KR20170027599A - Paraoxonase 1 mutants with enhanced hydrolytic proficiency for organophosphate paraoxone and and method for manufacturing the same - Google Patents

Abstract

The present invention relates to a paraoxonase 1 mutant having an enhanced hydrolytic activity of an organophosphate papraoxone compound, and to a method for producing the same. More specifically, according to the paraoxonase 1 mutant of the present invention, one or more of isoleucine (I74) which is 74th amino acid, histidine (H115) which is 115th amino acid, threonine (T332) which is 332th amino acid, and valine (V346) which is 346th amino acid from amino acid positions of human recombinant paraoxonase 1 having an amino acid sequence of SEQ ID NO: 1 are substituted by amino acid selected from the group consisting of phenylalanine (F), tryptophan (W), serine (S), and alanine (A), and an hydrolytic activity of an organophosphate papraoxone compound is enhanced compared to wild-type paraoxonase 1.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a paraxoxanthin first variant having increased hydrolytic activity of an organophosphorus paraoxone compound and a method for producing the same, and a method for producing the same. BACKGROUND ART < RTI ID = 0.0 > Paraoxonase < / RTI >

The present invention relates to a first step of increasing the hydrolytic activity of an organophosphorus compound, especially an organophosphorus paraxone compound, which can be harmful to the human body and human body, compared to wild type paraoxonase I, and a method for producing the same .

Organophosphates (OPs) inhibit acetylcholinesterase, an enzyme that plays a key role in the hydrolysis of the neurotransmitter acetylcholine in the nervous system, and cause excessive accumulation of acetylcholine, Is known to be a toxic substance that damages the body. Therefore, organophosphorous compounds are mainly used as chemical-inorganic materials such as pesticides, pesticides or nerve gases.

Examples of such organophosphorous compounds include paraoxon, parathion, azametiphos, azinphos, bensulide, malaoxon, malathion, But are not limited to, heptenophos, dichlorvos, dialifos, diazinon, dichlofenthion, dimethoate, dimethylvinphos, dioxa But are not limited to, dioxabenzofos, disulfoton, demethon S, edifenphos, ethephon, ethion, ethoprophos, chlorfenvinphos, chlorpyrifos, fenitrothion, fenthion, fonofos, formothion, heptenophos, iprobenfos, Isazofos, isoxathion, mecarbam, methamidophos, methamphetamine, But are not limited to, methidathion, monocrotophos, naled, omethoate, phoxim, pirimphos, profenofos, propaphos, Prothiofos, pyraclofos, pyrazophos, pyridaphenthion, quinalphos, terbufos, tetrachlorvinphos, tetrachlorophosphoric acid, Thiometon, tolclofos, triazophos, trichlorphon, vamidothion, coumaphos, and the like.

If the organophosphorus compound is exposed to such an organophosphorus compound, acetylcholine may not be decomposed in the body, resulting in serious damage to the nervous system. Therefore, a method for removing pesticides, pesticides, Several studies have been conducted.

Korean Patent Publication No. 0186932 relates to an adsorption / decomposition type powder composition for removing organophosphorus compounds, and more particularly, to a method for adsorbing / decomposing a high-performance activated carbon having a BET specific surface area of 2000 m ² / g as a cation exchange resin and an anion and an adsorbent And an adsorbing / decomposing type powder composition for removing organic phosphorus compounds contained as an active ingredient.

Paraoxonase 1 (PON1) exists in mammalian cells and is known as a serum enzyme capable of hydrolyzing various organophosphorus compounds and can be used as an antidote thereof. Representative organic phosphorus compounds that can be hydrolyzed by paraoxonase I include diethyl-paraoxone (EPO), dimethyl-paraoxone (MPO), diazinonoxone diazinon-oxone, DZO) and chloropyrifos-oxon (CPO). Paraoxonase I is also known to be capable of degrading sarin, soman and VX, which are well known chemical weapons gases.

Because of its suitability for mammals (i.e., non-toxic and minimal immune responses), paraoxonase I has been shown to be effective in in vivo treatment for sudden exposure to organophosphorus compounds, And are attracting attention as candidates for the removal of these compounds, that is, pesticide poisoning agents, residual pesticide poisoning agents and chemical-chemical poisoning agents.

However, despite many efforts, the structure and mechanism of action of paraoxonase is still unknown. The wild-type paraoxonase I is poorly commercialized due to its low hydrolytic activity on their well characterized substrates (A. Aharoni, L. Gaidukov, S. Yagur, L. Toker, I. Silman and DS Tawfik , Proc. Natl. Acad. Sci. USA, 2004, 101, 482-487).

Therefore, there is a need for studies to increase the activity of wild-type paraoxonase I so as to be commercially applicable.

KR 10-0186932 B1 (December 29, 2011)

Thus, the present inventors have found that, by changing the three-dimensional structure of the protein of paraoxonase I so that the paraoxonease 1 capable of hydrolyzing the paraoxone compound in the organophosphorus compound has industrial productivity, The present inventors have completed the present invention by designing and improving paraoxonase I.

Accordingly, it is an object of the present invention to provide a first paraoxonin A variant having increased hydrolytic activity of an organophosphorus paraoxone compound.

In addition, the present invention aims to provide a gene coding for the above-mentioned paraoxonase first variant.

It is another object of the present invention to provide a recombinant expression vector containing the gene.

It is still another object of the present invention to provide a process for producing the above-mentioned paraoxonase first variant.

In order to achieve the above object, the present invention provides a human recombinant pPO system having the amino acid sequence of SEQ ID NO: 1, wherein the isoleucine (I), histidine (H) Threonine (T) or amino acid 346 (valine) is substituted with any one amino acid selected from the group consisting of phenylalanine (F), tryptophan (W), serine (S) and alanine Wherein the hydrolytic activity of the organophosphorus paraoxone compound is increased.

In addition, the present invention provides a gene encoding the above-mentioned paraoxonase first variant.

The present invention also provides a recombinant expression vector comprising the gene.

(A) transforming the expression vector of the present invention into a microorganism to produce a recombinant microorganism; (b) culturing the produced recombinant microorganism to express a paraoxonase first variant; And (c) recovering the first paraoxonase first variant from the embryo.

According to the present invention, by selectively mutating in the amino acid sequence of the wild-type paraoxonase I, the hydrolytic activity of the organophosphorus paraoxone compound is increased by a stable and economical method, A first variant can be provided. The first paraoxonase mutant according to the present invention has a high rate of hydrolysis of various organophosphorus compounds, especially paraoxone compounds, thereby detoxifying the organophosphorus compounds. Therefore, And can be usefully used for manufacturing.

Fig. 1 is a schematic diagram showing the reaction mechanism of paraoxonase No. 1.
Fig. 2 is an image virtually showing the coupling structure of diethyl-paraoxone (EPO) and dimethyl-paraoxone (MPO), which are organophosphorus paraoxone compounds, and paraoxonase I. Fig. Here, the orange color represents EPO and the blue color represents MPO.
FIG. 3 is a graph showing the time-dependent reactivity of the first paraoxonase mutant according to the present invention and diethyl-paraoxone (EPO) as an organic phosphorus-based paraoxone compound.
FIG. 4 is a graph showing the time-dependent reactivity of the first paraoxonase mutant according to the present invention and dimethyl-paraoxone (MPO), which is an organic phosphorus-based paraoxone compound.

Hereinafter, the present invention will be described.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein and the experimental methods described below are well known and commonly used in the art.

nomenclature

In the description and claims of the present invention, conventional one letter and three letter codes are used with respect to amino acid residues.

Nomenclature of amino acids

A = Ala = alanine

V = Val = Valine

L = Leu = Leucine < RTI ID = 0.0 >

I = Ile = isoleucine < RTI ID = 0.0 >

F = Phe = phenylalanine < RTI ID = 0.0 >

W = Trp = Tryptophan

S = Ser = Serine

T = Thr = Threonine

D = Asp = Asparatic acid

H = His = Histidine

Nomenclature of Variants

In describing various enzyme variants produced or designed according to the present invention, the following nomenclature was used.

Original amino acid / position / substituted amino acid

Multiple mutations were separated by a plus sign (+).

(I74), histidine (H115) as the 115th amino acid, threonine (T332) as the 332nd amino acid, and 346th amino acid as the amino acid sequence of the human recombinant pfaxonase I having the amino acid sequence of SEQ ID NO: Wherein one or more of the amino acids valine (V346) is substituted with any one amino acid selected from the group consisting of phenylalanine (F), tryptophan (W), serine (S) and alanine (A) To provide a first paraoxonase variant with increased degradation activity.

The organic phosphorus-based paraoxone compound is not particularly limited, but may be a commercially available dimethyl-paraoxone (MPO), diethyl-paraoxone (EPO), diazinon-oxone, DZO) and chloropyrifos-oxone (CPO).

Currently, two different but complementary strategies have been implemented for protein optimization and redesign. This is known as 'rational design' and 'directed evolution', also commonly known as computer modeling.

Rational design is specifically used to create or modify molecules (proteins) by predicting amino acid sequences to produce proteins with desired properties, particularly selectivity, enantioselectivity, and thermal stability have. An artificial evolution is a technique to obtain a protein of optimal activity finally by repeating two steps of making a library by giving various mutations to a genetic circle, and then screening the mutant which is actively improved as desired in this library at a very high speed.

Although it is not necessary to have a special screening system because the enzymatic modification by rational design generates only a limited number of mutant enzymes, it is necessary to know in detail the catalytic mechanism of the target enzyme, the binding property of the enzyme or the determinants of the substrate specificity .

In the present invention, molecular docking for analyzing interactions by docking a ligand acting on a target protein is proposed for a reasonable design to improve the hydrolytic activity of the first organophosphorous compound of wild type p-dioxin Respectively. As an attempt to use the wild-type paraoxonase I from mammals, which has low catalytic efficiency for most of the organophosphorus compounds as a target protein, as a therapeutic catalyst scavenger, the human recombinant paraoxone produced by artificial evolution through DNA shuffling The first G3C9 rePON1 (the amino acid sequence of SEQ ID NO: 1, the nucleotide sequence of SEQ ID NO: 13) was used.

The present invention also uses diethyl-paraoxone (EPO) as a model ligand for the manipulation of G3C9 rePONl. Diethyl-paraoxone (EPO) is a lactonease derived from Geobacillus kaustophilus HTA426 against an organophosphorus compound due to structural similarity with other organophosphorus compounds (i.e., MPO, DZO and CPO) lactonase) as a ligand model for artificial evolution.

Accordingly, those skilled in the art have determined that an artificial mutation in paraoxonase I to improve the hydrolytic activity of diethyl-paraoxone (EPO) will also improve the hydrolytic activity of other organophosphorus compounds in a correlated manner. Thus, G3C9 Diethyl-paraoxone (EPO) was used as a model ligand for artificial evolution of rePON1.

O, O - dialkyl groups ( O, O - dialkyl groups) of G3C9 rePON1 and several organophosphorus paraoxone compounds were investigated in order to find selective pocket for different organophosphorus paraoxone compounds. A molecular docking between the two paraoxone compounds, diethyl-paraoxone (EPO) or dimethyl-paraoxone (MPO), and paraoxonase I was performed.

That is, the binding structure with G3C9 rePON1 was virtually created using a molecular structure modeling program (FIG. 2), and the diethyl-paraoxone (EPO) and dimethyl-paraoxone (MPO) Leucine (L69), the 74th amino acid, isoleucine (I74), the 115th amino acid histidine (H115), the 69th amino acid of G3C9 rePON1 provided as a binding site, and the 269th amino acid It was confirmed that aspartic acid (D269), threonine (T332) of 332 amino acid and valine (V346) of amino acid 346 were important for binding to the substrate.

Therefore, in order to prepare the first paroxonase variant having a high hydrolytic activity against the organophosphorus paraoxone compound, the 74th isoleucine (I74), the 115th histidine (H115), the 125th histidine (F), tryptophan (W), serine (S), and alanine (A) using a site-directed mutagenesis method using one or more of the 332 th threonine (T332) and the 346th valine (V346) ≪ / RTI >

The first paraoxonase mutant of the present invention can be produced as follows.

First, a plasmid containing a DNA encoding a first para-soybean mutant is prepared, and an arbitrary host cell is transformed using the plasmid to obtain a transformant or a cell line. Next, the above transformant or cell line is cultured to produce the first paraoxonase mutant.

The first Paraoxonase mutants prepared above were analyzed for their amino acid substitutions at each position by base analysis. As a result of measuring the activity after culturing the colonies containing this mutant, it was found that the organophosphorus paraxone compound (i.e., diethyl-paraoxone (EPO)) in which the first paraoxonase mutant was a substrate compared to wild- Or dimethyl-paraoxone (MPO)).

In the present invention, the first paraoxonase mutant is a mutant in which isoleucine (I74), which is the 74th amino acid of the amino acid sequence of human recombinant pALPO3NAA 1st amino acid sequence having the amino acid sequence of SEQ ID NO: 1, is substituted with phenylalanine (I74F). ≪ / RTI > Wherein said paraoxonase first variant may have the amino acid sequence of SEQ ID NO: 2.

In the present invention, the first paraoxonase mutant is one in which threonine (T332), which is the 332nd amino acid among the amino acid positions of the first human recombinant pAXO3 having the amino acid sequence of SEQ ID NO: 1, is substituted with serine (S) (T332S). ≪ / RTI > Here, the first paraoxonase mutant may have the amino acid sequence of SEQ ID NO: 3.

In the present invention, the first paraoxonase mutant is a variant in which valine (V346), which is the 346th amino acid in the amino acid position of the first human recombinant pAXO3 having the amino acid sequence of SEQ ID NO: 1, is substituted with alanine (V346A). ≪ / RTI > Here, the first paraoxonase variant may have the amino acid sequence of SEQ ID NO: 4.

In the present invention, the first paraoxonase mutant is substituted with phenylalanine (F), isoleucine (I74), which is the 74th amino acid of the amino acid sequence of the human recombinant pPAO gene having the amino acid sequence of SEQ ID NO: 1, (I74F + H115W), which is characterized in that the 115th amino acid histidine (H115) is substituted with tryptophan (W). Here, the first paraoxonase variant may have the amino acid sequence of SEQ ID NO: 5.

In the present invention, the first paraoxonase mutant is substituted with phenylalanine (F), isoleucine (I74), which is the 74th amino acid of the amino acid sequence of the human recombinant pPAO gene having the amino acid sequence of SEQ ID NO: 1, (I74F + T332S), wherein the threonine (T332), which is the 332nd amino acid, is substituted with serine (S). Here, the first paraoxonase variant may have the amino acid sequence of SEQ ID NO: 6.

In the present invention, the first paraoxonase mutant is substituted with phenylalanine (F), isoleucine (I74), which is the 74th amino acid of the amino acid sequence of the human recombinant pPAO gene having the amino acid sequence of SEQ ID NO: 1, (I74F + V346A) which is characterized in that the 346th amino acid valine (V346) is substituted with alanine (A). Here, the first paraoxonase variant may have the amino acid sequence of SEQ ID NO: 7.

In the present invention, the first paraoxonase mutant is substituted with tryptophan (W), histidine (H115), which is the 115th amino acid of the first amino acid position of the human recombinant pPAR gene having the amino acid sequence of SEQ ID NO: 1, (H115W + T332S), which is characterized in that threonine (T332) at position 332 is substituted with serine (S). Here, the first paraoxonase variant may have the amino acid sequence of SEQ ID NO: 8.

In the present invention, the first paraoxonase mutant is substituted with tryptophan (W), histidine (H115), which is the 115th amino acid of the first amino acid position of the human recombinant pPAR gene having the amino acid sequence of SEQ ID NO: 1, (H115W + V346A), which is characterized in that the 346th amino acid valine (V346) is substituted with alanine (A). Here, the first paraoxonase mutant may have the amino acid sequence of SEQ ID NO: 9.

In the present invention, the first paraoxonase mutant is substituted with serine (S), threonine (T332) which is the 332nd amino acid of the first amino acid position of the human recombinant pPO synthase having the amino acid sequence of SEQ ID NO: 1, (T332S + V346A), which is characterized in that the 346th amino acid valine (V346) is substituted with alanine (A). Here, the first paraoxonase variant may have the amino acid sequence of SEQ ID NO: 10.

In the present invention, the first paraoxonase mutant is an isoleucine (I74), which is the 74th amino acid of the amino acid sequence of the first human recombinant pPO synthase having the amino acid sequence of SEQ ID NO: 1, is substituted with phenylalanine (F) (I74F + H115W + H115W + H115W) wherein the 115th amino acid histidine (H115) is replaced by tryptophan (W), and the 332nd amino acid threonine (T332) T332S). Here, the first paraoxonase mutant may have the amino acid sequence of SEQ ID NO: 11.

In the present invention, the first paraoxonase mutant is an isoleucine (I74), which is the 74th amino acid of the amino acid sequence of the first human recombinant pPO synthase having the amino acid sequence of SEQ ID NO: 1, is substituted with phenylalanine (F) (I74F + H115W + H115W) which is characterized in that histidine (H115) as the 115th amino acid is substituted by tryptophan (W) and valine V346 as the 346th amino acid is substituted by alanine V346A). Here, the first paraoxonase variant may have the amino acid sequence of SEQ ID NO: 12.

That is, said first paraoxonase variant comprises a mutation at a position corresponding to at least one of the following mutations in the amino acid sequence of SEQ ID NO: 1:

I74F + H115W + V346A, I74F + H342A, I74F + H343A, I74F + H342A, I74F + H342A, I74F + H115W + V346A, I74F + H115W + T342A.

As described above, the present invention is based on the rational design based on an artificial evolution, and it is possible to produce a variety of para-isoanase (G3C9 rePON1) having enhanced hydrolytic activity against the organophosphorus paraxone compound by causing mutation in the amino acid of the human recombinant para- 1 < / RTI >

In a preferred embodiment of the present invention, diethyl-paraoxone (EPO) and dimethyl-para-oxone (H115W + T332S) of the paraoxonase first variant (H115W + T332S) and paraoxonase first variant (I74F + H115W + T332S) (MPO) was significantly higher than the hydrolytic activity of the wild type G3C9 rePON1 of the control group.

That is, the two paraoxonane first mutants dramatically increased catalytic efficiency for the hydrolysis of diethyl-paraoxone (EPO) and dimethyl-paraoxone (MPO) compared to the wild type.

The catalytic efficiencies (K _cat / K _m ) of H115W / T332S mutants and I74F / H115W / T332S mutants for the hydrolysis of diethyl-paraoxone (EPO) at 25 ° C were 1.01 × 10 ⁵ and 9.39 × 10 ⁴ M ^-1 · S ^-1 and these values were 39.0 and 36.1 times higher than the catalytic efficiency of the wild type (2.6 × 10 ³ M ^-1 · s ^-1 ), respectively. In addition, the catalytic efficiencies (K _cat / K _m ) of the G3C9 rePON1 H115W / T332S variants and I74F / H115W / T332S variants for the hydrolysis of dimethyl-paraoxone (MPO) at 25 ° C were 2.83 × 10 ⁴ and 2.01 × 10 ⁴ M ^-1 · S ^-1 , which were 40.0 and 28.5 times higher than the wild type catalytic efficiency (7.07 × 10 ² M ^-1 · s ^-1 ), respectively.

Thus, the first paraoxonase variant of the present invention comprises a mutation at a position corresponding to the amino acid sequence of the human recombinant pPAO gene having the amino acid sequence of SEQ ID NO: 1, particularly H115W + T332S or I74F + H115W + T332S Is more preferable.

The paraoxonase first variants of the present invention also include within its scope functional equivalents or functional derivatives having functional or substantially equivalent activity thereto. Such functional equivalents include variants by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, of the amino acid residues in the amino acid sequence shown in SEQ ID NOS: 2 to 12.

The first paraoxonase variant according to the present invention has a hydrolysis activity which is about 2.4 to 39 times greater than the hydrolysis activity of wild-type paraoxonase I.

In addition, the first paraoxonase variants of the invention and their wild type behaved similarly with respect to temperature change and have an optimum temperature of approximately 35 ° C. That is, the specific activity at 35 ° C was 1.4 to 1.6 times higher than at 25 ° C. Thus, it can be seen that variations in evolutionary mutants do not affect their thermal activity (data not shown).

It is expected that the first paraoxonase variants according to the present invention will also have a significantly increased hydrolytic activity against organophosphorus paraoxone compounds having large O, O -dialkyl groups such as DZO and CPO.

The present invention also provides a gene encoding said paraoxonase first variant.

The gene encoding the first paraoxonase first variants may preferably have a nucleotide sequence of any one of SEQ ID NOS: 13 to 24.

It is preferable that at least 70%, 80%, 90%, or 95% or more of the amino acid sequence or the base sequence of the present invention is substituted or deleted by substitution, deletion, insertion and addition of any one of the above sequences. An amino acid sequence or a base sequence having sequence identity may also be included in the present invention.

The term "sequence identity" refers to sequence similarity between two polynucleotide residues. Said "sequence identity" can be determined by comparing the two nucleotide sequences optimally aligned over the region of the base sequence to be compared. Here, the polynucleotide to be compared may have addition or deletion (e.g., gap, overhang, etc.) in comparison with a reference sequence (for example, consensus sequence or the like) for optimal alignment of two sequences. The numerical value of the sequence identity is determined by determining the same nucleotide base present in both sequences and determining the number of suitable sites and then dividing the number of sites by the total number of bases in the sequence region of the comparison subject, By multiplying it by the following equation. Sequence identity between the nucleic acid sequences is measured using, for example, sequence analysis software, specifically BLASTN, FASTA and the like. The BLASTN is available from http://www.ncbi.nlm.nih. gov / BLAST /.

The present invention also provides a recombinant expression vector comprising a gene encoding said paraoxonase first variant.

In the present invention, "vector" means a DNA product containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing the DNA in an appropriate host. The vector may be a plasmid, phage particle or simply a potential genome insert. Once transformed into the appropriate host, the vector may replicate and function independently of the host genome, or, in some cases, integrate into the genome itself. Because the plasmid is the most commonly used form of the current vector, the terms "plasmid" and "vector" are sometimes used interchangeably in the context of the present invention. For the purpose of the present invention, it is preferable to use a plasmid vector. Typical plasmid vectors that can be used for this purpose include (a) a cloning start point that allows replication to be efficiently made to include several hundred plasmid vectors per host cell, (b) a host cell transformed with the plasmid vector And (c) a restriction enzyme cleavage site allowing the foreign DNA fragment to be inserted. Although there is no appropriate restriction site for cleavage, the use of a synthetic oligonucleotide adapter or linker according to conventional methods allows for easy ligation of the vector and foreign DNA.

In addition, the gene is "operably linked" when placed in a functional relationship with another nucleic acid sequence. This may be the gene and regulatory sequence (s) linked in such a way as to enable gene expression when a suitable molecule (e. G., Transcriptional activator protein) is attached to the regulatory sequence (s). For example, DNA for a pre-sequence or secretory leader is operably linked to DNA for a polypeptide when expressed as a whole protein participating in the secretion of the polypeptide; A promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; Or the ribosome binding site is operably linked to a coding sequence if it affects the transcription of the sequence; Or a ribosome binding site is operably linked to a coding sequence if positioned to facilitate translation. Generally, "operably linked" means that the linked DNA sequences are in contact and, in the case of a secretory leader, are in contact and present in the reading frame. However, the enhancer need not be in contact. The linkage of these sequences is carried out by conjugation at convenient restriction sites. If such a site does not exist, a synthetic oligonucleotide adapter or a linker according to a conventional method is used.

Of course, not all vectors function equally well in expressing the DNA sequence of the present invention, and likewise all hosts do not function equally in the same expression system. However, those skilled in the art can appropriately select from among various vectors, expression control sequences, and hosts without departing from the scope of the present invention without undue experimentation. For example, in selecting a vector, the host should be considered, since the vector must be replicated within it, and the number of copies of the vector, the ability to control the number of copies, and other proteins encoded by the vector, The expression of the antibiotic marker should also be considered.

The expression vector that can be used in the present invention is not particularly limited, and a known expression vector may be used. Preferably, a vector containing the gene may be used.

More preferably, the expression vector has a promoter that regulates the expression of the inserted gene. In a preferred embodiment of the present invention, the expression vector has a T7 / lac promoter and pET-22T (+) expressing the inserted gene by the addition of IPTG was used.

The present invention also

(a) transforming the expression vector into a microorganism to produce a recombinant microorganism;

(b) culturing the produced recombinant microorganism to express a paraoxonase first variant; And

(c) recovering the first paraoxonase variant from said embryo

Or a pharmaceutically acceptable salt thereof.

The microorganism transformed with the expression vector of the present invention is not particularly limited, but any microorganism capable of producing the first Paraoxonase variant of the present invention can be used. Preferably, Escherichia coli, which is easy to obtain, have.

The transformed recombinant microorganism can be produced according to any transformation method. "Transformation" of the present invention refers to a phenomenon in which DNA is introduced as a host and DNA can be cloned as a chromosome factor or by chromosome integration completion, introducing an external DNA into a cell to cause an artificial genetic change . In general, transformation methods, particularly transformation of plasma cells, can be readily accomplished using the heat shock method with calcium chloride as described in Section 1.82 of Sambrook et al., &Quot; Molecular Cloning ". Alternatively, electroporaton (Neumann et al., EMBO J. 1: 841, 1982), calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, microinjection, lithium acetate -DMSO method and the like can be used. As a method for inserting the gene into the chromosome of the host cell, a commonly known gene manipulation method can be used in the present invention. Examples of the method include a retrovirus vector, an adenovirus vector, an adeno-associated virus vector, a herpes simplex A virus vector, a poxvirus vector, a lentivirus vector or a nonviral vector may be used.

Conditions for culturing the recombinant microorganism of the present invention are not particularly limited, but it is preferable that the temperature is 25 to 37 占 폚 and the pH condition is pH 6 to 8.

The recovery of the mutant from the cultured recombinant microorganism can be carried out by conventional biochemical separation techniques such as treatment with a protein precipitant (salting-out method), centrifugation, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration) , Adsorption chromatography, ion exchange chromatography, affinity chromatography and the like can be used. In order to separate proteins having high purity, they can be used in combination.

The first paraoxonase first variant according to the present invention can be used to hydrolyze various organophosphorus paraoxone compounds. The pH condition for hydrolyzing the organophosphorus paraoxone compound is not particularly limited, but it is preferably pH 7.5 to 11.5.

Hereinafter, the present invention will be described concretely with reference to Examples. However, the following Examples are intended to illustrate one embodiment of the present invention, but the scope of the present invention is not limited by the following Examples. Particularly, in the following examples, only the increase in the activity of diethyl-paraoxone (EPO) or dimethyl-paraoxone (MPO) among the organic phosphorus-based paraoxone compounds was confirmed, but the mutants of the present invention It will be apparent to those skilled in the art to have excellent hydrolytic activity.

[Example]

Example 1: Selection of mutation position of paraoxonase No. 1 through structural analysis

To prepare a new variant of wild-type paraoxonase I, the structure obtained by X-ray crystallography of human recombinant paraoxonase No. 1 G3C9 rePONl made by artificial evolution was used. The structure of the G3C9 rePON1 is registered in the protein data bank (PDB) as 3srg.

In order to select the position of the paraoxonase 1 mutation, the inventors of the present invention virtually synthesized diethyl-paraoxone (EPO) and dimethylparoxone (MPO) and paraoxonase I (PDB ID: 3srg) was subjected to molecular structure modeling using the software SYBYL? -X (Tripos, ver. 2.1) software of TRIFOS. The results are shown in Fig.

Referring to FIG. 2, when diethyl-paraoxone (EPO) or dimethylparoxone (MPO) in the organophosphorus paraoxone compound is combined with paraoxonase 1, the first paraoxonase 69 Leucine (L69), the 74th amino acid isoleucine (I74), the 115th amino acid histidine (H115), the 269th amino acid aspartic acid (D269), 332 Threonine (T332) and valine (V346), the 346th amino acid, were found to be important for binding to the substrate.

The diethyl-paraoxone (EPO) and dimethylparaxone (MPO) were similarly located in the active site of G3C9 rePON1 except for their O, O -dialkyl groups. (D269), which is the 269th amino acid residue (D269), which acts as a stabilizing factor for the hydroxyl group of the pentasaccharide oxy-anionic intermediate of paraoxone Threonine (T332) at amino acid 332 and valine (V346) at amino acid 346 were in close contact with O-methyl of dimethyl-paraoxone (MPO).

Dimethyl-p-O- oxone from methyl carbon _{(MPO) C γ1 (V346)} , C γ2 (V346) and the distance C was _γ2 respectively, 4.18, 3.5 and 4.85 Å up to (T332).

Diethyl - paraoxone (EPO) had a larger O, O - dialkyl group than dimethyl - paraoxone (MPO) and could not fit well to the active site composed of T332 and V346. Therefore, diethyl-paraoxone (EPO) must be further pushed counterclockwise from T334 and V346 and rotated compared to O-methyl of dimethyl-paraoxone (MPO).

This shifts the O ₄ of diethyl-paraoxone (EPO) to near OD ₁ of D269 and since the distance of 2.66 A between them is shorter than the Van der Waals diameter of the oxygen atom of 2.95 A, Causing van der Waals interactions with steric hindrance.

This steric hindrance is a calculated inhibition between the large values of paraoxonase I and diethyl-paraoxone (EPO) relative to the inhibition constant (0.801) calculated between paraoxonase I and dimethyl-paraoxone (MPO) It may be the cause of the inhibitor constant (which is the same as the Michaelis Menten constant, K _m ) (4.49).

The inventors then hypothesized that a site-directed mutagenesis to displace T332 and V346 into smaller residues could form a space to accommodate the O-ethyl of diethyl-paraoxone (EPO) , And these mutations were expected to improve the hydrolytic activity of G3C9 rePON1 on diethyl-paraoxone (EPO).

In addition, the respective distances from C _γ1 and C _γ2 of I74 to the O-ethyl carbon of diethyl-paraoxone (EPO) are 4.5 and 4.53 Å, respectively, while that of C _d2 (L69) and dimethyl-paraoxone The distance between O-methyl carbon was 2.85 Å. Thus, two residues in close contact with O-methyl of diethyl-paraoxone (EPO) and O-methyl of dimethyl-paraoxone (MPO), respectively, namely isoleucine (I74) and 69th amino acid Inrucine (L69) was also selected as a target for location-directed mutagenesis.

For the above reasons, 11 kinds of mutants as shown in the following Table 1 were selected.

Variant candidates No. Variant candidates One G3C9 rePON1 I74F (isoleucine → phenylalanine) 2 G3C9 rePON1 T332S (threonine → serine) 3 G3C9 rePON1 V346A (valine → alanine) 4 G3C9 rePON1 I74F + H115W (isoleucine → phenylalanine / histidine → tryptophan) 5 G3C9 rePON1 I74F + T332S (isoleucine → phenylalanine / threonine → serine) 6 G3C9 rePON1 I74F + V346A (isoleucine → phenylalanine / valine → alanine) 7 G3C9 rePON1 H115W + T332S (histidine → tryptophan / threonine → serine) 8 G3C9 rePON1 H115W + V346A (histidine → tryptophan / valine → alanine) 9 G3C9 rePON1 T332S + V346A (threonine → serine / valine → alanine) 10 G3C9 rePON1 I74F + H115W + T332S
(Isoleucine → phenylalanine / histidine → tryptophan / threonine → serine) 11 G3C9 rePON1 I74F + H115W + V346A
(Isoleucine → phenylalanine / histidine → tryptophan / valine → alanine)

Example 2: Preparation of paraoxonase first variant

A primer for preparing a paraoxonase 1 mutant using the G3C9 rePON1 wild type having the amino acid sequence of SEQ ID NO: 1 was prepared as shown in Table 2 below and shown in SEQ ID NOs: 25 to 54.

Primer sequence SEQ ID NO: primer order
(5'-seq-3 ') 25 MF_L69A AGT TCC GGC GCA AAA TAT CCG GGT ATT ATG TCC 26 MR_ L69A CGG ATA TTT TGC GCC GGA ACT GAT AAA CGC CAG 27 MF_L69V AGT TCC GGC GTT AAA TAT CCG GGT ATT ATG TCC 28 MR _L69V CGG ATA TTT AAC GCC GGA ACT GAT AAA CGC CAG 29 MF_L69I AGT TCC GGC ATT AAA TAT CCG GGT ATT ATG TCC 30 MR_L69I CGG ATA TTT AAT GCC GGA ACT GAT AAA CGC CAG 31 MF_I74A TAT CCG GGT GCT ATG TCC TTC GAT CCG GAC AAA 32 MR_I74A GAA GGA CAT AGC ACC CGG ATA TTT CAG GCC GGA 33 MF_I74V TAT CCG GGT GTT ATG TCC TTC GAT CCG GAC AAA 34 MR_I74V GAA GGA CAT AAC ACC CGG ATA TTT CAG GCC GGA 35 MF_I74L TAT CCG GGT CTT ATG TCC TTC GAT CCG GAC AAA 36 MR_I74L GAA GGA CAT AAG ACC CGG ATA TTT CAG GCC GGA 37 MF_I74F TAT CCG GGT TTC ATG TCC TTC GAT CCG GAC AAA TCA GGC 38 MR_I74F GAA GGA CAT GAA ACC CGG ATA TTT CAG GCC GGA ACT GAT 39 MF_I74W TAT CCG GGT TGG ATG TCC TTC GAT CCG GAC AAA TCA GGC 40 MR_I74W GAA GGA CAT CCA ACC CGG ATA TTT CAG GCC GGA ACT GAT 41 MF_H115W TTT AAT CCG TGG GGC ATT AGC ACC TTC ACG GAT G 42 MR_H115W GCT AAT GCC CCA CGG ATT AAA CGA TGA GAT ATC 43 MF_T332A CAA GGT TCC GCT GTG GCA GCA GTT TAT AAA GGT 44 MF_T332A TGC TGC CAC AGC GGA ACC TTG CAG GAC GGT ACC 45 MF_T332S CAA GGT TCC TCT GTG GCA GCA GTT TAT AAA GGT AAA CTG 46 MR_T332S TGC TGC CAC AGA GGA ACC TTG CAG GAC GGT ACC ATT TTC 47 MF_T332V CAA GGT TCC GTA GTG GCA GCA GTT TAT AAA GGT 48 MR_T332V TGC TGC CAC TAC GGA ACC TTG CAG GAC GGT ACC 49 MF_V346A ATT GGC ACC GCT TTC CAC AAA GCT CTG TAC TGT 50 MR_V346A TTT GTG GAA AGC GGT GCC AAT CAG CAG TTT ACC 51 MF_V346L: ATT GGC ACC CTT TTC CAC AAA GCT CTG TAC TGT 52 MR_V346L TTT GTG GAA AAG GGT GCC AAT CAG CAG TTT ACC 53 MF_V346I ATT GGC ACC ATT TTC CAC AAA GCT CTG TAC TGT 54 MR_V346I TTT GTG GAA AAT GGT GCC AAT CAG CAG TTT ACC

* MF: forward primer

MR: Reverse primer

Because of the known molecular biology knowledge, there may be several nucleotide combinations in one species of amino acid.

Site-directed mutagenesis was performed using the primers, and genes coding for the paraoxonase 1 variant were obtained by polymerase chain reaction using the primers. A fragment of the gene coding for the obtained paraoxonase 1 mutant was digested with restriction enzymes NdeI and XhoI to obtain a T7 / lac promoter according to a known molecular biology technique, and IPTG (isopropyl-1-thio-β-D-galactopyranoside ) Was added to the pET-22T (+) vector expressing the inserted gene to construct a first para-oxonase mutant vector, which was named mutant 1 - 11.

The mutants were transformed into Escherichia coli BL21 (DE3) to prepare recombinant E. coli.

Example 3: Determination of the hydrolytic activity of diaryl-paraoxone (EPO) of paraoxonase first variants

3-1. Measurement of hydrolysis rate for diethyl-paraoxone (EPO)

In order to confirm the degree of increase in the hydrolytic activity of diaryl parapoxone (EPO) of the first paraoxonase mutants, the kinetic parameters were measured using the prepared recombinant E. coli and G3C9 rePON1 as a control group Respectively.

The prepared recombinant E. coli were cultured at 37 ° C in LB medium containing ampicillin (1 mg / ml). 0.1 M IPTG was added to the culture solution to induce enzyme expression of the first variants of paraoxonase, followed by further culturing for 16 hours in a shaking incubator at 200 rpm.

The culture solution was centrifuged to recover the recombinant E. coli, and the recombinant E. coli was resuspended in a disruption buffer (50 mM NaH ₂ PO ₄ , 300 mM NaCl, 10 mM imidazole) and disrupted with an ultrasonic mill. The histidine-labeled paraoxonase first variants were selectively isolated using a Ni-NTA column in the shredded cell lysate.

0.5 mg / L of each of the separated paraoxonase first variants was added to a reaction solution containing 0.5 mM EPO, 50 mM sodium glycine buffer (pH 10.5) and 1 mM CaCl ₂ .

Paraoxonase 1 hydrolyzes paraoxone with p-nitrophenol and diethyl phosphate. When the reaction occurs, the pH of the reaction solution decreases and electrons (e ^- ) are produced . The resulting p-nitrophenol exhibits a yellow color, which can be analyzed by measuring the absorbance at a wavelength of 412 nm.

The hydrolysis activity of p-nitrophenol produced by hydrolysis of diethyl-paraoxone (EPO) was measured by measuring the absorbance at 412 nm. The extinction coefficient per mole of measured p-nitrophenol was 16,900 M ^-1 · cm ^-1 , and the results are shown in Table 3 below.

The kinetic parameters of the first paraoxonase variant for the hydrolysis of diethyl-paraoxone (EPO) G3C9 rePON1s The enzyme k _cat
(s ^-1 ) K _{m of} enzyme
(mM) K _cat / K _m of enzyme
(M ^-1 s ^-1 ) Control (wild type) 0.818 0.700 2.60 x 10 ³ Variant 1 (I74F) 3.162 0.349 9.06 × 10 ³ ( 3.49 ) Variant 2 (T332S) 5.906 0.312 1.89 x 10 < ⁴ > ( 7.29 ) Variant 3 (V346A) 3.402 0.598 5.69 x 10 ³ ( 2.19 ) Variant 4 (I74F + H115W) 7.671 0.479 1.60 x 10 ⁴ ( 6.16 ) Mutant 5 (I74F + T332S) 4.191 0.298 1.41 × 10 ⁴ ( 5.42 ) Variant 6 (I74F + V346A) 10.437 0.676 1.54 × 10 ⁴ ( 5.94 ) Variant 7 (H115W + T332S) 12.146 0.120 1.01 × 10 ⁵ (39.02) Variant 8 (H115W + V346A) 10.464 1.406 7.44 × 10 ³ ( 2.86 ) Variant 9 (T332S + V346A) 2.945 0.473 6.23 × 10 ³ ( 2.40 ) Variant 10 (I74F + H115W + T332S) 12.909 0.137 9.39 x 10 < ⁴ > ( 36.13 ) Variant 11 (I74F + H115W + V346A) 22.927 1.290 1.78 x 10 ⁴ ( 6.84 )

* The parentheses indicate the multiplication by which the activity is increased as compared to the wild type Paraoxonase No. 1 of the control group.

As shown in the above Table 3, it was confirmed that the first paraoxonase first variants expressed in Examples 1 to 11 significantly increased the activity as compared with wild type G3C9 rePON1 as the control group.

In particular, it was confirmed that the mutants 7 and 10 were substituted with the 74th amino acid, the 115th amino acid and the 332th amino acid, and these amino acid residues were important for the hydrolysis of diethyl-paraoxone (EPO).

3-2. Measurement of hydrolytic conversion of diethyl-paraoxone (EPO) over time

The degree of hydrolysis of diethyl-paraoxone (EPO) with time was measured using variants 7 and 10 which showed high reactivity with diethyl-paraoxone (EPO) in Example 3-1. The resulting p-nitrophenyl was measured in the same manner as described above, and the results are shown in Fig.

As shown in FIG. 3, the conversion of diethyl-paraoxone (EPO) catalyzed by paraoxonase first expressed in variants 7 and 10 reached approximately 100% after approximately 20 and 25 minutes at 25 ° C, respectively , Which was 42.0 and 33.6 times faster than the wild-type-catalyzed conversion (840 or 14 hours, respectively) (Fig. 3).

That is, it was confirmed that the paraoxonase 1 expressed in the mutants 7 and 10 hydrolyzes the substrate diethyl-paraoxone (EPO) remarkably rapidly according to the reaction time.

Example 4: Determination of hydrolytic activity of dimethyl para-oxone (MPO) of para-oxonase first variants

4-1. Measurement of hydrolysis rate for dimethyl-paraoxone (MPO)

The increase in the hydrolytic activity of the first paraoxonase first dimethyl-paraoxone (MPO) expressed in variants 7 and 10, which showed high hydrolytic activity against diethyl-paraoxone (EPO) For confirmation, a kinetic parameter was measured as mentioned in Example 3 above. The results are shown in Table 4.

The kinetic parameters of the first paraoxonase variant for the hydrolysis of dimethyl-paraoxone (MPO) G3C9 rePON1s The enzyme k _cat
(s ^-1 ) K _{m of} enzyme
(mM) K _cat / K _m of enzyme
(s ^-1 M ^-1 ) Control (wild type) 0.223 0.316 7.07 × 10 ² Variant 7
(H115W + T332S) 6.572 0.232 2.83 × 10 ⁴ ( 40.01 ) Variant 10
(I74F + H115W + T332S) 6.322 0.314 2.01 × 10 ⁴ ( 28.48 )

* The parentheses indicate the multiplication by which the activity is increased as compared to the wild type Paraoxonase No. 1 of the control group.

As a result, it was found that the two para-oxonaphtha first variants, i.e., variant 7 (H115W / T332S) and variant 10 (I74F / H115W / T332S) Significant improvements in hydrolysis are clearly shown.

Namely, as can be seen from the above Table 4, the enzyme activity of paraoxonase I expressed in mutants 7 and 10 was greatly increased not only for diethyl-paraoxone (EPO) but also for dimethyl-paraoxone (MPO) .

It was confirmed that paraoxonase 1 expressed in the mutants 7 and 10 was substituted with the 74th amino acid, the 115th amino acid and the 332st amino acid, and these amino acid residues were also important for the hydrolysis of dimethyl-paraoxone (MPO) .

4-2. Measurement of hydrolytic conversion of dimethyl-paraoxone (MPO) over time

The degree of hydrolysis to dimethyl-paraoxone (MPO) was measured over time using variants 7 and 10 which showed high reactivity to dimethyl-paraoxone (MPO) in Example 4-1. The resulting p-nitrophenyl was measured in the same manner as described above, and the results are shown in Fig.

As shown in FIG. 4, the conversion of dimethyl-paraoxone (MPO) catalyzed by paraoxonane first expressed in variants 7 and 10 reached 82-85%. The time for conversion of approximately 82% dimethyl-paraoxone (MPO) catalyzed by variant 7 (H115W / T332S) and variant 10 (I74F / H115W / T332S) was approximately 10 min. 600 hours (10 hours).

<110> Kwangwoon University Industry-Academic Collaboration Foundation <120> PARAOXONASE 1 MUTANTS WITH ENHANCED HYDROLYTIC PROFICIENCY FOR          ORGANOPHOSPHATE PARAOXONE AND METHOD FOR MANUFACTURING THE SAME <130> 0001 <160> 54 <170> Kopatentin 2.0 <210> 1 <211> 355 <212> PRT <213> Human <400> 1 Met Ala Lys Leu Thr Ala Leu Thr Leu Leu Gly Leu Gly Leu Ala Leu   1 5 10 15 Phe Asp Gly Gln Lys Ser Ser Phe Gln Thr Arg Phe Asn Val His Arg              20 25 30 Glu Val Thr Pro Val Glu Leu Pro Asn Cys Asn Leu Val Lys Gly Val          35 40 45 Asp Asn Gly Ser Glu Asp Leu Glu Ile Leu Pro Asn Gly Leu Ala Phe      50 55 60 Ile Ser Ser Gly Leu Lys Tyr Pro Gly Ile Met Ser Phe Asp Pro Asp  65 70 75 80 Lys Ser Gly Lys Ile Leu Leu Met Asp Leu Asn Glu Glu Asp Pro Val                  85 90 95 Val Leu Glu Leu Gly Ile Thr Gly Asn Thr Leu Asp Ile Ser Ser Phe             100 105 110 Asn Pro His Gly Ile Ser Thr Phe Thr Asp Glu Asp Asn Thr Val Tyr         115 120 125 Leu Leu Val Val Asn His Pro Asp Ser Ser Ser Thr Val Glu Val Phe     130 135 140 Lys Phe Gln Glu Glu Glu Lys Ser Leu Leu His Leu Lys Thr Ile Arg 145 150 155 160 His Lys Leu Leu Pro Ser Val Asn Asp Ile Val Ala Val Gly Pro Glu                 165 170 175 His Phe Tyr Ala Thr Asn Asp His Tyr Phe Ala Asp Pro Tyr Leu Lys             180 185 190 Ser Trp Glu Met His Leu Gly Leu Ala Trp Ser Phe Val Thr Tyr Tyr         195 200 205 Ser Pro Asn Val Val Val Ala Glu Gly Phe Asp Phe Ala Asn     210 215 220 Gly Ile Asn Ile Ser Pro Asp Gly Lys Tyr Val Tyr Ile Ala Glu Leu 225 230 235 240 Leu Ala His Lys Ile His Val Tyr Glu Lys His Ala Asn Trp Thr Leu                 245 250 255 Thr Pro Leu Lys Ser Leu Asp Phe Asp Thr Leu Val Asp Asn Ile Ser             260 265 270 Val Asp Pro Val Thr Gly Asp Leu Trp Val Gly Cys His Pro Asn Gly         275 280 285 Met Arg Ile Phe Tyr Tyr Asp Pro Lys Asn Pro Pro Gly Ser Glu Val     290 295 300 Leu Arg Ile Gln Asp Ile Leu Ser Glu Glu Pro Lys Val Thr Val Val 305 310 315 320 Tyr Ala Glu Asn Gly Thr Val Leu Gln Gly Ser Thr Val Ala Ala Val                 325 330 335 Tyr Lys Gly Lys Leu Leu Ile Gly Thr Val Phe His Lys Ala Leu Tyr             340 345 350 Cys Glu Leu         355 <210> 2 <211> 355 <212> PRT <213> Human <220> <221> VARIANT <74> <223> Isoleucine -> Phenylalanine <400> 2 Met Ala Lys Leu Thr Ala Leu Thr Leu Leu Gly Leu Gly Leu Ala Leu   1 5 10 15 Phe Asp Gly Gln Lys Ser Ser Phe Gln Thr Arg Phe Asn Val His Arg              20 25 30 Glu Val Thr Pro Val Glu Leu Pro Asn Cys Asn Leu Val Lys Gly Val          35 40 45 Asp Asn Gly Ser Glu Asp Leu Glu Ile Leu Pro Asn Gly Leu Ala Phe      50 55 60 Ile Ser Ser Gly Leu Lys Tyr Pro Gly Phe Met Ser Phe Asp Pro Asp  65 70 75 80 Lys Ser Gly Lys Ile Leu Leu Met Asp Leu Asn Glu Glu Asp Pro Val                  85 90 95 Val Leu Glu Leu Gly Ile Thr Gly Asn Thr Leu Asp Ile Ser Ser Phe             100 105 110 Asn Pro His Gly Ile Ser Thr Phe Thr Asp Glu Asp Asn Thr Val Tyr         115 120 125 Leu Leu Val Val Asn His Pro Asp Ser Ser Ser Thr Val Glu Val Phe     130 135 140 Lys Phe Gln Glu Glu Glu Lys Ser Leu Leu His Leu Lys Thr Ile Arg 145 150 155 160 His Lys Leu Leu Pro Ser Val Asn Asp Ile Val Ala Val Gly Pro Glu                 165 170 175 His Phe Tyr Ala Thr Asn Asp His Tyr Phe Ala Asp Pro Tyr Leu Lys             180 185 190 Ser Trp Glu Met His Leu Gly Leu Ala Trp Ser Phe Val Thr Tyr Tyr         195 200 205 Ser Pro Asn Val Val Val Ala Glu Gly Phe Asp Phe Ala Asn     210 215 220 Gly Ile Asn Ile Ser Pro Asp Gly Lys Tyr Val Tyr Ile Ala Glu Leu 225 230 235 240 Leu Ala His Lys Ile His Val Tyr Glu Lys His Ala Asn Trp Thr Leu                 245 250 255 Thr Pro Leu Lys Ser Leu Asp Phe Asp Thr Leu Val Asp Asn Ile Ser             260 265 270 Val Asp Pro Val Thr Gly Asp Leu Trp Val Gly Cys His Pro Asn Gly         275 280 285 Met Arg Ile Phe Tyr Tyr Asp Pro Lys Asn Pro Pro Gly Ser Glu Val     290 295 300 Leu Arg Ile Gln Asp Ile Leu Ser Glu Glu Pro Lys Val Thr Val Val 305 310 315 320 Tyr Ala Glu Asn Gly Thr Val Leu Gln Gly Ser Thr Val Ala Ala Val                 325 330 335 Tyr Lys Gly Lys Leu Leu Ile Gly Thr Val Phe His Lys Ala Leu Tyr             340 345 350 Cys Glu Leu         355 <210> 3 <211> 355 <212> PRT <213> Human <220> <221> VARIANT <222> <223> Threonine -> Serine <400> 3 Met Ala Lys Leu Thr Ala Leu Thr Leu Leu Gly Leu Gly Leu Ala Leu   1 5 10 15 Phe Asp Gly Gln Lys Ser Ser Phe Gln Thr Arg Phe Asn Val His Arg              20 25 30 Glu Val Thr Pro Val Glu Leu Pro Asn Cys Asn Leu Val Lys Gly Val          35 40 45 Asp Asn Gly Ser Glu Asp Leu Glu Ile Leu Pro Asn Gly Leu Ala Phe      50 55 60 Ile Ser Ser Gly Leu Lys Tyr Pro Gly Ile Met Ser Phe Asp Pro Asp  65 70 75 80 Lys Ser Gly Lys Ile Leu Leu Met Asp Leu Asn Glu Glu Asp Pro Val                  85 90 95 Val Leu Glu Leu Gly Ile Thr Gly Asn Thr Leu Asp Ile Ser Ser Phe             100 105 110 Asn Pro His Gly Ile Ser Thr Phe Thr Asp Glu Asp Asn Thr Val Tyr         115 120 125 Leu Leu Val Val Asn His Pro Asp Ser Ser Ser Thr Val Glu Val Phe     130 135 140 Lys Phe Gln Glu Glu Glu Lys Ser Leu Leu His Leu Lys Thr Ile Arg 145 150 155 160 His Lys Leu Leu Pro Ser Val Asn Asp Ile Val Ala Val Gly Pro Glu                 165 170 175 His Phe Tyr Ala Thr Asn Asp His Tyr Phe Ala Asp Pro Tyr Leu Lys             180 185 190 Ser Trp Glu Met His Leu Gly Leu Ala Trp Ser Phe Val Thr Tyr Tyr         195 200 205 Ser Pro Asn Val Val Val Ala Glu Gly Phe Asp Phe Ala Asn     210 215 220 Gly Ile Asn Ile Ser Pro Asp Gly Lys Tyr Val Tyr Ile Ala Glu Leu 225 230 235 240 Leu Ala His Lys Ile His Val Tyr Glu Lys His Ala Asn Trp Thr Leu                 245 250 255 Thr Pro Leu Lys Ser Leu Asp Phe Asp Thr Leu Val Asp Asn Ile Ser             260 265 270 Val Asp Pro Val Thr Gly Asp Leu Trp Val Gly Cys His Pro Asn Gly         275 280 285 Met Arg Ile Phe Tyr Tyr Asp Pro Lys Asn Pro Pro Gly Ser Glu Val     290 295 300 Leu Arg Ile Gln Asp Ile Leu Ser Glu Glu Pro Lys Val Thr Val Val 305 310 315 320 Tyr Ala Glu Asn Gly Thr Val Leu Gln Gly Ser Ser Val Ala Ala Val                 325 330 335 Tyr Lys Gly Lys Leu Leu Ile Gly Thr Val Phe His Lys Ala Leu Tyr             340 345 350 Cys Glu Leu         355 <210> 4 <211> 355 <212> PRT <213> Human <220> <221> VARIANT <222> (346) <223> Valine -> Alanine <400> 4 Met Ala Lys Leu Thr Ala Leu Thr Leu Leu Gly Leu Gly Leu Ala Leu   1 5 10 15 Phe Asp Gly Gln Lys Ser Ser Phe Gln Thr Arg Phe Asn Val His Arg              20 25 30 Glu Val Thr Pro Val Glu Leu Pro Asn Cys Asn Leu Val Lys Gly Val          35 40 45 Asp Asn Gly Ser Glu Asp Leu Glu Ile Leu Pro Asn Gly Leu Ala Phe      50 55 60 Ile Ser Ser Gly Leu Lys Tyr Pro Gly Ile Met Ser Phe Asp Pro Asp  65 70 75 80 Lys Ser Gly Lys Ile Leu Leu Met Asp Leu Asn Glu Glu Asp Pro Val                  85 90 95 Val Leu Glu Leu Gly Ile Thr Gly Asn Thr Leu Asp Ile Ser Ser Phe             100 105 110 Asn Pro His Gly Ile Ser Thr Phe Thr Asp Glu Asp Asn Thr Val Tyr         115 120 125 Leu Leu Val Val Asn His Pro Asp Ser Ser Ser Thr Val Glu Val Phe     130 135 140 Lys Phe Gln Glu Glu Glu Lys Ser Leu Leu His Leu Lys Thr Ile Arg 145 150 155 160 His Lys Leu Leu Pro Ser Val Asn Asp Ile Val Ala Val Gly Pro Glu                 165 170 175 His Phe Tyr Ala Thr Asn Asp His Tyr Phe Ala Asp Pro Tyr Leu Lys             180 185 190 Ser Trp Glu Met His Leu Gly Leu Ala Trp Ser Phe Val Thr Tyr Tyr         195 200 205 Ser Pro Asn Val Val Val Ala Glu Gly Phe Asp Phe Ala Asn     210 215 220 Gly Ile Asn Ile Ser Pro Asp Gly Lys Tyr Val Tyr Ile Ala Glu Leu 225 230 235 240 Leu Ala His Lys Ile His Val Tyr Glu Lys His Ala Asn Trp Thr Leu                 245 250 255 Thr Pro Leu Lys Ser Leu Asp Phe Asp Thr Leu Val Asp Asn Ile Ser             260 265 270 Val Asp Pro Val Thr Gly Asp Leu Trp Val Gly Cys His Pro Asn Gly         275 280 285 Met Arg Ile Phe Tyr Tyr Asp Pro Lys Asn Pro Pro Gly Ser Glu Val     290 295 300 Leu Arg Ile Gln Asp Ile Leu Ser Glu Glu Pro Lys Val Thr Val Val 305 310 315 320 Tyr Ala Glu Asn Gly Thr Val Leu Gln Gly Ser Thr Val Ala Ala Val                 325 330 335 Tyr Lys Gly Lys Leu Leu Ile Gly Thr Ala Phe His Lys Ala Leu Tyr             340 345 350 Cys Glu Leu         355 <210> 5 <211> 355 <212> PRT <213> Human <220> <221> VARIANT <74> <223> Isoleucine -> Phenylalanine <220> <221> VARIANT <115> <223> Histidine -> Tryptophan <400> 5 Met Ala Lys Leu Thr Ala Leu Thr Leu Leu Gly Leu Gly Leu Ala Leu   1 5 10 15 Phe Asp Gly Gln Lys Ser Ser Phe Gln Thr Arg Phe Asn Val His Arg              20 25 30 Glu Val Thr Pro Val Glu Leu Pro Asn Cys Asn Leu Val Lys Gly Val          35 40 45 Asp Asn Gly Ser Glu Asp Leu Glu Ile Leu Pro Asn Gly Leu Ala Phe      50 55 60 Ile Ser Ser Gly Leu Lys Tyr Pro Gly Phe Met Ser Phe Asp Pro Asp  65 70 75 80 Lys Ser Gly Lys Ile Leu Leu Met Asp Leu Asn Glu Glu Asp Pro Val                  85 90 95 Val Leu Glu Leu Gly Ile Thr Gly Asn Thr Leu Asp Ile Ser Ser Phe             100 105 110 Asn Pro Trp Gly Ile Ser Thr Phe Thr Asp Glu Asp Asn Thr Val Tyr         115 120 125 Leu Leu Val Val Asn His Pro Asp Ser Ser Ser Thr Val Glu Val Phe     130 135 140 Lys Phe Gln Glu Glu Glu Lys Ser Leu Leu His Leu Lys Thr Ile Arg 145 150 155 160 His Lys Leu Leu Pro Ser Val Asn Asp Ile Val Ala Val Gly Pro Glu                 165 170 175 His Phe Tyr Ala Thr Asn Asp His Tyr Phe Ala Asp Pro Tyr Leu Lys             180 185 190 Ser Trp Glu Met His Leu Gly Leu Ala Trp Ser Phe Val Thr Tyr Tyr         195 200 205 Ser Pro Asn Val Val Val Ala Glu Gly Phe Asp Phe Ala Asn     210 215 220 Gly Ile Asn Ile Ser Pro Asp Gly Lys Tyr Val Tyr Ile Ala Glu Leu 225 230 235 240 Leu Ala His Lys Ile His Val Tyr Glu Lys His Ala Asn Trp Thr Leu                 245 250 255 Thr Pro Leu Lys Ser Leu Asp Phe Asp Thr Leu Val Asp Asn Ile Ser             260 265 270 Val Asp Pro Val Thr Gly Asp Leu Trp Val Gly Cys His Pro Asn Gly         275 280 285 Met Arg Ile Phe Tyr Tyr Asp Pro Lys Asn Pro Pro Gly Ser Glu Val     290 295 300 Leu Arg Ile Gln Asp Ile Leu Ser Glu Glu Pro Lys Val Thr Val Val 305 310 315 320 Tyr Ala Glu Asn Gly Thr Val Leu Gln Gly Ser Thr Val Ala Ala Val                 325 330 335 Tyr Lys Gly Lys Leu Leu Ile Gly Thr Val Phe His Lys Ala Leu Tyr             340 345 350 Cys Glu Leu         355 <210> 6 <211> 355 <212> PRT <213> Human <220> <221> VARIANT <74> <223> Isoleucine -> Phenylalanine <220> <221> VARIANT <222> <223> Threonine -> Serine <400> 6 Met Ala Lys Leu Thr Ala Leu Thr Leu Leu Gly Leu Gly Leu Ala Leu   1 5 10 15 Phe Asp Gly Gln Lys Ser Ser Phe Gln Thr Arg Phe Asn Val His Arg              20 25 30 Glu Val Thr Pro Val Glu Leu Pro Asn Cys Asn Leu Val Lys Gly Val          35 40 45 Asp Asn Gly Ser Glu Asp Leu Glu Ile Leu Pro Asn Gly Leu Ala Phe      50 55 60 Ile Ser Ser Gly Leu Lys Tyr Pro Gly Phe Met Ser Phe Asp Pro Asp  65 70 75 80 Lys Ser Gly Lys Ile Leu Leu Met Asp Leu Asn Glu Glu Asp Pro Val                  85 90 95 Val Leu Glu Leu Gly Ile Thr Gly Asn Thr Leu Asp Ile Ser Ser Phe             100 105 110 Asn Pro His Gly Ile Ser Thr Phe Thr Asp Glu Asp Asn Thr Val Tyr         115 120 125 Leu Leu Val Val Asn His Pro Asp Ser Ser Ser Thr Val Glu Val Phe     130 135 140 Lys Phe Gln Glu Glu Glu Lys Ser Leu Leu His Leu Lys Thr Ile Arg 145 150 155 160 His Lys Leu Leu Pro Ser Val Asn Asp Ile Val Ala Val Gly Pro Glu                 165 170 175 His Phe Tyr Ala Thr Asn Asp His Tyr Phe Ala Asp Pro Tyr Leu Lys             180 185 190 Ser Trp Glu Met His Leu Gly Leu Ala Trp Ser Phe Val Thr Tyr Tyr         195 200 205 Ser Pro Asn Val Val Val Ala Glu Gly Phe Asp Phe Ala Asn     210 215 220 Gly Ile Asn Ile Ser Pro Asp Gly Lys Tyr Val Tyr Ile Ala Glu Leu 225 230 235 240 Leu Ala His Lys Ile His Val Tyr Glu Lys His Ala Asn Trp Thr Leu                 245 250 255 Thr Pro Leu Lys Ser Leu Asp Phe Asp Thr Leu Val Asp Asn Ile Ser             260 265 270 Val Asp Pro Val Thr Gly Asp Leu Trp Val Gly Cys His Pro Asn Gly         275 280 285 Met Arg Ile Phe Tyr Tyr Asp Pro Lys Asn Pro Pro Gly Ser Glu Val     290 295 300 Leu Arg Ile Gln Asp Ile Leu Ser Glu Glu Pro Lys Val Thr Val Val 305 310 315 320 Tyr Ala Glu Asn Gly Thr Val Leu Gln Gly Ser Ser Val Ala Ala Val                 325 330 335 Tyr Lys Gly Lys Leu Leu Ile Gly Thr Val Phe His Lys Ala Leu Tyr             340 345 350 Cys Glu Leu         355 <210> 7 <211> 355 <212> PRT <213> Human <220> <221> VARIANT <74> <223> Isoleucine -> Phenylalanine <220> <221> VARIANT <222> (346) <223> Valine -> Alanine <400> 7 Met Ala Lys Leu Thr Ala Leu Thr Leu Leu Gly Leu Gly Leu Ala Leu   1 5 10 15 Phe Asp Gly Gln Lys Ser Ser Phe Gln Thr Arg Phe Asn Val His Arg              20 25 30 Glu Val Thr Pro Val Glu Leu Pro Asn Cys Asn Leu Val Lys Gly Val          35 40 45 Asp Asn Gly Ser Glu Asp Leu Glu Ile Leu Pro Asn Gly Leu Ala Phe      50 55 60 Ile Ser Ser Gly Leu Lys Tyr Pro Gly Phe Met Ser Phe Asp Pro Asp  65 70 75 80 Lys Ser Gly Lys Ile Leu Leu Met Asp Leu Asn Glu Glu Asp Pro Val                  85 90 95 Val Leu Glu Leu Gly Ile Thr Gly Asn Thr Leu Asp Ile Ser Ser Phe             100 105 110 Asn Pro His Gly Ile Ser Thr Phe Thr Asp Glu Asp Asn Thr Val Tyr         115 120 125 Leu Leu Val Val Asn His Pro Asp Ser Ser Ser Thr Val Glu Val Phe     130 135 140 Lys Phe Gln Glu Glu Glu Lys Ser Leu Leu His Leu Lys Thr Ile Arg 145 150 155 160 His Lys Leu Leu Pro Ser Val Asn Asp Ile Val Ala Val Gly Pro Glu                 165 170 175 His Phe Tyr Ala Thr Asn Asp His Tyr Phe Ala Asp Pro Tyr Leu Lys             180 185 190 Ser Trp Glu Met His Leu Gly Leu Ala Trp Ser Phe Val Thr Tyr Tyr         195 200 205 Ser Pro Asn Val Val Val Ala Glu Gly Phe Asp Phe Ala Asn     210 215 220 Gly Ile Asn Ile Ser Pro Asp Gly Lys Tyr Val Tyr Ile Ala Glu Leu 225 230 235 240 Leu Ala His Lys Ile His Val Tyr Glu Lys His Ala Asn Trp Thr Leu                 245 250 255 Thr Pro Leu Lys Ser Leu Asp Phe Asp Thr Leu Val Asp Asn Ile Ser             260 265 270 Val Asp Pro Val Thr Gly Asp Leu Trp Val Gly Cys His Pro Asn Gly         275 280 285 Met Arg Ile Phe Tyr Tyr Asp Pro Lys Asn Pro Pro Gly Ser Glu Val     290 295 300 Leu Arg Ile Gln Asp Ile Leu Ser Glu Glu Pro Lys Val Thr Val Val 305 310 315 320 Tyr Ala Glu Asn Gly Thr Val Leu Gln Gly Ser Thr Val Ala Ala Val                 325 330 335 Tyr Lys Gly Lys Leu Leu Ile Gly Thr Ala Phe His Lys Ala Leu Tyr             340 345 350 Cys Glu Leu         355 <210> 8 <211> 355 <212> PRT <213> Human <220> <221> VARIANT <115> <223> Histidine -> Tryptophan <220> <221> VARIANT <222> <223> Threonine -> Serine <400> 8 Met Ala Lys Leu Thr Ala Leu Thr Leu Leu Gly Leu Gly Leu Ala Leu   1 5 10 15 Phe Asp Gly Gln Lys Ser Ser Phe Gln Thr Arg Phe Asn Val His Arg              20 25 30 Glu Val Thr Pro Val Glu Leu Pro Asn Cys Asn Leu Val Lys Gly Val          35 40 45 Asp Asn Gly Ser Glu Asp Leu Glu Ile Leu Pro Asn Gly Leu Ala Phe      50 55 60 Ile Ser Ser Gly Leu Lys Tyr Pro Gly Ile Met Ser Phe Asp Pro Asp  65 70 75 80 Lys Ser Gly Lys Ile Leu Leu Met Asp Leu Asn Glu Glu Asp Pro Val                  85 90 95 Val Leu Glu Leu Gly Ile Thr Gly Asn Thr Leu Asp Ile Ser Ser Phe             100 105 110 Asn Pro Trp Gly Ile Ser Thr Phe Thr Asp Glu Asp Asn Thr Val Tyr         115 120 125 Leu Leu Val Val Asn His Pro Asp Ser Ser Ser Thr Val Glu Val Phe     130 135 140 Lys Phe Gln Glu Glu Glu Lys Ser Leu Leu His Leu Lys Thr Ile Arg 145 150 155 160 His Lys Leu Leu Pro Ser Val Asn Asp Ile Val Ala Val Gly Pro Glu                 165 170 175 His Phe Tyr Ala Thr Asn Asp His Tyr Phe Ala Asp Pro Tyr Leu Lys             180 185 190 Ser Trp Glu Met His Leu Gly Leu Ala Trp Ser Phe Val Thr Tyr Tyr         195 200 205 Ser Pro Asn Val Val Val Ala Glu Gly Phe Asp Phe Ala Asn     210 215 220 Gly Ile Asn Ile Ser Pro Asp Gly Lys Tyr Val Tyr Ile Ala Glu Leu 225 230 235 240 Leu Ala His Lys Ile His Val Tyr Glu Lys His Ala Asn Trp Thr Leu                 245 250 255 Thr Pro Leu Lys Ser Leu Asp Phe Asp Thr Leu Val Asp Asn Ile Ser             260 265 270 Val Asp Pro Val Thr Gly Asp Leu Trp Val Gly Cys His Pro Asn Gly         275 280 285 Met Arg Ile Phe Tyr Tyr Asp Pro Lys Asn Pro Pro Gly Ser Glu Val     290 295 300 Leu Arg Ile Gln Asp Ile Leu Ser Glu Glu Pro Lys Val Thr Val Val 305 310 315 320 Tyr Ala Glu Asn Gly Thr Val Leu Gln Gly Ser Ser Val Ala Ala Val                 325 330 335 Tyr Lys Gly Lys Leu Leu Ile Gly Thr Val Phe His Lys Ala Leu Tyr             340 345 350 Cys Glu Leu         355 <210> 9 <211> 355 <212> PRT <213> Human <220> <221> VARIANT <115> <223> Histidine -> Tryptophan <220> <221> VARIANT <222> (346) <223> Valine -> Alanine <400> 9 Met Ala Lys Leu Thr Ala Leu Thr Leu Leu Gly Leu Gly Leu Ala Leu   1 5 10 15 Phe Asp Gly Gln Lys Ser Ser Phe Gln Thr Arg Phe Asn Val His Arg              20 25 30 Glu Val Thr Pro Val Glu Leu Pro Asn Cys Asn Leu Val Lys Gly Val          35 40 45 Asp Asn Gly Ser Glu Asp Leu Glu Ile Leu Pro Asn Gly Leu Ala Phe      50 55 60 Ile Ser Ser Gly Leu Lys Tyr Pro Gly Ile Met Ser Phe Asp Pro Asp  65 70 75 80 Lys Ser Gly Lys Ile Leu Leu Met Asp Leu Asn Glu Glu Asp Pro Val                  85 90 95 Val Leu Glu Leu Gly Ile Thr Gly Asn Thr Leu Asp Ile Ser Ser Phe             100 105 110 Asn Pro Trp Gly Ile Ser Thr Phe Thr Asp Glu Asp Asn Thr Val Tyr         115 120 125 Leu Leu Val Val Asn His Pro Asp Ser Ser Ser Thr Val Glu Val Phe     130 135 140 Lys Phe Gln Glu Glu Glu Lys Ser Leu Leu His Leu Lys Thr Ile Arg 145 150 155 160 His Lys Leu Leu Pro Ser Val Asn Asp Ile Val Ala Val Gly Pro Glu                 165 170 175 His Phe Tyr Ala Thr Asn Asp His Tyr Phe Ala Asp Pro Tyr Leu Lys             180 185 190 Ser Trp Glu Met His Leu Gly Leu Ala Trp Ser Phe Val Thr Tyr Tyr         195 200 205 Ser Pro Asn Val Val Val Ala Glu Gly Phe Asp Phe Ala Asn     210 215 220 Gly Ile Asn Ile Ser Pro Asp Gly Lys Tyr Val Tyr Ile Ala Glu Leu 225 230 235 240 Leu Ala His Lys Ile His Val Tyr Glu Lys His Ala Asn Trp Thr Leu                 245 250 255 Thr Pro Leu Lys Ser Leu Asp Phe Asp Thr Leu Val Asp Asn Ile Ser             260 265 270 Val Asp Pro Val Thr Gly Asp Leu Trp Val Gly Cys His Pro Asn Gly         275 280 285 Met Arg Ile Phe Tyr Tyr Asp Pro Lys Asn Pro Pro Gly Ser Glu Val     290 295 300 Leu Arg Ile Gln Asp Ile Leu Ser Glu Glu Pro Lys Val Thr Val Val 305 310 315 320 Tyr Ala Glu Asn Gly Thr Val Leu Gln Gly Ser Thr Val Ala Ala Val                 325 330 335 Tyr Lys Gly Lys Leu Leu Ile Gly Thr Ala Phe His Lys Ala Leu Tyr             340 345 350 Cys Glu Leu         355 <210> 10 <211> 355 <212> PRT <213> Human <220> <221> VARIANT <222> <223> Threonine -> Serine <220> <221> VARIANT <222> (346) <223> Valine -> Alanine <400> 10 Met Ala Lys Leu Thr Ala Leu Thr Leu Leu Gly Leu Gly Leu Ala Leu   1 5 10 15 Phe Asp Gly Gln Lys Ser Ser Phe Gln Thr Arg Phe Asn Val His Arg              20 25 30 Glu Val Thr Pro Val Glu Leu Pro Asn Cys Asn Leu Val Lys Gly Val          35 40 45 Asp Asn Gly Ser Glu Asp Leu Glu Ile Leu Pro Asn Gly Leu Ala Phe      50 55 60 Ile Ser Ser Gly Leu Lys Tyr Pro Gly Ile Met Ser Phe Asp Pro Asp  65 70 75 80 Lys Ser Gly Lys Ile Leu Leu Met Asp Leu Asn Glu Glu Asp Pro Val                  85 90 95 Val Leu Glu Leu Gly Ile Thr Gly Asn Thr Leu Asp Ile Ser Ser Phe             100 105 110 Asn Pro His Gly Ile Ser Thr Phe Thr Asp Glu Asp Asn Thr Val Tyr         115 120 125 Leu Leu Val Val Asn His Pro Asp Ser Ser Ser Thr Val Glu Val Phe     130 135 140 Lys Phe Gln Glu Glu Glu Lys Ser Leu Leu His Leu Lys Thr Ile Arg 145 150 155 160 His Lys Leu Leu Pro Ser Val Asn Asp Ile Val Ala Val Gly Pro Glu                 165 170 175 His Phe Tyr Ala Thr Asn Asp His Tyr Phe Ala Asp Pro Tyr Leu Lys             180 185 190 Ser Trp Glu Met His Leu Gly Leu Ala Trp Ser Phe Val Thr Tyr Tyr         195 200 205 Ser Pro Asn Val Val Val Ala Glu Gly Phe Asp Phe Ala Asn     210 215 220 Gly Ile Asn Ile Ser Pro Asp Gly Lys Tyr Val Tyr Ile Ala Glu Leu 225 230 235 240 Leu Ala His Lys Ile His Val Tyr Glu Lys His Ala Asn Trp Thr Leu                 245 250 255 Thr Pro Leu Lys Ser Leu Asp Phe Asp Thr Leu Val Asp Asn Ile Ser             260 265 270 Val Asp Pro Val Thr Gly Asp Leu Trp Val Gly Cys His Pro Asn Gly         275 280 285 Met Arg Ile Phe Tyr Tyr Asp Pro Lys Asn Pro Pro Gly Ser Glu Val     290 295 300 Leu Arg Ile Gln Asp Ile Leu Ser Glu Glu Pro Lys Val Thr Val Val 305 310 315 320 Tyr Ala Glu Asn Gly Thr Val Leu Gln Gly Ser Ser Val Ala Ala Val                 325 330 335 Tyr Lys Gly Lys Leu Leu Ile Gly Thr Ala Phe His Lys Ala Leu Tyr             340 345 350 Cys Glu Leu         355 <210> 11 <211> 355 <212> PRT <213> Human <220> <221> VARIANT <74> <223> Isoleucine -> Phenylalanine <220> <221> VARIANT <115> <223> Histidine -> Tryptophan <220> <221> VARIANT <222> <223> Threonine -> Serine <400> 11 Met Ala Lys Leu Thr Ala Leu Thr Leu Leu Gly Leu Gly Leu Ala Leu   1 5 10 15 Phe Asp Gly Gln Lys Ser Ser Phe Gln Thr Arg Phe Asn Val His Arg              20 25 30 Glu Val Thr Pro Val Glu Leu Pro Asn Cys Asn Leu Val Lys Gly Val          35 40 45 Asp Asn Gly Ser Glu Asp Leu Glu Ile Leu Pro Asn Gly Leu Ala Phe      50 55 60 Ile Ser Ser Gly Leu Lys Tyr Pro Gly Phe Met Ser Phe Asp Pro Asp  65 70 75 80 Lys Ser Gly Lys Ile Leu Leu Met Asp Leu Asn Glu Glu Asp Pro Val                  85 90 95 Val Leu Glu Leu Gly Ile Thr Gly Asn Thr Leu Asp Ile Ser Ser Phe             100 105 110 Asn Pro Trp Gly Ile Ser Thr Phe Thr Asp Glu Asp Asn Thr Val Tyr         115 120 125 Leu Leu Val Val Asn His Pro Asp Ser Ser Ser Thr Val Glu Val Phe     130 135 140 Lys Phe Gln Glu Glu Glu Lys Ser Leu Leu His Leu Lys Thr Ile Arg 145 150 155 160 His Lys Leu Leu Pro Ser Val Asn Asp Ile Val Ala Val Gly Pro Glu                 165 170 175 His Phe Tyr Ala Thr Asn Asp His Tyr Phe Ala Asp Pro Tyr Leu Lys             180 185 190 Ser Trp Glu Met His Leu Gly Leu Ala Trp Ser Phe Val Thr Tyr Tyr         195 200 205 Ser Pro Asn Val Val Val Ala Glu Gly Phe Asp Phe Ala Asn     210 215 220 Gly Ile Asn Ile Ser Pro Asp Gly Lys Tyr Val Tyr Ile Ala Glu Leu 225 230 235 240 Leu Ala His Lys Ile His Val Tyr Glu Lys His Ala Asn Trp Thr Leu                 245 250 255 Thr Pro Leu Lys Ser Leu Asp Phe Asp Thr Leu Val Asp Asn Ile Ser             260 265 270 Val Asp Pro Val Thr Gly Asp Leu Trp Val Gly Cys His Pro Asn Gly         275 280 285 Met Arg Ile Phe Tyr Tyr Asp Pro Lys Asn Pro Pro Gly Ser Glu Val     290 295 300 Leu Arg Ile Gln Asp Ile Leu Ser Glu Glu Pro Lys Val Thr Val Val 305 310 315 320 Tyr Ala Glu Asn Gly Thr Val Leu Gln Gly Ser Ser Val Ala Ala Val                 325 330 335 Tyr Lys Gly Lys Leu Leu Ile Gly Thr Val Phe His Lys Ala Leu Tyr             340 345 350 Cys Glu Leu         355 <210> 12 <211> 355 <212> PRT <213> Human <220> <221> VARIANT <74> <223> Isoleucine -> Phenylalanine <220> <221> VARIANT <115> <223> Histidine -> Tryptophan <220> <221> VARIANT <222> (346) <223> Valine -> Alanine <400> 12 Met Ala Lys Leu Thr Ala Leu Thr Leu Leu Gly Leu Gly Leu Ala Leu   1 5 10 15 Phe Asp Gly Gln Lys Ser Ser Phe Gln Thr Arg Phe Asn Val His Arg              20 25 30 Glu Val Thr Pro Val Glu Leu Pro Asn Cys Asn Leu Val Lys Gly Val          35 40 45 Asp Asn Gly Ser Glu Asp Leu Glu Ile Leu Pro Asn Gly Leu Ala Phe      50 55 60 Ile Ser Ser Gly Leu Lys Tyr Pro Gly Phe Met Ser Phe Asp Pro Asp  65 70 75 80 Lys Ser Gly Lys Ile Leu Leu Met Asp Leu Asn Glu Glu Asp Pro Val                  85 90 95 Val Leu Glu Leu Gly Ile Thr Gly Asn Thr Leu Asp Ile Ser Ser Phe             100 105 110 Asn Pro Trp Gly Ile Ser Thr Phe Thr Asp Glu Asp Asn Thr Val Tyr         115 120 125 Leu Leu Val Val Asn His Pro Asp Ser Ser Ser Thr Val Glu Val Phe     130 135 140 Lys Phe Gln Glu Glu Glu Lys Ser Leu Leu His Leu Lys Thr Ile Arg 145 150 155 160 His Lys Leu Leu Pro Ser Val Asn Asp Ile Val Ala Val Gly Pro Glu                 165 170 175 His Phe Tyr Ala Thr Asn Asp His Tyr Phe Ala Asp Pro Tyr Leu Lys             180 185 190 Ser Trp Glu Met His Leu Gly Leu Ala Trp Ser Phe Val Thr Tyr Tyr         195 200 205 Ser Pro Asn Val Val Val Ala Glu Gly Phe Asp Phe Ala Asn     210 215 220 Gly Ile Asn Ile Ser Pro Asp Gly Lys Tyr Val Tyr Ile Ala Glu Leu 225 230 235 240 Leu Ala His Lys Ile His Val Tyr Glu Lys His Ala Asn Trp Thr Leu                 245 250 255 Thr Pro Leu Lys Ser Leu Asp Phe Asp Thr Leu Val Asp Asn Ile Ser             260 265 270 Val Asp Pro Val Thr Gly Asp Leu Trp Val Gly Cys His Pro Asn Gly         275 280 285 Met Arg Ile Phe Tyr Tyr Asp Pro Lys Asn Pro Pro Gly Ser Glu Val     290 295 300 Leu Arg Ile Gln Asp Ile Leu Ser Glu Glu Pro Lys Val Thr Val Val 305 310 315 320 Tyr Ala Glu Asn Gly Thr Val Leu Gln Gly Ser Thr Val Ala Ala Val                 325 330 335 Tyr Lys Gly Lys Leu Leu Ile Gly Thr Ala Phe His Lys Ala Leu Tyr             340 345 350 Cys Glu Leu         355 <210> 13 <211> 1065 <212> DNA <213> Human <400> 13 atggcaaaac tgaccgctct gacgctgctg ggtctgggtc tggcactgtt tgatggtcag 60 aaaagctctt ttcaaacccg tttcaacgtg catcgcgaag tgacgccggt tgaactgccg 120 aactgcaatc tggtcaaagg cgtggataac ggttctgaag acctggaaat tctgccgaat 180 ggtctggcgt ttatcagttc cggcctgaaa tatccgggta ttatgtcctt cgatccggac 240 aaatcaggca aaatcctgct gatggatctg aatgaagaag acccggtggt tctggaactg 300 ggcattaccg gtaacacgct ggatatctca tcgtttaatc cgcatggcat tagcaccttc 360 acggatgaag acaacaccgt gtatctgctg gtcgtgaatc acccggatag ctctagtacg 420 gtcgaagtgt tcaaattcca ggaagaagaa aaatcgctgc tgcatctgaa aaccattcgt 480 cacaaactgc tgccgagcgt taacgatatc gttgcggtcg gtccggaaca tttttacgcg 540 acgaatgatc actatttcgc cgacccgtac ctgaaatctt gggaaatgca tctgggcctg 600 gcctggtcgt ttgtcaccta ttacagcccg aacgacgtgc gcgttgtcgc agaaggtttt 660 gatttcgcta acggcattaa tatcagtccg gatggtaaat atgtgtacat tgcggaactg 720 ctggcccata aaatccacgt ttatgaaaaa catgccaact ggaccctgac gccgctgaaa 780 tccctggatt ttgacaccct ggtcgataat atttcagttg acccggtcac gggcgatctg 840 tgggttggtt gccacccgaa cggcatgcgt atcttctatt acgacccgaa aaatccgccg 900 ggctctgaag tgctgcgcat tcaggatatc ctgagtgaag aaccgaaagt taccgtggtt 960 tacgcagaaa atggtaccgt cctgcaaggt tccacggtgg cagcagttta taaaggtaaa 1020 ctgctgattg gcaccgtttt ccacaaagct ctgtactgtg aactg 1065 <210> 14 <211> 1065 <212> DNA <213> Human <400> 14 atggcaaaac tgaccgctct gacgctgctg ggtctgggtc tggcactgtt tgatggtcag 60 aaaagctctt ttcaaacccg tttcaacgtg catcgcgaag tgacgccggt tgaactgccg 120 aactgcaatc tggtcaaagg cgtggataac ggttctgaag acctggaaat tctgccgaat 180 ggtctggcgt ttatcagttc cggcctgaaa tatccgggtt tcatgtcctt cgatccggac 240 aaatcaggca aaatcctgct gatggatctg aatgaagaag acccggtggt tctggaactg 300 ggcattaccg gtaacacgct ggatatctca tcgtttaatc cgcatggcat tagcaccttc 360 acggatgaag acaacaccgt gtatctgctg gtcgtgaatc acccggatag ctctagtacg 420 gtcgaagtgt tcaaattcca ggaagaagaa aaatcgctgc tgcatctgaa aaccattcgt 480 cacaaactgc tgccgagcgt taacgatatc gttgcggtcg gtccggaaca tttttacgcg 540 acgaatgatc actatttcgc cgacccgtac ctgaaatctt gggaaatgca tctgggcctg 600 gcctggtcgt ttgtcaccta ttacagcccg aacgacgtgc gcgttgtcgc agaaggtttt 660 gatttcgcta acggcattaa tatcagtccg gatggtaaat atgtgtacat tgcggaactg 720 ctggcccata aaatccacgt ttatgaaaaa catgccaact ggaccctgac gccgctgaaa 780 tccctggatt ttgacaccct ggtcgataat atttcagttg acccggtcac gggcgatctg 840 tgggttggtt gccacccgaa cggcatgcgt atcttctatt acgacccgaa aaatccgccg 900 ggctctgaag tgctgcgcat tcaggatatc ctgagtgaag aaccgaaagt taccgtggtt 960 tacgcagaaa atggtaccgt cctgcaaggt tccacggtgg cagcagttta taaaggtaaa 1020 ctgctgattg gcaccgtttt ccacaaagct ctgtactgtg aactg 1065 <210> 15 <211> 1065 <212> DNA <213> Human <400> 15 atggcaaaac tgaccgctct gacgctgctg ggtctgggtc tggcactgtt tgatggtcag 60 aaaagctctt ttcaaacccg tttcaacgtg catcgcgaag tgacgccggt tgaactgccg 120 aactgcaatc tggtcaaagg cgtggataac ggttctgaag acctggaaat tctgccgaat 180 ggtctggcgt ttatcagttc cggcctgaaa tatccgggta ttatgtcctt cgatccggac 240 aaatcaggca aaatcctgct gatggatctg aatgaagaag acccggtggt tctggaactg 300 ggcattaccg gtaacacgct ggatatctca tcgtttaatc cgcatggcat tagcaccttc 360 acggatgaag acaacaccgt gtatctgctg gtcgtgaatc acccggatag ctctagtacg 420 gtcgaagtgt tcaaattcca ggaagaagaa aaatcgctgc tgcatctgaa aaccattcgt 480 cacaaactgc tgccgagcgt taacgatatc gttgcggtcg gtccggaaca tttttacgcg 540 acgaatgatc actatttcgc cgacccgtac ctgaaatctt gggaaatgca tctgggcctg 600 gcctggtcgt ttgtcaccta ttacagcccg aacgacgtgc gcgttgtcgc agaaggtttt 660 gatttcgcta acggcattaa tatcagtccg gatggtaaat atgtgtacat tgcggaactg 720 ctggcccata aaatccacgt ttatgaaaaa catgccaact ggaccctgac gccgctgaaa 780 tccctggatt ttgacaccct ggtcgataat atttcagttg acccggtcac gggcgatctg 840 tgggttggtt gccacccgaa cggcatgcgt atcttctatt acgacccgaa aaatccgccg 900 ggctctgaag tgctgcgcat tcaggatatc ctgagtgaag aaccgaaagt taccgtggtt 960 tacgcagaaa atggtaccgt cctgcaaggt tcctctgtgg cagcagttta taaaggtaaa 1020 ctgctgattg gcaccgtttt ccacaaagct ctgtactgtg aactg 1065 <210> 16 <211> 1065 <212> DNA <213> Human <400> 16 atggcaaaac tgaccgctct gacgctgctg ggtctgggtc tggcactgtt tgatggtcag 60 aaaagctctt ttcaaacccg tttcaacgtg catcgcgaag tgacgccggt tgaactgccg 120 aactgcaatc tggtcaaagg cgtggataac ggttctgaag acctggaaat tctgccgaat 180 ggtctggcgt ttatcagttc cggcctgaaa tatccgggta ttatgtcctt cgatccggac 240 aaatcaggca aaatcctgct gatggatctg aatgaagaag acccggtggt tctggaactg 300 ggcattaccg gtaacacgct ggatatctca tcgtttaatc cgcatggcat tagcaccttc 360 acggatgaag acaacaccgt gtatctgctg gtcgtgaatc acccggatag ctctagtacg 420 gtcgaagtgt tcaaattcca ggaagaagaa aaatcgctgc tgcatctgaa aaccattcgt 480 cacaaactgc tgccgagcgt taacgatatc gttgcggtcg gtccggaaca tttttacgcg 540 acgaatgatc actatttcgc cgacccgtac ctgaaatctt gggaaatgca tctgggcctg 600 gcctggtcgt ttgtcaccta ttacagcccg aacgacgtgc gcgttgtcgc agaaggtttt 660 gatttcgcta acggcattaa tatcagtccg gatggtaaat atgtgtacat tgcggaactg 720 ctggcccata aaatccacgt ttatgaaaaa catgccaact ggaccctgac gccgctgaaa 780 tccctggatt ttgacaccct ggtcgataat atttcagttg acccggtcac gggcgatctg 840 tgggttggtt gccacccgaa cggcatgcgt atcttctatt acgacccgaa aaatccgccg 900 ggctctgaag tgctgcgcat tcaggatatc ctgagtgaag aaccgaaagt taccgtggtt 960 tacgcagaaa atggtaccgt cctgcaaggt tccacggtgg cagcagttta taaaggtaaa 1020 ctgctgattg gcaccgcttt ccacaaagct ctgtactgtg aactg 1065 <210> 17 <211> 1065 <212> DNA <213> Human <400> 17 atggcaaaac tgaccgctct gacgctgctg ggtctgggtc tggcactgtt tgatggtcag 60 aaaagctctt ttcaaacccg tttcaacgtg catcgcgaag tgacgccggt tgaactgccg 120 aactgcaatc tggtcaaagg cgtggataac ggttctgaag acctggaaat tctgccgaat 180 ggtctggcgt ttatcagttc cggcctgaaa tatccgggtt tcatgtcctt cgatccggac 240 aaatcaggca aaatcctgct gatggatctg aatgaagaag acccggtggt tctggaactg 300 ggcattaccg gtaacacgct ggatatctca tcgtttaatc cgtggggcat tagcaccttc 360 acggatgaag acaacaccgt gtatctgctg gtcgtgaatc acccggatag ctctagtacg 420 gtcgaagtgt tcaaattcca ggaagaagaa aaatcgctgc tgcatctgaa aaccattcgt 480 cacaaactgc tgccgagcgt taacgatatc gttgcggtcg gtccggaaca tttttacgcg 540 acgaatgatc actatttcgc cgacccgtac ctgaaatctt gggaaatgca tctgggcctg 600 gcctggtcgt ttgtcaccta ttacagcccg aacgacgtgc gcgttgtcgc agaaggtttt 660 gatttcgcta acggcattaa tatcagtccg gatggtaaat atgtgtacat tgcggaactg 720 ctggcccata aaatccacgt ttatgaaaaa catgccaact ggaccctgac gccgctgaaa 780 tccctggatt ttgacaccct ggtcgataat atttcagttg acccggtcac gggcgatctg 840 tgggttggtt gccacccgaa cggcatgcgt atcttctatt acgacccgaa aaatccgccg 900 ggctctgaag tgctgcgcat tcaggatatc ctgagtgaag aaccgaaagt taccgtggtt 960 tacgcagaaa atggtaccgt cctgcaaggt tccacggtgg cagcagttta taaaggtaaa 1020 ctgctgattg gcaccgtttt ccacaaagct ctgtactgtg aactg 1065 <210> 18 <211> 1065 <212> DNA <213> Human <400> 18 atggcaaaac tgaccgctct gacgctgctg ggtctgggtc tggcactgtt tgatggtcag 60 aaaagctctt ttcaaacccg tttcaacgtg catcgcgaag tgacgccggt tgaactgccg 120 aactgcaatc tggtcaaagg cgtggataac ggttctgaag acctggaaat tctgccgaat 180 ggtctggcgt ttatcagttc cggcctgaaa tatccgggtt tcatgtcctt cgatccggac 240 aaatcaggca aaatcctgct gatggatctg aatgaagaag acccggtggt tctggaactg 300 ggcattaccg gtaacacgct ggatatctca tcgtttaatc cgcatggcat tagcaccttc 360 acggatgaag acaacaccgt gtatctgctg gtcgtgaatc acccggatag ctctagtacg 420 gtcgaagtgt tcaaattcca ggaagaagaa aaatcgctgc tgcatctgaa aaccattcgt 480 cacaaactgc tgccgagcgt taacgatatc gttgcggtcg gtccggaaca tttttacgcg 540 acgaatgatc actatttcgc cgacccgtac ctgaaatctt gggaaatgca tctgggcctg 600 gcctggtcgt ttgtcaccta ttacagcccg aacgacgtgc gcgttgtcgc agaaggtttt 660 gatttcgcta acggcattaa tatcagtccg gatggtaaat atgtgtacat tgcggaactg 720 ctggcccata aaatccacgt ttatgaaaaa catgccaact ggaccctgac gccgctgaaa 780 tccctggatt ttgacaccct ggtcgataat atttcagttg acccggtcac gggcgatctg 840 tgggttggtt gccacccgaa cggcatgcgt atcttctatt acgacccgaa aaatccgccg 900 ggctctgaag tgctgcgcat tcaggatatc ctgagtgaag aaccgaaagt taccgtggtt 960 tacgcagaaa atggtaccgt cctgcaaggt tcctctgtgg cagcagttta taaaggtaaa 1020 ctgctgattg gcaccgtttt ccacaaagct ctgtactgtg aactg 1065 <210> 19 <211> 1065 <212> DNA <213> Human <400> 19 atggcaaaac tgaccgctct gacgctgctg ggtctgggtc tggcactgtt tgatggtcag 60 aaaagctctt ttcaaacccg tttcaacgtg catcgcgaag tgacgccggt tgaactgccg 120 aactgcaatc tggtcaaagg cgtggataac ggttctgaag acctggaaat tctgccgaat 180 ggtctggcgt ttatcagttc cggcctgaaa tatccgggtt tcatgtcctt cgatccggac 240 aaatcaggca aaatcctgct gatggatctg aatgaagaag acccggtggt tctggaactg 300 ggcattaccg gtaacacgct ggatatctca tcgtttaatc cgcatggcat tagcaccttc 360 acggatgaag acaacaccgt gtatctgctg gtcgtgaatc acccggatag ctctagtacg 420 gtcgaagtgt tcaaattcca ggaagaagaa aaatcgctgc tgcatctgaa aaccattcgt 480 cacaaactgc tgccgagcgt taacgatatc gttgcggtcg gtccggaaca tttttacgcg 540 acgaatgatc actatttcgc cgacccgtac ctgaaatctt gggaaatgca tctgggcctg 600 gcctggtcgt ttgtcaccta ttacagcccg aacgacgtgc gcgttgtcgc agaaggtttt 660 gatttcgcta acggcattaa tatcagtccg gatggtaaat atgtgtacat tgcggaactg 720 ctggcccata aaatccacgt ttatgaaaaa catgccaact ggaccctgac gccgctgaaa 780 tccctggatt ttgacaccct ggtcgataat atttcagttg acccggtcac gggcgatctg 840 tgggttggtt gccacccgaa cggcatgcgt atcttctatt acgacccgaa aaatccgccg 900 ggctctgaag tgctgcgcat tcaggatatc ctgagtgaag aaccgaaagt taccgtggtt 960 tacgcagaaa atggtaccgt cctgcaaggt tccacggtgg cagcagttta taaaggtaaa 1020 ctgctgattg gcaccgcttt ccacaaagct ctgtactgtg aactg 1065 <210> 20 <211> 1065 <212> DNA <213> Human <400> 20 atggcaaaac tgaccgctct gacgctgctg ggtctgggtc tggcactgtt tgatggtcag 60 aaaagctctt ttcaaacccg tttcaacgtg catcgcgaag tgacgccggt tgaactgccg 120 aactgcaatc tggtcaaagg cgtggataac ggttctgaag acctggaaat tctgccgaat 180 ggtctggcgt ttatcagttc cggcctgaaa tatccgggta ttatgtcctt cgatccggac 240 aaatcaggca aaatcctgct gatggatctg aatgaagaag acccggtggt tctggaactg 300 ggcattaccg gtaacacgct ggatatctca tcgtttaatc cgtggggcat tagcaccttc 360 acggatgaag acaacaccgt gtatctgctg gtcgtgaatc acccggatag ctctagtacg 420 gtcgaagtgt tcaaattcca ggaagaagaa aaatcgctgc tgcatctgaa aaccattcgt 480 cacaaactgc tgccgagcgt taacgatatc gttgcggtcg gtccggaaca tttttacgcg 540 acgaatgatc actatttcgc cgacccgtac ctgaaatctt gggaaatgca tctgggcctg 600 gcctggtcgt ttgtcaccta ttacagcccg aacgacgtgc gcgttgtcgc agaaggtttt 660 gatttcgcta acggcattaa tatcagtccg gatggtaaat atgtgtacat tgcggaactg 720 ctggcccata aaatccacgt ttatgaaaaa catgccaact ggaccctgac gccgctgaaa 780 tccctggatt ttgacaccct ggtcgataat atttcagttg acccggtcac gggcgatctg 840 tgggttggtt gccacccgaa cggcatgcgt atcttctatt acgacccgaa aaatccgccg 900 ggctctgaag tgctgcgcat tcaggatatc ctgagtgaag aaccgaaagt taccgtggtt 960 tacgcagaaa atggtaccgt cctgcaaggt tcctctgtgg cagcagttta taaaggtaaa 1020 ctgctgattg gcaccgtttt ccacaaagct ctgtactgtg aactg 1065 <210> 21 <211> 1065 <212> DNA <213> Human <400> 21 atggcaaaac tgaccgctct gacgctgctg ggtctgggtc tggcactgtt tgatggtcag 60 aaaagctctt ttcaaacccg tttcaacgtg catcgcgaag tgacgccggt tgaactgccg 120 aactgcaatc tggtcaaagg cgtggataac ggttctgaag acctggaaat tctgccgaat 180 ggtctggcgt ttatcagttc cggcctgaaa tatccgggta ttatgtcctt cgatccggac 240 aaatcaggca aaatcctgct gatggatctg aatgaagaag acccggtggt tctggaactg 300 ggcattaccg gtaacacgct ggatatctca tcgtttaatc cgtggggcat tagcaccttc 360 acggatgaag acaacaccgt gtatctgctg gtcgtgaatc acccggatag ctctagtacg 420 gtcgaagtgt tcaaattcca ggaagaagaa aaatcgctgc tgcatctgaa aaccattcgt 480 cacaaactgc tgccgagcgt taacgatatc gttgcggtcg gtccggaaca tttttacgcg 540 acgaatgatc actatttcgc cgacccgtac ctgaaatctt gggaaatgca tctgggcctg 600 gcctggtcgt ttgtcaccta ttacagcccg aacgacgtgc gcgttgtcgc agaaggtttt 660 gatttcgcta acggcattaa tatcagtccg gatggtaaat atgtgtacat tgcggaactg 720 ctggcccata aaatccacgt ttatgaaaaa catgccaact ggaccctgac gccgctgaaa 780 tccctggatt ttgacaccct ggtcgataat atttcagttg acccggtcac gggcgatctg 840 tgggttggtt gccacccgaa cggcatgcgt atcttctatt acgacccgaa aaatccgccg 900 ggctctgaag tgctgcgcat tcaggatatc ctgagtgaag aaccgaaagt taccgtggtt 960 tacgcagaaa atggtaccgt cctgcaaggt tccacggtgg cagcagttta taaaggtaaa 1020 ctgctgattg gcaccgcttt ccacaaagct ctgtactgtg aactg 1065 <210> 22 <211> 1065 <212> DNA <213> Human <400> 22 atggcaaaac tgaccgctct gacgctgctg ggtctgggtc tggcactgtt tgatggtcag 60 aaaagctctt ttcaaacccg tttcaacgtg catcgcgaag tgacgccggt tgaactgccg 120 aactgcaatc tggtcaaagg cgtggataac ggttctgaag acctggaaat tctgccgaat 180 ggtctggcgt ttatcagttc cggcctgaaa tatccgggta ttatgtcctt cgatccggac 240 aaatcaggca aaatcctgct gatggatctg aatgaagaag acccggtggt tctggaactg 300 ggcattaccg gtaacacgct ggatatctca tcgtttaatc cgcatggcat tagcaccttc 360 acggatgaag acaacaccgt gtatctgctg gtcgtgaatc acccggatag ctctagtacg 420 gtcgaagtgt tcaaattcca ggaagaagaa aaatcgctgc tgcatctgaa aaccattcgt 480 cacaaactgc tgccgagcgt taacgatatc gttgcggtcg gtccggaaca tttttacgcg 540 acgaatgatc actatttcgc cgacccgtac ctgaaatctt gggaaatgca tctgggcctg 600 gcctggtcgt ttgtcaccta ttacagcccg aacgacgtgc gcgttgtcgc agaaggtttt 660 gatttcgcta acggcattaa tatcagtccg gatggtaaat atgtgtacat tgcggaactg 720 ctggcccata aaatccacgt ttatgaaaaa catgccaact ggaccctgac gccgctgaaa 780 tccctggatt ttgacaccct ggtcgataat atttcagttg acccggtcac gggcgatctg 840 tgggttggtt gccacccgaa cggcatgcgt atcttctatt acgacccgaa aaatccgccg 900 ggctctgaag tgctgcgcat tcaggatatc ctgagtgaag aaccgaaagt taccgtggtt 960 tacgcagaaa atggtaccgt cctgcaaggt tcctctgtgg cagcagttta taaaggtaaa 1020 ctgctgattg gcaccgcttt ccacaaagct ctgtactgtg aactg 1065 <210> 23 <211> 1065 <212> DNA <213> Human <400> 23 atggcaaaac tgaccgctct gacgctgctg ggtctgggtc tggcactgtt tgatggtcag 60 aaaagctctt ttcaaacccg tttcaacgtg catcgcgaag tgacgccggt tgaactgccg 120 aactgcaatc tggtcaaagg cgtggataac ggttctgaag acctggaaat tctgccgaat 180 ggtctggcgt ttatcagttc cggcctgaaa tatccgggtt tcatgtcctt cgatccggac 240 aaatcaggca aaatcctgct gatggatctg aatgaagaag acccggtggt tctggaactg 300 ggcattaccg gtaacacgct ggatatctca tcgtttaatc cgtggggcat tagcaccttc 360 acggatgaag acaacaccgt gtatctgctg gtcgtgaatc acccggatag ctctagtacg 420 gtcgaagtgt tcaaattcca ggaagaagaa aaatcgctgc tgcatctgaa aaccattcgt 480 cacaaactgc tgccgagcgt taacgatatc gttgcggtcg gtccggaaca tttttacgcg 540 acgaatgatc actatttcgc cgacccgtac ctgaaatctt gggaaatgca tctgggcctg 600 gcctggtcgt ttgtcaccta ttacagcccg aacgacgtgc gcgttgtcgc agaaggtttt 660 gatttcgcta acggcattaa tatcagtccg gatggtaaat atgtgtacat tgcggaactg 720 ctggcccata aaatccacgt ttatgaaaaa catgccaact ggaccctgac gccgctgaaa 780 tccctggatt ttgacaccct ggtcgataat atttcagttg acccggtcac gggcgatctg 840 tgggttggtt gccacccgaa cggcatgcgt atcttctatt acgacccgaa aaatccgccg 900 ggctctgaag tgctgcgcat tcaggatatc ctgagtgaag aaccgaaagt taccgtggtt 960 tacgcagaaa atggtaccgt cctgcaaggt tcctctgtgg cagcagttta taaaggtaaa 1020 ctgctgattg gcaccgtttt ccacaaagct ctgtactgtg aactg 1065 <210> 24 <211> 1065 <212> DNA <213> Human <400> 24 atggcaaaac tgaccgctct gacgctgctg ggtctgggtc tggcactgtt tgatggtcag 60 aaaagctctt ttcaaacccg tttcaacgtg catcgcgaag tgacgccggt tgaactgccg 120 aactgcaatc tggtcaaagg cgtggataac ggttctgaag acctggaaat tctgccgaat 180 ggtctggcgt ttatcagttc cggcctgaaa tatccgggtt tcatgtcctt cgatccggac 240 aaatcaggca aaatcctgct gatggatctg aatgaagaag acccggtggt tctggaactg 300 ggcattaccg gtaacacgct ggatatctca tcgtttaatc cgtggggcat tagcaccttc 360 acggatgaag acaacaccgt gtatctgctg gtcgtgaatc acccggatag ctctagtacg 420 gtcgaagtgt tcaaattcca ggaagaagaa aaatcgctgc tgcatctgaa aaccattcgt 480 cacaaactgc tgccgagcgt taacgatatc gttgcggtcg gtccggaaca tttttacgcg 540 acgaatgatc actatttcgc cgacccgtac ctgaaatctt gggaaatgca tctgggcctg 600 gcctggtcgt ttgtcaccta ttacagcccg aacgacgtgc gcgttgtcgc agaaggtttt 660 gatttcgcta acggcattaa tatcagtccg gatggtaaat atgtgtacat tgcggaactg 720 ctggcccata aaatccacgt ttatgaaaaa catgccaact ggaccctgac gccgctgaaa 780 tccctggatt ttgacaccct ggtcgataat atttcagttg acccggtcac gggcgatctg 840 tgggttggtt gccacccgaa cggcatgcgt atcttctatt acgacccgaa aaatccgccg 900 ggctctgaag tgctgcgcat tcaggatatc ctgagtgaag aaccgaaagt taccgtggtt 960 tacgcagaaa atggtaccgt cctgcaaggt tccacggtgg cagcagttta taaaggtaaa 1020 ctgctgattg gcaccgcttt ccacaaagct ctgtactgtg aactg 1065 <210> 25 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer MF_L69A <400> 25 agttccggcg caaaatatcc gggtattatg tcc 33 <210> 26 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer MR_L69A <400> 26 cggatatttt gcgccggaac tgataaacgc cag 33 <210> 27 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer MF_L69V <400> 27 agttccggcg ttaaatatcc gggtattatg tcc 33 <210> 28 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer MR_L69V <400> 28 cggatattta acgccggaac tgataaacgc cag 33 <210> 29 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer MF_L69I <400> 29 agttccggca ttaaatatcc gggtattatg tcc 33 <210> 30 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer MR_L69I <400> 30 cggatattta atgccggaac tgataaacgc cag 33 <210> 31 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer MF_I74A <400> 31 tatccgggtg ctatgtcctt cgatccggac aaa 33 <210> 32 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer MR_I74A <400> 32 gaaggacata gcacccggat atttcaggcc gga 33 <210> 33 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer MF_I74V <400> 33 tatccgggtg ttatgtcctt cgatccggac aaa 33 <210> 34 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer MR_I74V <400> 34 gaaggacata acacccggat atttcaggcc gga 33 <210> 35 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer MF_I74L <400> 35 tatccgggtc ttatgtcctt cgatccggac aaa 33 <210> 36 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer MR_I74L <400> 36 gaaggacata agacccggat atttcaggcc gga 33 <210> 37 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer MF_I74F <400> 37 tatccgggtt tcatgtcctt cgatccggac aaatcaggc 39 <210> 38 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer MR_I74F <400> 38 gaaggacatg aaacccggat atttcaggcc ggaactgat 39 <210> 39 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer MF_I74W <400> 39 tatccgggtt ggatgtcctt cgatccggac aaatcaggc 39 <210> 40 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer MR_I74W <400> 40 gaaggacatc caacccggat atttcaggcc ggaactgat 39 <210> 41 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> primer MF_H115W <400> 41 tttaatccgt ggggcattag caccttcacg gatg 34 <210> 42 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer MR_H115W <400> 42 gctaatgccc cacggattaa acgatgagat atc 33 <210> 43 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer MF_T332A <400> 43 caaggttccg ctgtggcagc agtttataaa ggt 33 <210> 44 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer MF_T332A <400> 44 tgctgccaca gcggaacctt gcaggacggt acc 33 <210> 45 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer MF_T332S <400> 45 caaggttcct ctgtggcagc agtttataaa ggtaaactg 39 <210> 46 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer MR_T332S <400> 46 tgctgccaca gaggaacctt gcaggacggt accattttc 39 <210> 47 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer MF_T332V <400> 47 caaggttccg tagtggcagc agtttataaa ggt 33 <210> 48 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer MR_T332V <400> 48 tgctgccact acggaacctt gcaggacggt acc 33 <210> 49 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer MF_V346A <400> 49 attggcaccg ctttccacaa agctctgtac tgt 33 <210> 50 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer MR_V346A <400> 50 tttgtggaaa gcggtgccaa tcagcagttt acc 33 <210> 51 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer MF_V346L <400> 51 attggcaccc ttttccacaa agctctgtac tgt 33 <210> 52 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer MR_V346L <400> 52 tttgtggaaa agggtgccaa tcagcagttt acc 33 <210> 53 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer MF_V346I <400> 53 attggcacca ttttccacaa agctctgtac tgt 33 <210> 54 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer MR_V346I <400> 54 tttgtggaaa atggtgccaa tcagcagttt acc 33

Claims (13)

(I74), histidine (H115) which is the 115th amino acid, threonine (T332) which is the 332nd amino acid and threonine (T332) which is the amino acid of the 346th amino acid Wherein at least one of valine V346 is substituted with any one amino acid selected from the group consisting of phenylalanine (F), tryptophan (W), serine (S) and alanine (A) Increased paraoxonase first variant compared to wild type paraoxonase I. The method according to claim 1,
Wherein said first paraoxonase variant is a mutation at a position corresponding to at least one of the following mutations in the amino acid sequence of SEQ ID NO:
I74F + H115W + V346A, I74F + T342A, I74F + T342A, I74F + T342A, T342A +
/ RTI &gt; a first variant of para-oxonase. The method according to claim 1,
Wherein said first paraoxonase variant comprises a mutation at a position corresponding to H115W + T332S, I74F + H115W + T332S in the amino acid sequence of SEQ ID NO: 1. The method according to claim 1,
Wherein said variant has an amino acid sequence of any one of SEQ ID NOS: 2 to 12. The method according to claim 1,
Wherein said paraoxonase first variant has a hydrolytic activity that is 2.4-39 times greater than the hydrolysis activity of wild-type paraoxonase I. &lt; RTI ID = 0.0 &gt; 1. &lt; / RTI &gt; The method according to claim 1,
The organophosphorus paraoxone compound may be selected from the group consisting of dimethyl-paraoxone (MPO), diethyl-paraoxone (EPO), diazinon-oxone (DZO), chloropyrimidine- chloropyrifos-oxon, CPO). &lt; / RTI &gt; The method according to claim 6,
Wherein said organophosphorus paraoxone compound is dimethyl-paraoxone (MPO). The method according to claim 6,
Wherein said organophosphorus paraoxone compound is diethyl-paraoxone (EPO). A gene encoding the first paraoxonase variant of claim 1. 10. The method of claim 9,
Wherein the gene has the nucleotide sequence of any one of SEQ ID NOS: 13 to 24. A recombinant expression vector comprising the gene of claim 9. (a) transforming the expression vector of claim 10 into a microorganism to produce a recombinant microorganism;
(b) culturing the produced recombinant microorganism to express a paraoxonase first variant; And
(c) recovering the first paraoxonase variant from said embryo
&Lt; / RTI &gt; of claim 1, wherein the first variant of paraoxonase is selected from the group consisting of: 13. The method of claim 12,
Wherein the microorganism is Escherichia coli.

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