoresis, 17(8), 1319-24) confirmed the formation of tetramers high-affinity TM2 (with high affinity) to non-denatured state, as in the case of TM2 with a low pI. The results showed a band of the same size as the strip TM2 wild type in high-affinity TM2, which showed the formation of tetramers. The expression level of p�starimage TM2 protein was 20 mg per 1 l of culture solution.

2-4) Purification of high-affinity TM2.( TM2 with high affinity)

High-affinity TM2 was purified by the method of Hofmann, et al. (1980), as described above. As a result, the amount of purified TM2 mutant protein was essentially at the same level as the amount expressed inEscherichia coliprotein, with a purity of 95% or more.

2-5) Measurement of activity using fluorescent Biotin

Biotissues the ability of purified TM2 mutant was confirmed using the method published in Biochim. Biophys. Acta, 1427, 44-48 (1999). The results showed that the fluorescence intensity decreases in proportion to the increased quantity of the solution of the mutant TM2. This confirms that the D40N mutation does not significantly inhibits the binding TM2 with Biotin-like connection.

2-6) Electrophoresis with isoelectric focusing

Isoelectric point of the high-affinity TM2 were measured by electrophoresis with isoelectric focusing using the XCell SureLock Mini-Cell (Invitrogen). Table 6 shows the results of the analysis of high-affinity TM2 (200 ng) obtained in accordance with the instructions. Electric point TM2 D40N containing a substitution of basic amino acids was higher electric point of the wild type TM2 also during electrophoresis with isoelectric focusing.

Table 6
The isoelectric point of the high-affinity TM2
Name of templateCalculated pIThe observed pI
TM2 D40N8,69,7
TM27,4from 8.5 to 8.8

2-7) Measurement biotinidase ability

Intermolecular interactions were analyzed using the test of the binding of iminobiotin and test of Biotin binding high-affinity TM2 using a Biacore 3000 (Biacore).

2-7-1) Test the binding of iminobiotin

High-affinity TM2 and TM2, which are used as ligands to attach to the sensor chips, purified using the 2-iminobiotin-agarose in accordance with the traditional way and were dialyzed against 20 mm KPi (pH 7) overnight. These samples were diluted with 10 mm acetate buffer (pH 5, Biacore) to approximately 50 μg/ml.

The immobilization was carried out at 25°C and a flow rate of 10 μl/min using HBS-EP (Biacore) as the working buffer. TM2 and TM2 mutant (approximately 4000 to 8000 RU) immobilizovana on sensor chip CM5 (Biacore) by attaching amine. The activation time was set to 10 minutes.

Specific interaction was measured using iminobiotin-BSA as the analyte (the substance flowing through the flow channel) at 25°C and a flow rate of 20 µl/min and using CAPS buffer (50 mm CAPS, 150 mm NaCl, 0.005% tween 20, pH 11) as the working buffer. Iminobiotin-BSA was prepared as follows: Highly purified BSA (Sigma, 2 mg) and NHS-iminobiotin (Pierce, 1 mg) was dissolved in 50 mm sodium borate (pH 8,0, 1 ml), followed by incubation at 4°C for 2 hours. The solution was placed in a dialysis tube (MWCO 6-8,000) and were dialyzed against 50 mm sodium carbonate (pH6,7) at 4°With during the night. The obtained conjugate iminobiotin-BSA (mol. weight: 67 kDa, 30 μm) was used as the analyte to the biosensor Biacore (registered trademark). The time of the introduction of iminobiotin-BSA was 2 minutes and dissociation time was 10 minutes. The measurement was carried out by stepwise increasing concentration, starting with a low concentration, without stages of recovery. First introduced (2 minutes) 40 ál BSA, diluted in working buffer to 9,375 nm, 18.75 per nm to 37.5 nm, 75 nm, 150 nm, 300 nm and 600 nm, starting with the lower concentration in the flow cell, which was immobilized target protein, to measure dissociation. Then in the same flow cell similarly measured iminobiotin-BSA, obtained using the process,�Sanogo above.

Each constant for samples exhibiting interaction, calculated using the analysis program BIAevaluation ver. 4.1. Sensogram obtained for BSA as benchmarks in the same concentration as iminobiotin-BSA, subtracted from sensogram obtained for each concentration of iminobiotin-BSA, and received sensogram subjected to the analysis of the kinetics of interaction using the model binding 1:1 (Langmuir) to calculate the rate constants of binding (ka) and the rate constants of dissociation (kd). The dissociation constant (KD) were determined by kd/ka. In the case where the reduction step is not used, Rmax (the maximum number of bindable analyte) is reduced at each measurement. Accordingly, in the analysis of Rmax was calculated by fitting for each concentration and used only the results at concentrations (mainly from 18,75 to 75 nm) is close to the model binding 1:1 (Langmuir).

As a result, the rate constant of binding (ka) TM2 D40N with iminobiotin was increased by 40%, and the rate constant of dissociation (kd) was reduced by 45% compared to TM2, which resulted in a decrease in KD of approximately 60%. That is, the affinity TM2 D40N to iminobiotin was 2.5 times higher than the affinity TM2 (table 7).

2-7-2) Test of Biotin binding

Highly purified BSA (Sigma, 2 mg) and NHS-LC-Biotin (Pierce, 1 mg) was dissolved in 50 mm sodium borate (pH 8,0, 1 ml) and incubated at 4°C for 2 hours. The solution was placed in a dialysis tube (MWCO 6-8000) and were dialyzed against 50 mm sodium carbonate (pH of 6.7) at 4°With during the night. The resulting conjugate Biotin-LC-BSA (mol. weight: 67 kDa, 30 μm) was used as a ligand to the biosensor Biacore (registered trademark). In addition, high-affinity TM2 and TM2 D40N purified using the 2-iminobiotin-agarose as described above and were dialyzed against 20 mm KPi (pH 7)overnight to obtain analytes.

Biotin-LC-BSA and BSA, serving as negative control, were immobilizovana on sensor chip CM5 method of attaching amine. The level of immobilization was adjusted to approximately 200 RU. Chips on which was immobilized BSA was placed in flow cells 1 and 3, and chips, which was immobilized Biotin-LC-BSA, was placed in flow cells 2 and 4. Flow cells 1 and 2 were loaded TM2, and flow cells 3 and 4 were loaded high-affinity TM2 (D40N) at a flow rate of 20 µl/min for 2 minutes using the working buffer [10 mm HEPES, pH 7,4, 150 mm NaCl, 3 mm EDTA, 0.005% surfactant 20 (Biacore)].

After that monitored the dissociation of the samples for 60 minutes, but the associated high-affinity TM2 and TM2 (D40N) not dissociatively. Respectively, conducted a seven-speed measurement(3,125, 6,25, 12,5, 25, 50, 100 and 200 nm), starting with the lowest concentration, without stages Voss�of yavlenie. Data for BSA as a benchmark is subtracted from the data for Biotin-LC-BSA. The measurement was performed at 25°C. the resulting sensorgram subjected to the analysis of the kinetics of interaction using analysis program BIAevaluation ver. 4.1 for the model binding 1:1 to calculate the rate constants of binding (ka) and the rate constants of dissociation (kd). The dissociation constant (KD) were determined by kd/ka. In the case where the reduction step is not used, Rmax (the maximum number of bindable analyte) is reduced at each measurement. Accordingly, in the analysis of Rmax was calculated by fitting for each concentration and used only the results for concentration of analyte close to the model binding 1:1.

As a result, the rate constant of binding (ka) TM2 D40N with iminobiotin was increased, and the rate constant of dissociation (kd) was reduced by 45% compared to TM2 (table 7).

These results show that biotinidase ability (affinity) TM2 D40N was increased.

Table 7
The analysis of the interaction between high-affinity TM2, iminobiotin and Biotin
Name of templateka(1/MS) kd(1/s)
Iminobiotin-BSA
TM2D40N1,8±0,6×1056,9±1,0×10-4
TM21,3±0,5×1051,3±0,6×10-3
Biotin-LC-BSA
TM2D40NOf 1.1×106(1,8×10-8)
TM2Of 9.9×105(1,2×10-6)

Based on the results of example 2 properties of the high-affinity TM2 of the present invention are summarized in table 8.

Table 8
Summary of properties of high-affinity TM2
The binding of fluorescent BiotinAnalysis of affinity using Biacore (KD (M) = kd/ka)
KD for them�of abietina KD for Biotin
D40N+4,0±1,2×10-9Of 1.7×10-14
TM2+9,8±4,7×10-9Of 1.3×10-12

Example 3: Design and analysis of high-affinity TM2 with low non-specific binding

3-1)Designing high-affinity TM2 with low non-specific binding (HALU TM2: high affinity and low nonspecific character)

In TM2 was introduced mutation to reduce nonspecific binding and increase affinity to Biotin.

Based on the results of example 1 as mutations to reduce nonspecific binding mutations were introduced R104E and K141E as amino acid mutations to reduce nonspecific binding. Based on the results of measurement biotinidase abilities in example 2, as mutations to increase the affinity to Biotin was introduced D40N mutation. That is, were designed TM2 mutant containing mutations D40N and R104E (hereinafter in this description referred to as "TM2 D40NR104E"; its nucleotide sequence is presented in SEQ ID NO:21 and amino acid sequence in SEQ ID NO:22) and the TM2 mutant containing m�orientation D40N and R104EK141E (hereinafter in this description referred to as "TM2 D40NR104EK141E"; its nucleotide sequence is presented in SEQ ID NO:23, and amino acid sequence in SEQ ID NO:24).

3-2) PCR and cloning

To construct HALU TM2 were used the same primers as the primers used for the introduction of each mutation. PCR conditions and cloning techniques were the same as described above.

The gene encoding TM2 D40NR104E, was constructed by obtaining the vector TM2 D40NR104E/pTrc99A for protein expression TM2 D40NR104E by introducing mutations by PCR reaction in two stages using vector TM2 D40N/pTrc99A as a matrix and using a pair of primers Tm2NtermPci and Tm2 R104E R and a pair of primers Tm2 R104E F and Tm2CtermBam. The gene encoding TM2 D40NR104EK141E was constructed by obtaining the vector TM2 D40NR104EK141E/pTrc99A for protein expression TM2 D40NR104EK141E by introducing mutations by PCR reaction in a single stage using vector TM2 D40NR104E/pTrc99A as template and pairs of primers Tm2NtermPci and Tm2 K141E Bam.

3-3) the Expression of HALU TM2 inEscherichia coli

Escherichia coliTB1 transformed with the vector pTrc99A containing any of the mutant TM2, inoculable in LB culture medium (6 ml) containing an antibiotic, ampicillin (final concentration: 100 μg/ml), and cultured with shaking at 37°C or 25°C until the absorbance at 600 nm, OD600equal to 0.5. Added 1 mm IPTG, and the culture is additionally built�chevali at 37°C or 25°C during the night. Escherichia colicollected from 1 ml of culture solution by centrifugation and suspended in 20 mm phosphate buffer (pH 7, 400 μl) followed by destruction of bacterial cells by sonication. The solution is the destruction centrifuged (15,000 rpm) to collect the soluble fraction in the supernatant. This soluble fraction and an equal volume of 2×SDS buffer for the sample was mixed and heated at 95°C for 10 minutes. Proteins were separated by SDS-PAGE and detected by CBB staining.

The result was revealed strip of about 15.5 kDa in each of Escherichia coli transformed with the vector pTrc99A containing the mutant TM2, but was not detected in Escherichia coli transformed with the vector pTrc99A containing no mutant TM2. The sizes of these bands were in accordance with the molecular mass of 15.5 kDa monomer, predicted based on the amino acid sequence of TM2. Then according to the method of Bayer et al. (1996, Electrophoresis, 17(8), 1319-24) confirmed the formation of tetramers HALU TM2 in Undenatured condition, as in the case of TM2 with low pI and high-affinity TM2. The results showed a band of the same size as the strip of wild-type TM2 in HALU TM2, which showed the formation of tetramers. The level of expression of soluble mutant proteins TM2 was 24 mg per 1 l of the culture solution in the culture TM2 D40NR104E at 37°C, 10 mg in the culture TM2 D40NR104EK141E at 37°C and 32 mg in cul�round TM2 D40NR104E at 25°C. As for TM2 D40NR104EK141E, replacement of host cells in BL21 (DE3) increased the expression level of the soluble fraction in culture at 25°C to 43 mg.

3-4) Purification of HALU TM2

HALU TM2 was purified by the method of Hofmann et al. (1980), as described above. As a result, the amount of each purified protein mutant TM2 was essentially at the same level as the amount expressed inEscherichia coliprotein, with a purity of 90% or more.

3-5) Measurement of activity using fluorescent Biotin

Biotissues the ability of each purified TM2 mutant was confirmed using the method published in Biochim. Biophys. Acta, 1427, 44-48 (1999). The results showed that the fluorescence intensity decreases in proportion to the increase in the number of mutant solution HALU TM2. This confirms that the mutant HALU TM2 binds to the Biotin-like connection.

3-6) Electrophoresis with isoelectric focusing

Isoelectric point HALU TM2 were measured by electrophoresis with isoelectric focusing using the XCell SureLock Mini-Cell (Invitrogen). In accordance with the instruction page of each HALU TM2 (4 µg) were detected by CBB staining. The results are shown in table 9. The observed isoelectric point TM2 D40N was equal to 9.7 and was reduced to 8.9 after the introduction of mutations R104E and decreased to from 7.3 to 7.5 after the introduction of additional mutations K141E.

Table 9
Isoelectric point HALU TM2
Name of templateCalculated pIThe observed pI
TM2 D40NR104E6,38,9
TM2 D40NR104EK141E5,4from 7.3 to 7.5

2-7) Measurement biotinidase ability

The test of Biotin binding high-affinity TM2 was performed using a Biacore 3000 (Biacore).

Used as a ligand Biotin-BSA were prepared as described in section 2-7-2). HALU TM2 and TM2 used as analytes, were obtained by treatment with 2-iminobiotin-agarose in accordance with the ordinary method and were dialyzed against 20 mm KPi (pH 7) overnight. Each analyte was adjusted to approximately 50 μg/ml in 10 mm acetate buffer (pH 5, Biacore).

The immobilization of the ligand and the measurement and analysis of specific interaction with the analyzed substances were performed as described in section 2-7-2). As a result, the rate constant of binding (ka) with Biotin was increased in both mutants HALU TM2 compared to TM2. The rate constant of dissociation (kd) in TM2 D40NR104EK141 was reduced, which demonstrates an additional increase biotinidase ability (see tab. 10).

Table 10
The analysis of the interaction between high-affinity TM2 and Biotin
Biotin-BSA (of 22.4 Å)
Name of templatekakdKD
TM2 D40NR104EOf 1.4×106(2.9 x 10-9)(Of 2.2×10-15)
TM2 D40NR104EK141EOf 1.5×106(Of 1.4×10-8)(9,3×10-15)
TM2Of 9.9×105(1,2×10-6)(Of 1.3×10-12)

3-8) thermostability of protein structure HALU TM2

Each of the solutions of 0.2 μg/μl of the mutant TM2 (10 ál (2 ág)) was heated at room temperature, 50, 60, 70, 80, 90 or 99°C for 20 minutes. Then the solution was centrifuged at 15,000 rpm for 10 minutes, and soluble protein supernatant was suspended in an equal volume of 2×SDS buffer for about�sample (250 mm Tris-HCl, pH of 6.8, 20% 2-mercaptoethanol, 20% SDS, 20% glycerol). The suspension was heated at 95°C for 10 minutes followed by SDS-PAGE. Protein bands were detected by CBB staining. A standard curve was obtained using a quantitative marker (LMW ELECTROPHORESIS CALIBRATION KIT; Pharmacia Biotech) using Las-3000 (FUJIFILM) for the quantification of protein bands.

As a result, the temperature at which lost 50% protein D40NR104E was 78°C. in contrast, 78% protein D40NR104EK141E preserved, even if the protein was heated at 99°C. the Temperature at which lost 50% protein, amounted to 87.5°C for TM2 and 70°C for streptavidin.

3-9) Nonspecific binding of HALU TM2 with the serum of a person.

In this example investigated the nonspecific adsorption of serum protein on magnetic beads with immobilized HALU TM2.

TM2 D40NR104E and TM2 D40NR104EK141E covalently attached to magnetic beads (Dynabeads M-270 carboxylic acid Acid, Dynal) by the method described in paragraph 1-9) of example 1, and measured the amount of protein human serum adsorbed on the beads. The amount of each HALU TM2, immobilized on magnetic beads was adjusted to 10 μg/100 μl beads. A person's serum (CHEMICON) was diluted 800 times with PBS buffer and diluted human serum was added magnetic beads (50 µl), which was a certain amount of immobilized protein HALU TM2, with subsequent mixing of perivolaki�up against the bottom of the tube, containing the beads at room temperature for 15 minutes. Magnetic beads were washed with PBS buffer containing 0.1% tween 20 (PBST, 500 ml) four times and subjected to the interaction of antigen-antibody at room temperature for 15 minutes with HRP labeled antibody against mouse IgG (100 μl), diluted 5000 times in PBST containing 0.5% BSA. Then the beads were washed with PBST (500 ml) five times with the subsequent manifestation of staining using 1-Step Ultra TMB-ELISA (100 µl). After stopping the existence of staining of 2 M sulfuric acid (100 μl) magnetic beads were collected with a magnet. Was measured the absorbance of the supernatant at 450 nm using a microplate reader Infinite 200.

The results are shown in Fig.4. As can be seen in Fig.4, non-specific binding was low and TM2 D40NR104E, and for TM2 D40NR104EK141E compared with magnetic beads with immobilized wild-type TM2.

3-10) Nonspecific binding to fibronectin

Nonspecific binding of HALU TM2 with fibronectin was investigated in the same way as in paragraph 1-10) of example 1.

The results showed that the binding TM2 D40NR104EK141E with fibronectin was also very low and, thus, significantly lower compared to TM2 (figure 5). Inhibitory effect D40NR104EK141E on binding to fibronectin was comparable with the effect of TM2 R104EK141E exhibiting the highest activity among the TM2 mutants with n�scoi pI.

These results showed that the D40N mutation has no effect on the binding R104EK141E with fibronectin.

3-11) non-specific DNA binding

Nonspecific binding of HALU TM2 with DNA was analyzed using the method of paragraph 1-11) of example 1. The results showed that the binding TM2 D40NR104EK141 with 10 μg of DNA was below the threshold, as in the case of TM2 R104EK141E, whereas wild-type TM2 weakly associated (Fig.6). This shows that the D40N mutation does not affect DNA binding.

Based on the results of example 3 in table 11 summarizes the properties of high-affinity TM2 with low non-specific binding of the present invention.

Table 11
The final properties of high-affinity TM2 with low non-specific binding
The binding of fluorescent BiotinpIThe Biacore analysis
(KD)
Temperature stability (temp., in which lost 50% protein)Nonspecific binding to serum protein (A450)Binding to fibronectin (A450)Nonspecific binding�s DNA
D40NR104E+8,9Of 2.2×10-1578°C0,56NTNT
D40NR104EK141E+from 7.3 to 7.59,3×10-1599°C or more0,450,12-
TM2WT+from 8.5 to 8.8Of 1.3×10-1286°C0,67To 1.34±
NT: not defined

1. Modified biotinidase protein comprising the amino acid sequence represented by SEQ ID NO: 2, or amino acid sequence identical to the sequence represented by SEQ ID NO: 2, 98% or more, and having biotinidase activity, where
one or more residues selected from the group consisting of:
1) the arginine residue at position 104 of SEQ ID NO: 2;
2) the lysine residue at position 141 of SEQ ID NO: 2;
3) the lysine residue in position�AI 26 SEQ ID NO: 2, and
4) the lysine residue in position 73 of SEQ ID NO: 2
replaced by the balance of acidic amino acid or a residue of a neutral amino acid.

2. Modified biotinidase protein according to claim 1, wherein the amino acid residue is selected from 1) to 4), substituted amino acid residue having a hydrophobicity index of 2 or less.

3. Modified biotinidase protein of claim 1, wherein 1) the arginine residue at position 104 of SEQ ID NO: 2 and/or 2) a lysine residue at position 141 of SEQ ID NO: 2 is replaced with an acidic amino acid residue or a neutral amino acid residue.

4. Modified biotinidase protein according to claim 3, where 1) the arginine residue at position 104 of SEQ ID NO: 2 and/or 2) a lysine residue at position 141 of SEQ ID NO: 2 is replaced with an acidic amino acid residue.

5. Modified biotinidase protein according to claim 3 or 4, where 1) the arginine residue at position 104 of SEQ ID NO: 2 and/or 2) a lysine residue at position 141 of SEQ ID NO: 2 is replaced with a glutamic acid residue.

6. Modified biotinidase protein according to any one of claims.1-4, where the aspartic acid residue in position 40 of SEQ ID NO: 2 is replaced with an asparagine residue.

7. Modified biotinidase protein according to any one of claims.1-4, which is selected from the group consisting of:
modified bytesmessage protein (R104E-CE), in which the arginine residue at position 104 of SEQ ID NO: 2 is replaced with a glutamic acid residue and a lysine residue at position 141 is replaced by glutamic acid residue;
modified bytesmessage protein (D40N-R104E) in which the aspartic acid residue in position 40 of SEQ ID NO: 2 is replaced with an asparagine residue and the arginine residue at position 104 is replaced by a glutamic acid residue;
modified bytesmessage protein (D40N-K141E) in which the aspartic acid residue in position 40 of SEQ ID NO: 2 is replaced with an asparagine residue, and the lysine residue at position 141 is replaced by the residue of glutamic acid; and
modified bytesmessage protein (D40N-R104E-CE) in which the aspartic acid residue in position 40 of SEQ ID NO: 2 is replaced with an asparagine residue, the arginine residue at position 104 is replaced by a glutamic acid residue and a lysine residue at position 141 is replaced by a glutamic acid residue.

8. Modified biotinidase protein according to any one of claims.1-4, which satisfies at least one requirement selected from the following requirements from (a) to l):
a) aspartic residue at position 14 of SEQ ID NO: 2 is not modified or is replaced by glutamine or aspartic acid;
(b) the serine residue at position 18 of SEQ ID NO: 2 is not modified or is replaced with threonine or tyrosine;
(c) the tyrosine residue at position 34 of SEQ ID NO: 2 is not modified or is replaced by serine, threonine or phenylalanine;
(d) a serine residue at position 36 of SEQ ID NO: 2 is not modified or is replaced with threonine Il� a tyrosine;
(e) an aspartic acid residue in position 40 of SEQ ID NO: 2 is not modified or is replaced by asparagine;
(f) the tryptophan residue at position 69 of SEQ ID NO: 2 is not modified;
(g) a serine residue at position 76 of SEQ ID NO: 2 is not modified or is replaced with threonine or tyrosine;
(h) a threonine residue at position 78 of SEQ ID NO: 2 is not modified or is replaced by serine or tyrosine;
(i) the tryptophan residue at position 80 of SEQ ID NO: 2 is not modified;
(j) the tryptophan residue at position 96 of SEQ ID NO: 2 is not modified;
(k) a tryptophan residue at position 108 of SEQ ID NO: 2 is not modified; and
l) residue aspartic acid at position 116 of SEQ ID NO: 2 is not modified or is replaced with glutamic acid or asparagine.

9. Modified biotinidase protein according to any one of claims.1-4, which satisfies at least one property selected from the following properties (i) to (iv):
(i) having an isoelectric point lower than the isoelectric point of the protein consisting of the amino acid sequence represented by SEQ ID NO: 2;
(ii) exhibiting low non-specific binding to nucleic acids and/or proteins in comparison to a protein consisting of the amino acid sequence represented by SEQ ID NO: 2;
(iii) exhibiting lower fibrocartilaginous activity compared to a protein consisting of the amino acid� sequence, represented by SEQ ID NO: 2; and
(iv) exhibiting higher biotinidase activity compared to a protein consisting of the amino acid sequence represented by SEQ ID NO: 2.

10. Modified biotinidase protein comprising the amino acid sequence represented by SEQ ID NO: 2, or amino acid sequence that is identical to 98% or more sequence represented by SEQ ID NO: 2, and possessing biotinidase activity, where
the aspartic acid residue in position 40 of SEQ ID NO: 2 is replaced with an asparagine residue.

11. Modified biotinidase protein according to claim 10, where biotinidase activity is higher than the protein consisting of the amino acid sequence represented by SEQ ID NO: 2.

12. Nucleic acid that encodes the protein according to any one of claims.1-11.

13. A vector containing a nucleic acid according to claim 12, for expression of the protein according to any one of claims.1-11.

14. The carrier for binding of Biotin, which is immobilized protein according to any one of claims.1-11.



 

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16 cl, 7 dwg, 13 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: present invention refers to immunology. Presented is an antibody able to bind to an amplified epidermal growth factor receptor (EGFR) and to de2-7 EGFR, a truncated version of EGFR, and characterised by sequences of variable domains. There are also disclosed a kit for diagnosing a tumour, an immunoconjugate, pharmaceutical compositions and methods of treating a malignant tumour based on using the antibody according to the invention, as well as a single-cell host to form the antibody according to the present invention.

EFFECT: invention can find further application in diagnosing and treating cancer.

43 cl, 98 dwg, 20 tbl, 26 ex

FIELD: medicine.

SUBSTANCE: claimed invention relates to biotechnology and represents an expression plasmid without resistance to an antibiotic, containing a polynucleotide, coding a repressor protein cI. The expression of the said repressor protein regulates the expression of a toxic gene product, embedded into a non-essential section of a host genome. The claimed invention also discloses a constructed host cell, belonging to Gram-negative bacteria, which contains the said plasmid. The host cell is used to obtain the plasmid or to obtain a protein or an immunogen, if the expression plasmid additionally contains genes, coding the said protein or immunogen. The method of obtaining the expression plasmid and the method of obtaining the protein or immunogen are carried out in several stages. First, a strain of host cells, belonging to the Gram-negative bacteria, is created by embedding by the allele substitution of the gene, coding the toxic product, into the non-essential section of a host chromosome. After that, construction of the DNA-plasmid, which contains the gene, coding the repressor protein cI, and in case of necessity the gene, coding the protein or immunogen, is performed. Then, the obtained host cells are transformed by the said plasmid and grown in the presence of sucrose at a temperature of 30-42°C.

EFFECT: invention makes it possible to realise control of the toxic gene, localised on a chromosome, by means of the repressor, localised on the plasmid in the absence of selective pressure by an antibiotic.

33 cl, 31 dwg, 8 tbl, 10 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to immunology. Presented are variants of anti-CD20 modified antibody or its antigen-binding fragment. Each of the variants is characterised by the fact that it contains a variable light and heavy chain domain, and induces a higher apoptosis level as compared to anti-B-Ly1 chimeric antibody. There are presented: a mixture of antibodies, wherein at least 20% of oligosaccharides in Fc domain have a branched chain and are not fucosylated, as well as a pharmaceutical composition for producing a therapeutic agent for a malignant haematological or autoimmune disease by using the antibodies or the mixture of antibodies. Described are: an expression vector, a based host cell, variants of coding polynucleotides, as well as a method for producing the antibody in the cell.

EFFECT: using these inventions provides the new antibodies with the improved therapeutic properties, including with increased binding of Fc receptor, and with the increased effector function that can find application for treating the malignant haematological or autoimmune disease.

32 cl, 3 ex, 9 tbl, 26 dwg

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and gene engineering. A method for selecting at least one transfected eukaryotic host cell expressing a target product, the eukaryotic host cells comprise at least an introduced polynucleotide encoding the target product, an introduced polynucleotide encoding a DHFR enzyme using at least one expression vector, providing a plurality of eukaryotic host cells, whose viability is dependent upon folate uptake, wherein the said host cells comprise at least a foreign polynucleotide encoding the target product, a foreign polynucleotide encoding a DHFR enzyme, culturing the said plurality of the eukaryotic host cells in a selective culture medium comprising folic acid in a concentration of 12.5-50 nM combined with a concentration of MTX of 2.3-500 nM, selecting at least one eukaryotic host cell expressing the target product.

EFFECT: described is a method of the target product and culture medium preparation.

11 cl, 2 tbl, 2 ex

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