KR100444431B1 - A method for screening amino acid residues of IGFBP-3 which are necessary for biding ALS - Google Patents

Abstract

PURPOSE: A screening method of an amino acid residue of IGFBP-3 required for binding with ALS using an IGFBP-3 mutant is provided, thereby rapidly and easily screening the amino acid residue of IGFBP-3 in the absence of a ligand IFG. CONSTITUTION: The screening method of an amino acid residue of IGFBP-3 required for binding with ALS using an IGFBP-3 mutant comprises the steps of: (1) preparing mRNA encoding the IGFBP-3 mutant having one nucleotide sequence selected from SEQ ID NO:2 to SEQ ID NO:15 using a primer, and mRNA encoding a natural type ALS(acid labile subunit); (2) introducing the prepared two mRNAs and radiation-labeled amino acids into Xenopus oocyte; (3) incubating the Xenopus oocyte to form ALS/IGFBP-3 mutant complex; and (4) isolating the complex and measuring the amount of the complex, wherein the primer has one nucleotide sequence selected from SEQ ID NO:17 to SEQ ID NO:23.

Description

Method for screening amino acid residues of IGFBP-3 which are necessary for biding ALS}

The present invention relates to IGFBP variants and methods of screening amino acid residues that bind ALS using the same. More specifically, the present invention comprises the steps of (1) preparing mRNA encoding the IGFBP-3 variant and mRNA encoding the native type ALS (Acid Labile Subunit), (2) the two mRNAs prepared above and radiolabeled amino acids Injecting into a frog egg (Xenopus Oocyte), (3) culturing the frog egg to form a complex of ALS / IGFBP-3 variants, and (4) separating the complex and detecting the amount thereof. It relates to a method for screening amino acid residues involved in binding to ALS in IGFBP-3. Further, the present invention relates to the above IGFBP-3 variants and nucleic acid sequences encoding them.

Growth factors are polypeptides that stimulate a wide range of biological responses (eg, DNA synthesis, cell division, expression of specific genes, etc.) in a defined population of target cells. Transformation growth factor β1 (TGF-β1), TGF-β2, TGF-β3, TGF-β4, TGF-β5, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF) ), Various growth factors have already been identified, including insulin-like growth factor-I (IGF-I) and IGF-II.

IGF-I and IGF-II are related in amino acid sequence and structure, and each polypeptide has a molecular weight of approximately 7.5 kilodaltons (Kd). IGF-I regulates the main effects of growth hormone and is the primary regulator of postnatal growth. In addition, IGF-I seems to increase the production of IGF-I when the cells are treated with various other growth factors, and it is believed that IGF-II plays a major role in fetal growth. IGF-I and IGF-II both have insulin-like activity and promote cell division for cells of nerves, muscles, skeleton and various other tissues.

IGF circulates in the form of non-covalent ternary complexes consisting of larger protein subunits called IGF-I or IGF-II, IGFBP-3, acid variable subunits (ALS). These ternary complexes each consist of three components of the same molar amount. ALS does not have direct binding activity to IGF, but appears to bind only to the IGF / IGFBP-3 binary complex. The IGF / IGFBP-3 / ALS ternary complex has a molecular weight of approximately 150 Kd. This ternary complex is believed to act as a reservoir and buffer for IGF-I and IGF-II, which prevents abrupt changes in free IGF concentration in the circulation.

Since most circulating IGF-I, IGF-II and IGFBP-3 are in the form of complexes, there are few detectable free IGFs. Moreover, it is undesirable to have high levels of free IGF in the blood. High blood levels of free IGF cause severe hypoglycemia due to insulin-like activity of IGF. In contrast to IGF and IGFBP-3, there is a substantial pool of free ALS in plasma that ensures that all IGF / IGFBP-3 complexes entering the circulation immediately form ternary complexes.

IGFBP-3 is the most common IGF binding protein in this circulation system, while at least five different distinguishing IGF binding proteins (IGFBPs) have been identified in various tissues and body fluids. However, although these proteins bind IGF, they each have distinct amino acid sequences that originate from separate genes. Thus binding proteins are not simply analogs or derivatives of common precursors. Unlike IGFBP-3, other circulating IGFBPs are not saturated with IGFs. No IGFBP other than IGFBP-3 can form a 150Kd ternary complex.

On the other hand, information available regarding the amino acid residues or domains of IGFBP-3 that bind IGF and ALS is extremely limited. NMR and positional mutation studies have revealed that 56Ile, 57Tyr, 69Pro and 75Arg to 81Leu make a hydrophobic patch for binding to IGF, while at the same time the basic residues bind to the negative charge of IGF. By swapping between the N-terminal, central and C-terminal domains of IGFBP-2 and IGFBP-3, the center and carboxy of IGFBP-3 for binding to ALS independent of the IGF ligand It has been found that the terminal domain is essential. In addition, analysis of IGFBP-3 mutations lacking the central and / or C-terminal domains also confirmed that both the central and C-terminal domains are essential for ALS binding. The researchers identified Lys-Gly-Arg-Lys-Arg (from amino acids 228 to 232), a basic domain located within the heparine binding domain of the C-terminal domain, to Met-Asp-Gly-Glu-Ala. IGFBP-3 variant was found to reduce the binding affinity with ALS by 90%.

However, the researchers studied the binding activity of ALS and IGFBP-3 in the presence of IGF ligand. In addition, a lot of time and effort was previously required for IGFBP-3, in order to identify the amino acid residues involved in binding to ALS, each of the ALS and IGFBP-3 variants had to be expressed and purified in the cell and then screened for their interactions. There was a problem.

Accordingly, the present inventors attempted to screen the amino acid residue of another IGFBP-3 involved in binding to ALS in the absence of the IGF ligand, as well as to screen in a more efficient manner than the prior art. To this end, IGFBP-3 amino acids are suspected to be involved in binding to ALS, and DNAs encoding the IGFBP-3 variants in which such amino acids are mutated to prepare mRNA from them, which are then encoded by native ALS The amino acid residues involved in binding to ALS in IGFBP-3 were screened by injecting the mRNA into frog eggs and incubating them to form complexes of the ALS / IGFBP-3 variant.

According to one aspect of the present invention, there is provided a method for preparing an mRNA comprising: (1) preparing an mRNA encoding an IGFBP-3 variant and an mRNA encoding a native type ALS (Acid Labile Subunit); Injecting into a frog egg (Xenopus Oocyte), (3) culturing the frog egg to form a complex of ALS / IGFBP-3 variants, and (4) separating the complex and detecting the amount thereof. It provides a method for screening amino acid residues involved in binding to ALS in IGFBP-3.

In another aspect, the present invention provides an IGFBP-3 variant comprising the amino acid sequence of any one of SEQ ID NO: 2 to SEQ ID NO: 15.

In another aspect, the present invention provides a nucleic acid sequence encoding the IGFBP-3 variant.

Figure 1 shows the results of the pulse and chase (pulse & chase) experiment injecting the natural type IGFBP-3 mRNA, ALS mRNA or natural type IGFBP-3 mRNA and ALS mRNA to the frog eggs used in the present invention (○ : When only IGFBP-3 mRNA was injected, Δ: When only ALS mRNA was injected, when IGFBP-3 mRNA (●) and ALS mRNA (▲) were injected simultaneously).

Figure 2 shows the results of measuring the expression of the protein produced by injecting and incubating ALS mRNA, IGFBP-3 variant mRNA, or ALS mRNA and native IGFBP-3 mRNA in frog eggs (lane NI: Frog eggs not injected with any mRNA externally, Lane 1: Frog eggs injected with all N-terminal deleted IGFBP-3 variant mRNAs (Δ11-40, Δ11-60, Δ11-88, respectively, from above), Lane 2: Frog eggs injected with both ALS mRNA and native IGFBP-3 mRNA, lane 3: frog eggs injected with ALS mRNA alone).

3 (A) shows the results of measuring the amount of ALS / IGFBP-3 complexes formed in the medium when the N-terminal-deleted IGFBP-3 variant mRNA and ALS mRNA were injected and cultured at the same time in frog eggs (FIG. Lane W: native type IGFBP-3, lane C: C-terminal substituted IGFBP-3 variant, lane 1: Δ11-40, lane 2: Δ11-60, lane 3: Δ11-88), (B) Injecting and incubating the N-terminal substituted IGFBP-3 variant mRNA and ALS mRNA at the same time, the result of measuring the amount of ALS / IGFBP-3 complex formed in the medium (lane Ova: Ovalbumin, Lane 1: I56S + Y57A, lane 2: L80S + L81S, lane 3: D72N + E73G, lane 4: C87W + V88A).

4 is a result of measuring the amount of ALS / IGFBP-3 complex formed in the medium immediately after incubation (0 hours), 3 hours, and 6 hours after injecting IGFBP-3 variant mRNA and ALS mRNA into frog eggs simultaneously. (Lane W: native IGFBP-3, lane 1: Δ11-40, lane 2: Δ11-60, lane 3: Δ11-88, lane 3 ': free Δ11-88, lane 4: I56S + Y57A) Lane 5: L80S + L81S, lane 6: D72N + E73G, lane 7: C87W + V88A, lane 7 ′: free C87W + V88A).

As used herein, the amino acid single letter means the following amino acids in accordance with standard abbreviation provisions in the field of biochemistry:

A: alanine; B: asparagine or aspartic acid; C: cysteine;

D: aspartic acid; E: glutamic acid; F: phenylalanine;

G: glycine; H: histidine; I: isoleucine; K: lysine; L: leucine;

M: methionine; N: asparagine; P: proline; Q: glutamine;

R: arginine; S: serine; T: threonine; V: valine;

W: tryptophan; Y: tyrosine; Z: glutamine or glutamic acid.

As used herein, the term “IGFBP-3 variant” refers to the substitution, deletion or insertion of one or more codons in a gene encoding IGFBP-3, resulting in unmodified or native form ( native) refers to a protein with a change in amino acid sequence compared to IGFBP-3.

As used herein, "Δamino acid position-amino acid position" refers to an IGFBP-3 variant deleted from the amino acid of the preceding amino acid position to the amino acid of the following amino acid position in the amino acid sequence of native type IGFBP-3. For example, Δ11-40 refers to the IGFBP-3 variant from which the 11th to 40th amino acids are deleted. Also, "(amino acid one character) (amino acid position) (amino acid one character)" means an IGFBP-3 variant subsubstituted with an amino acid previously indicated at the amino acid position previously indicated at the corresponding amino acid position in the natural type IGFBP-3 amino acid sequence. . For example, I56S refers to a variant in which the 56th amino acid residue isoleucine is substituted with serine. In addition, "(amino acid one character) (amino acid position) (amino acid one character) + (amino acid one character) (amino acid position) (amino acid one character)" means that the above-mentioned amino acid substitution occurred simultaneously at each corresponding amino acid position. Means 3 variants. For example, 56S + Y57A refers to the IGFBP-3 variant in which the 56th amino acid isoleucine (I) is substituted with serine (S) and the 57th amino acid tyrosine (Y) is substituted with alanine (A). Further, as used herein, "[228-232] ^S" is KGRKR which is the amino acid corresponding to the 228th to 232th amino acids known in the art to be involved in binding to ALS in the amino acid sequence of IGFBP-3. It refers to IGFBP-3 variants substituted with MDGEA. For convenience the present invention encompasses all of these variants into a single IGFBP-3 variant.

Binding of ALS at the same time deleted from the amino acid at the preceding amino acid position to the amino acid at the following amino acid position in the amino acid sequence of "Δamino acid position-amino acid position / [228-232] ^S native type IGFBP-3 as used herein; Refers to an IGFBP-3 variant in which the amino acid KGRKR, which is known to be involved in, is substituted with MDGEA, (amino acid letter) (amino acid position) (amino acid letter) + (amino acid letter) (amino acid position) (amino acid letter) ) Refers to an IGFBP-3 variant in which the amino acid KGRKR, which is known to be involved in binding to ALS, is substituted from the previously indicated amino acid at the corresponding amino acid position in the corresponding amino acid position in the native IGFBP-3 amino acid sequence and substituted with MDGEA. For convenience the present invention encompasses all of these variants as dual IGFBP-3 variants.

"IGFBP-3" is largely divided into (1) N-terminal domain: amino acids 1 to 89 of the amino acid sequence of native IGFBP-3, and (2) central domain: amino acid 90 of the amino acid sequence of native IGFBP-3. To 186th amino acid and (3) C-terminal domain: 187 amino acids to 264 amino acids in the amino acid sequence of the native type IGFBP-3, thus consisting of three domains. More specifically, when analyzing the amino acid sequence of IGFBP-3, lysine-glycine-arginine-glycine-arginine (KGRKR) forms the site (ALS-binding site) that binds to ALS and is 56th iso Leucine (I) and 57th tyrosine (Y) are amino acid residues that bind IGF, and 72th aspartic acid (D) and 73rd glutamic acid (E) are variable loop regions in the binding patch with IGF. amino acid residues forming a loop region), 80th leucine (L) and 81st leucine (L) is another amino acid residue that binds IGF, 87th cysteine (C) and 88th valine (V) is 12th It contains conserved cysteine residues.

Based on this sequence analysis, the C-terminus including KGRKR corresponding to amino acids 228 to 232 of the amino acid sequence of IGFBP-3, known in the art as a binding site for ALS, remains intact. In one case, mutation (substitution or deletion) of amino acids in the N-terminal domain was induced. The present invention includes such IGFBP-3 variants.

In one embodiment, the present invention provides (1) Δ11-40: IGFBP-3 variant deleted from the 11th amino acid to the 40th amino acid, and (2) Δ11-60: the IGFBP-3 variant deleted from the 11th amino acid to 60th amino acid And (3) Δ11-88: an N-terminal deletion from which the specific amino acid at the N-terminus of the native IGFBP-3 amino acid sequence, including the IGFBP-3 variant deleted from the 11th to 88th amino acids, is removed. ) IGFBP-3 variants.

In another embodiment, (1) I56S + Y57A: IGFBP-3 variant in which isoleucine (I), the 56th amino acid, is substituted with serine (S), and tyrosine (Y), the 57th amino acid, with alanine (A). , (2) D72N + E73G: IGFBP-3 variant in which the 72th amino acid aspartic acid (D) is substituted with asparagine (N), and the 73rd amino acid glutamic acid (E) is substituted with glycine (G), (3) L80S + L81S: IGFBP-3 variant in which leucine (L), the 80th amino acid, was substituted with serine (S), and (4) C87W + V88A: the cysteine, the 87th amino acid, substituted with leucine (L), the 81st amino acid, with serine (S). A specific amino acid at the N-terminus of the native IGFBP-3 amino acid sequence including the IGFBP-3 variant in which (C) is substituted with tryptophan (W) and 88-amino acid valine (V) with alanine (A) Provided are N-substituted IGFBP-3 variants substituted with other amino acids.

The present invention induces a variation of a specific amino acid at the N-terminus noted above, and further substituted KGEKR with MDGEA corresponding to amino acids 228 to 232 known in the art as binding sites for ALS. The present invention also includes such IGFBP-3 mutants.

In one aspect, the present invention provides an IGFBP wherein KGRKR corresponding to amino acids 228 to 232 known to be related to binding between ALS and IGFBP-3 is substituted with MDGEA, and at the same time the specific amino acid at the N-terminus previously known is deleted. -3 variants are provided. Specifically, this embodiment includes (1) Δ11-40 / [228-232] ^S : an IGFBP-3 variant in which KGRKR corresponding to amino acids 228 to 232 is deleted from 11th to 40th amino acids and substituted with MDGEA, (2) Δ11-60 / [228-232] ^S : IGFBP-3 variant wherein KGRKR corresponding to 228-232 amino acids deleted from the 11th to 60th amino acids is substituted with MDGEA, and (3) Δ11-88 / [228-232] ^S : IGFBP-3 variant having KGRKR corresponding to 228-232 amino acids deleted from 11th to 88th amino acid and substituted with MDGEA.

In another aspect, the present invention provides an IGFBP in which KGRKR corresponding to amino acids 228 to 232 known to be related to binding between ALS and IGFBP-3 is substituted with MDGEA, and at the same time a specific amino acid at the N-terminus previously mentioned is substituted. -3 variants are provided. Specifically, in this embodiment, (1) I56S + Y57A / [228-232] ^S : 56th amino acid I is replaced with S, 57th amino acid Y is substituted with A, and KGRKR corresponding to amino acids 228-232 is IGFBP-3 variant substituted with MDGEA, (2) D72N + E73G / [228-232] ^S : 72th amino acid D replaces N, 73th amino acid E replaces G and corresponds to amino acids 228-232 IGFBP-3 variant wherein KGRKR is substituted with MDGEA. (3) L80S + L81S / [228-232] ^S : IGFBP- wherein L, the 80th amino acid, is substituted by S, 81th amino acid, L is replaced by S, and KGRKR corresponding to the 228th to 232nd amino acids is substituted by MDGEA. Variant 3, (4) C87W + V88A / [228-232] ^S : The 87th amino acid C is replaced by W, the 88th amino acid V is replaced by A and the KGRKR corresponding to amino acids 228-232 is substituted by MDGEA. IGFBP-3 variants and the like.

IGFBP-3 variants prepared in the present invention are summarized in Table 1 below.

Nucleic acid sequences encoding IGFBP-3 variants according to the invention also form an aspect of the invention. In the present invention, since the DNA encoding the IGFBP-3 variant is prepared and then its transcription product mRNA is used in the screening method according to the present invention, both RNA and DNA are included in the scope of the nucleic acid sequence according to the present invention. DNA encoding the variants of the invention can be prepared by a variety of methods known in the art. Such methods include, but are not limited to, oligonucleotide-mediated mutations (oligonucleotide-mediated mutagenesis) and cassette mutations (casette mutagenesis).

In particular, mutation methods mediated by oligonucleotides are preferred in preparing DNA sequences encoding IGFBP-3 deletions and / or substitutional variants according to the invention. Such techniques are well known in the art and are disclosed by Zoler et al. Supra. In brief, the IGFBP-3 gene can be mutated by hybridizing oligonucleotides encoding the desired variant in the DNA template. Preferably, the DNA template is a plasmid comprising DNA encoding IGFBP-3 which is unmuted or native. After hybridization, the DNA polymerase is used to synthesize a complete second strand complementary to the template, which will encode the mutation selected from the IGFBP-3 gene.

Typically, oligonucleotides consisting of about 25 nucleotides in length are used. Shorter oligonucleotides may be introduced but the optimal oligonucleotide is one having 12 to 15 nucleotides complementary to the template on both sides of the nucleotide encoding the mutation. This is because such oligonucleotides can sufficiently hybridize to template DNA. Oligonucleotides can be synthesized by techniques known in the art (Crea et al. Proc. Natl. Acad. Sci. USA, 1978, vol. 75, p.5765).

In one embodiment, the present invention provides a deleted IGFBP-3 variant from which a specific amino acid at the N-terminus of the native IGFBP-3 amino acid sequence is removed, more specifically Δ11-40, Δ11-60, Δ11-88. It provides a nucleic acid sequence encoding the. Δ11-40 can be prepared by PCR using oligonucleotides encoding the 41st to about 45th amino acids as primers as a template using DNA encoding the native type IGFBP-3. DNA encoding IGFBP-3 can be used as a template, and oligonucleotides encoding the 61st to about 65th amino acids can be prepared by PCR using a primer. Finally, Δ11-88 can also be prepared by PCR using oligonucleotides encoding the 89th to about 94th amino acids as primers, using DNA encoding native type IGFBP-3 as a template. The oligonucleotides consisting of nucleotide sequences corresponding to 1 to 10 of the natural type IGFBP-3 amino acid sequence are synthesized and linked to the deletion variants according to the present invention using DNA synthesizers widely used in the art. It can manufacture.

In another aspect, the invention provides a substituted IGFBP-3 variant in which a specific amino acid at the N-terminus of a native IGFBP-3 amino acid sequence is substituted with another amino acid, specifically I56S + Y57A, D72N + E73G, L80S + Nucleic acid sequences encoding L81S or C87W + V88A are provided. Thus, if two or more amino acids to be mutated are located close on the polypeptide, they can be mutated simultaneously using one oligonucleotide encoding all of the desired amino acid variants (substitutions). This method of preparation is identical to the method of gene preparation in which one nucleotide is well known in the art, except that oligonucleotides comprising two or more amino acid variations are used as primers.

In another aspect, the present invention provides an IGFBP-3 variant wherein KGRKR corresponding to amino acids 228 to 232 known to be related to binding between ALS and IGFBP-3 is substituted with MDGEA, and at the same time the preceding amino acid is removed. To Δ11-40 / [228-232] ^S , Δ11-60 / [228-232] ^S or Δ11-88 / [228-232] ^S.

In still another aspect, the present invention provides an IGFBP-3 variant wherein KGRKR corresponding to amino acids 228 to 232 known to be related to binding between ALS and IGFBP-3 is substituted with MDGEA, and at the same time, the amino acid specified above is substituted. Nucleic acid sequences encoding I56S + Y57A / [228-232] ^S , S72 / 72 / [228-232] ^S , L80S + L81S / [228-232] ^S or C87W + V88A / [228-232] ^S to provide.

As described above, if two or more amino acids to be mutated are located far away from the polypeptide (if separated by more than 10 amino acids), it is impossible to prepare one oligonucleotide encoding all of the desired mutations. Instead, other methods should be introduced. The first method is to construct individual oligonucleotides in which each amino acid comprises a variation. If the oligonucleotides are annealed to single-strand template DNA simultaneously, the second strand DNA synthesized from the template will encode all of the desired amino acid variants. Another method involves more than one mutagenesis to produce such a variant. In primary mutagenesis, a native DNA is used as a template, and a heteroduplex DNA is produced when an oligonucleotide containing the first desired amino acid variation is annealed to this template. In secondary mutagenesis, the mutated DNA generated from primary (above) mutagenesis is used as a template. Therefore, this template already contains at least one variant. When oligonucleotides containing additional amino acid mutations are annealed to this template, the resulting DNA encodes both primary and secondary mutagenesis mutations. In summary, the method of mutating two or more nucleotides described above is equivalent to repeating the method of mutating one nucleotide many times.

Cassette mutant methods are also the preferred method for preparing sequences of nucleic acids encoding IGFBP-3 mutants according to the present invention. This method is based on the technique disclosed by well et al. Supra. The starting material is a plasmid (or other vector) containing the IGFBP-3 gene that is intended to cause the mutation. In the cassette mutation method, it is preferable to use when there is a restriction enzyme specifically present only at the position to be mutated. However, this is not required. If such restriction enzyme sites do not exist, it is possible to introduce them into appropriate positions of the IGFBP-3 gene using mutation methods mediated by the oligonucleotides described above. After restriction enzyme sites have been introduced into the plasmid, the enzyme is linearized by treating the plasmid. Double-stranded oligonucleotides having DNA sequences that contain mutations of interest but are located between restriction enzyme sites can be synthesized using conventional methods. These two strands are synthesized separately and then hybridized together using conventional techniques. Such double stranded oligonucleotides are named as cassettes. Such cassettes must be prepared to have 3- and 5-terminals compatible with both ends of the linearized plasmid, and they can be directly linked to the plasmid. This plasmid will contain DNA encoding the desired IGFBP-3 mutant through the above procedure.

Another method for preparing all of the DNA encoding the IGFBP-3 variant according to the present invention will be chemical synthesis. For example, nucleic acid sequences encoding IGFBP-3 variants, in particular DNA, can be synthesized by chemical methods using oligonucleotide synthesizers. Such oligonucleotides are made based on the amino acid sequence of the desired IGFBP-3 variant, and preferably by selecting the desired codons in the host cell (Frog eggs in the present invention) in which the IGFBP-3 variant is expressed.

With regard to the nucleic acid sequences according to the invention, it is well known that degenerate of the genetic code, ie the amino acids can be encoded by one or more codons. Accordingly, there will be a number of degenerate nucleic acid sequences encoding variants according to the invention, all of which are considered to be within the scope of the invention.

As noted above, the IGFBP-3 variant according to the present invention can be usefully used to identify amino acid residues or domains involved in binding to ALS in IGFBP-3. If any of the IGFBP-3 variants according to the present invention are complexed with ALS to a degree similar to that of native type IGFBP-3, the amino acids mutated in the IGFBP-3 variants are amino acids that do not participate in binding to ALS. If another IGFBP-3 variant does not complex with ALS when compared to native IGFBP-3, it will be appreciated that the amino acid mutated in the IGFBP-3 variant is an amino acid residue involved in binding to ALS.

Accordingly, the present invention comprises the steps of (1) preparing the mRNA encoding the IGFBP-3 variant and the mRNA encoding the native type ALS (Acid Labile Subunit), (2) the two mRNAs prepared above and the radiolabeled amino acid frog Injecting into an egg (Xenopus Oocyte), (3) culturing the frog egg to form a complex of ALS / IGFBP-3 variants, and (4) separating the complex and detecting the amount thereof. Screening amino acid residues involved in binding to ALS at -3.

In the screening method, mRNA encoding the IGFBP-3 variant and mRNA encoding the native type ALS (Acid Labile Subunit) can be prepared by in vivo or in vitro transcription. However, since several genes are simultaneously transcribed in vivo, it may be difficult to obtain only the desired mRNA. Therefore, the mRNA according to the present invention will be preferably prepared by an in vitro transcription method.

In vitro transcription methods (1) preparing a DNA sequence encoding an IGFBP-3 variant, operably linking the DNA sequence to a promoter to produce a recombinant vector, and transforming a host cell with the recombinant vector, Selecting the transformed host cell to separate the recombinant vector therefrom; (2) synthesizing RNA from the isolated recombinant vector as a template; (3) digesting the recombinant vector, which acted as a template, using DNase, and (4) separating the synthesized RNA transcript.

The vector used to prepare the mRNA according to the present invention by an in vitro transcription method should include a promoter which is a regulatory sequence essential for transcription to occur. In the present invention, it would be desirable to use a promoter capable of strongly promoting transcription of the gene. Such promoters include, but are not limited to, T3, T7, SP6 promoters, and the like. Since such a promoter is included on a vector, vectors which can be usefully used in the present invention include, for example, pSP64 poly (A), pSP72-pSP73, pGEM, pGEMEX, pALTER, pSl, pCl, pTARGET and the like. Of course, promoters such as the T3, T7, and SP6 promoters may be inserted and used in other suitable vectors.

On the other hand, the term "promoter" as used in the present specification may be defined as an array of nucleic acid regulatory sequences that direct the transcription of a nucleic acid. The promoter is located near the initiation site of transcription and includes all necessary sequences, including polymerase binding sites. In the present invention, the promoter may optionally include an enhancer that is thousands or more bases away from the start site of transcription. Promoters can be constitutive (ie, active in most environmental and developmental conditions) or inducible (ie, active only in certain environmental and developmental conditions).

As used herein, another term “operably linked” refers to a state in which a nucleic acid is placed in a functional relationship with another nucleic acid sequence. This may be genes and regulatory sequence (s) linked in such a way as to enable gene expression when appropriate molecules (eg, transcriptional activating proteins) bind to regulatory sequence (s). For example, if a promoter regulates the transcription of a coding sequence, it is operably linked to that sequence. In general, “operably linked” means that the linked DNA sequences are in contact, and in the case of a secretory leader, are in contact and present within the reading frame. Linking of these sequences is performed by ligation (linking) at suitable restriction enzyme sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers according to conventional methods are used.

In vitro, mRNA synthesis is performed by incorporating the recombinant vector containing DNA isolated in step (1), which serves as a template, by adding and mixing various components (RNA polymerase, NTP, etc.) necessary for RNA synthesis. According to the mechanism, RNA is synthesized. That is, the RNA polymerase is coupled to the promoter of the recombinant vector serving as a template, and then moved along the template DNA to select and polymerize NTP complementary to its sequence.

In the ex vivo transcriptional method of the present invention, a preferred embodiment is to use a recombinant vector containing a DNA that serves as a template to be linearized by cleavage with an appropriate restriction enzyme. This is because only the desired portion (ie, DNA encoding ALS or DNA encoding IGFBP-3 variant) can be transcribed. Linearized vectors can be obtained by digesting the cyclic recombinant vector with appropriate restriction enzymes, followed by extraction using phenol, chloroform, isoamyl in order and precipitation with ethanol.

Another preferred embodiment is to perform capping so that mRNA is effectively translated in frog eggs and produces a biologically active protein. Cap formation for mRNA produced by ex vivo transcription can be achieved in two ways, one using guanylyltransferase to deliver the 5-terminal cap structure to the transcript in vitro. The other is to add the 7-methyl guanosine cap structure (m7GpppG) to the in vitro transcriptional reactant and insert it directly into the RNA during the transcription reaction. At this time, the addition of excess GTP at the beginning of the transfer can be more effectively formed cap.

Finally, the mRNA of interest produced in in vitro transcription can be isolated according to methods known in the art. IGFBP-3 of the present invention is a gene derived from a eukaryotes, the polyadenine (A) tail is linked to the mRNA 3 '-terminus of its transcription product. This molecule allows for easy separation of mRNA. The in vitro transcription product is applied (or passed through) to a solid matrix carrier having a polyuredine (U) or polythymidine (T) tail, causing the poly A tail of the mRNA to adhere to the solid matrix carrier. Subsequently, washing with a solution containing a high concentration of salt (typically 1.5 M) can elute the pure form of mRNA. In addition to these methods, the desired mRNA can also be isolated using phenol extraction and ethanol precipitation or appropriate mRNA separation kits.

On the other hand, frog eggs to be injected with the mRNA thus prepared can be obtained using a method known in the art. First, the frog is anesthetized using an appropriate anesthetic until about 30 minutes or no response to gentle prodding. Here, the anesthetics include, but are not limited to, amethocaine, benzocaine, benzocaine, butacaine, butoxycaine, butoxicaine, chloroprocaine, procaine, procaine, Tricaine may be used. Preferably, tricaine is selected and used by dissolving about 1.0 g in 500 ml distilled water.

The lower abdomen of the anesthetized frog is incised and the ovarian lobe is removed and placed on a large culture plate (60 mm or 125 mm) filled with modified OR-2 medium (see Table 2). The ovary lobe is cut into thin pieces and placed in OR-2 medium (see Table 3), and then gently shaken at room temperature (about 20 ° C.) or 18 ° C. for 1.5 to 2 hours to form a red-veinfollicular coat. Make sure is removed. When about 50% of the shell is removed, the OR-2 medium is discarded, washed several times with fresh OR-2 medium and transferred to culture plates filled with modified OR-2 medium to incubate and select good eggs.

Good eggs are large eggs in the 5-6 development stage, which are symmetrical and should have a uniform distribution of pigmentation. Good eggs also consist of two hemispheres that occupy nearly the same area. In the case of a good egg, the animal hemisphere should be dark chocolate brown to black and free of any irregular white spots, swirls or stained patches. Vegitable hemispheres are creamy yellow to pale white, ideally having no dark spots.

Modified OR-2 (MOR-2) Medium ingredient Concentration (mM) NaCl 25 KCl 1.0 CaCl ₂ 1.0 NaOH 3.8 Na2HPO4 1.0 HEPES, 5.0 Remarks: pH 7.8

OR-2 badge ingredient Concentration (mM) NaCl 82.5 KCl 2.5 CaCl ₂ 1.0 MgCl ₂ 1.0 NaOH 3.8 Na ₂ HPO ₄ 1.0 HEPES 5.0 Remarks: pH 7.8

Prior to injecting mRNA into the selected frog eggs, if the composition containing the previously prepared mRNA was stored frozen, it is desirable to add the process of thawing and rotating salt crystals at about 10,000 rpm for about 1 hour to precipitate salt crystals. . The RNA composition is inhaled in an appropriate amount using a microinjector and then injected into the frog eggs. Total mRNA in one frog egg is preferably injected into the plant hemisphere in an amount of about 40-50 ng. Frog eggs injected with mRNA according to the present invention are cultured in humidified air at 15 to 20 ° C. Depending on the gene, it will be expressed after about 1/2 to 4 days.

SDS-PAGE is preferably used to separate ALS and IGFBP-3 variants that do not form complexes or complexes from the cultures (frog eggs and medium). This is because SDS-PAGE (polyacrylamide gel electrophoresis) is useful when the protein is to be separated only by size and not by other physical properties as in the present invention. Sodium dodecyl sulfate (SDS) is a type of surfactant that allows all proteins to have only a primary structure and contains a large amount of negative charge that can migrate to the anode when placed in an electric field. Thus, SDS prevents proteins from being separated by shape or charge. Proteins treated with SDS are now electrophoresed on polyacrylamide gels consisting of networks of pores of various sizes. As can be expected, the complex (ALS / IGFBP-3) has a higher molecular weight than ALS or IGFBP-3 and moves slowly so that the ALS or IGFBP-3 which does not form a complex with the complex can be separated, respectively.

Meanwhile, since the complex formed in the culture process may be separated during the SDD-PAGE process, it is preferable to first process the cross-linking agent in the culture before performing the SDS-PAGE. The cross-linking factor is a substance that makes the intermolecular covalent bond more robust, and the cross-linking factor available in the present invention is not limited thereto, but is not limited thereto, such as bis (sulphosuccinimidyl) sebacate, Bis (sulphosuccinimidyl) suberate, Disuccinimidyl sebacate, Disuccinimidyl suberate (DSS), Disuccinimidyl tartrate : DST)], disulphosuccinimidyl tartrate [sulpho DST], dithiobis (succinimidyl propionate): sulpho DSP], ethylene glycobis (succinimidyl succinate ) [Ethylene glycolbis (succinimidylsuccinate): EGS] and the like. On the other hand, this cross linking reaction can be terminated using Tris or glycine containing an amine group.

Proteins isolated according to size through SDS-PAGE can be visualized by radiograph. Stronger band intensities mean that the substituted or deleted amino acid residues of the IGFBP-3 variants constituting the complex have little effect on binding to ALS, whereas a weaker band strength of the complex constitutes the complex. Substituted or deleted amino acid residues of the IGFBP-3 variant have a significant effect on the binding to ALS.

Hereinafter, the examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples in accordance with the gist of the present invention, those skilled in the art. Will be self-evident.

<Example 1>

Sample Preparation

Phenylmethylsufonylfluoride (PMSF), EDTA, SDS, Triton X-100, deoxycholate and hydroxyethylpiperazine ethanesulfonate (HEPES) were purchased from Sigma Chemical Company (St. Louis, MO). . Recombinant human insulin-like growth factor-I (IGF-I) was obtained from Gropep (Adelaide, Australia) and rabbit antiserum for hIGFBP-3 and ovalbumin, respectively. Upstate Biotechnology, Inc. (Lake Placid, NY) and Sigma Chemical Company (St. Louis, Mo.). Deoxyribonucleoside triphosphate (dNTP), Taq polymerase and other polymerase chain reaction (PCR) reagents and Wizard DNA purification kits were purchased from Promega (Madison, WI). pCR2.1 plasmid vector, pSP6 in vitro transcription vector, mutant pAlter plasmid vector and pPS plasmid cloning vector were all purchased from Promega. TA cloning kit pCR2.1 was purchased from Invitrogen (San Diego, Calif.). Disuccinimidyl suberate (DSS) was purchased from Pierce Chemical Co. (Rockford, IL). N-glycae was purchased from Genzyne Corp., Boston, Mass.

<Example 2>

(1) Preparation of a recombinant vector comprising DNA encoding ALS

The pCR2.1 plasmid containing the 915-bp ALS exon 2 fragment and the pCR2.1 plasmid containing the pALS gene encoding the C-terminal half of the 1058-bp ALS protein have a 155-bp overlapping sequence, This sequence contains an Nsp I recognition site. The 915-bp pCR2.1 plasmid and 1058-bp pCR2.1 plasmid were digested with Bam HI / Nsp I and Nsp I / Not I, digested, gel separated and ligation then inserted into the pBS plasmid vector.

(2) Preparation of DNA Sequences Encoding IGFBP-3 Variants

IGFBP-3 variants prepared in the present invention are briefly summarized in Table 1 above. The DNA encoding the IGFBP-3 variant is a position-directed mutation using an oligonucleotide containing the desired mutation as a primer as a template a cDNA encoding human native IGFBP-3 inserted at the Eco RI site of the pAlter plasmid vector. Prepared using the method (site-directed mutagenesis).

DNA sequences encoding single IGFBP-3 variants were prepared as follows. DNA sequences encoding the N-terminal deletion variants Δ11-40, Δ11-60, Δ11-88 of IGFBP-3 were prepared using primers ACCCAGAACTTCTCC, AAGAAGGGATTTTAT, GACGTGCACTGCTA, respectively. [228-232] DNA sequences encoding ^S variants were prepared using ATGGACGGGGAGGCG oligonucleotides. DNA sequences encoding the N-terminal substitution variants of IGFBP-3, I56S + Y57A, D72N + E73G, L80S + L81S, C87W + V88A, were prepared using primers GACAGGCAGGCATAC, GGCACAATTATATCA, ATAGGCCTAGCATATC, CATGATCCGATACGAC, respectively.

DNA sequences encoding double IGFBP-3 variants were prepared as follows. The DNA sequence encoding Δ11-40 / [228-232] ^S is prepared as described above, and the primer used in preparing [228-232] ^S using the mutated DNA sequence as a template. It was prepared by performing a site-directed mutagenesis using. The DNA sequence encoding Δ11-60 / [228-232] ^S is prepared as described above, and the primer used in preparing [228-232] ^S using the mutated DNA sequence as a template. It was prepared by performing a site-directed mutagenesis using. The DNA sequence encoding Δ11-88 / [228-232] ^S is prepared as described above, and the primer used in preparing [228-232] ^S using the mutated DNA sequence as a template. It was prepared by performing a site-directed mutagenesis using.

Subsequently, I56S + Y57A / [228-232] DNA sequence encoding the ^S is manufactured a DNA sequence encoding a I56S + Y57A as described above, and then this mutated DNA sequence as a template [228-232] produced when ^S Prepared by site-directed mutagenesis using the primers used in D72N + E73G / [228-232] DNA sequence encoding the ^S + D72N is to prepare a DNA sequence encoding the E73G and then this mutated DNA sequence as a template [228-232] using primers prepared as described above at the time of ^S It was prepared by performing a site-directed mutagenesis using. The DNA sequence encoding L80S + L81S / [228-232] ^S is prepared as described above, and the primer used in preparing the [228-232] ^S using the mutated DNA sequence as a template. It was prepared by performing a site-directed mutagenesis using. C87W + V88A / [228-232] DNA sequence encoding the ^S + C87W was prepared a DNA sequence encoding the following V88A so mutated DNA sequence as a template [228-232] using primers prepared as described above at the time of ^S It was prepared by performing a site-directed mutagenesis using.

The DNA sequences encoding the thus prepared IGFBP-3 variants were inserted into the pSP65 V vector (Taxonomy ID: 40277) after confirming that the desired mutations were achieved by dideoxy chain termination.

Primer used to prepare DNA encoding the IGFBP-3 variant prepared in the present invention Variant primer SEQ ID NO: [228-232] ^S 5’-ATGGACGGGGAGGCG-3 ’ 16 Δ11-40 5’-ACCCAGAACTTCTCC-3 ’ 17 Δ11-60 5’-AAGAAGGGATTTTAT-3 ’ 18 Δ11-88 5’-GACGTGCACTGCTA-3 ’ 19 I56S + Y57A 5’-GACAGGCAGGCATAC-3 ’ 20 D72N + E73G 5’-GGCACAATTATATCA-3 ’ 21 L80S + L81S 5’-ATAGGCCTAGCATATC-3 ’ 22 C87W + V88A 5’-CATGATCCGATACGAC-3 ’ 23

<Example 3>

MRNA synthesis encoding ALS or IGFBP-3 variants in vitro

MRNAs encoding ALS or IGFBP-3 variants were obtained by following the manufacturer's instructions using a kit for in vitro transcription (Riboprobe in vitro Transcription system, Promega). In in vitro transcription, 7-methyl guanosine cap structure (m7GpppG) was added in 10-fold excess of GTP to allow effective capping of RNA transcripts during the initial 45 min of culture, and as the reaction proceeded, GTP Was added at the same concentration as CAP. After transcription was completed, the DNA template was removed by incubation for 15 minutes at 37 ℃ with DNaseI excluding RNase function, the desired RNA transcript was isolated by phenol extraction and ethanol precipitation.

<Example 4>

Cultivation and Screening of Frog Eggs

OR-2 medium (MOR2; 25 mM NaCl, 1.0 mM KCl, 1.0 mM CaCl ₂₎ modified with ovary leaves from adult frogs, anesthetized for about 30 minutes using tricaine (dissolved about 1.0 g in 500 ml distilled water). , 2.0 mM) the large culture plates (60mm or 125mm), and then, the I cut the leaflets into thin slices oR-2 medium (82.5mM NaCl in _{place, 2.5mM KCl, 1.0mMCaCl 2, 1.0mM} MgCl 2, 3.8 filled with mM NaOH, 1.0 mM Na ₂ HPO ₄ , 5.0 mM HEPES, pH 7.8) and gently shake at room temperature for 2 hours to see if the red-veinfollicular coat was removed. When the 50% shell was removed, discard the OR-2 medium, wash several times with fresh OR-2 medium, transfer to a culture plate filled with MOR-2 medium, incubate at 14 ° C. for 2 hours and select good eggs. It was.

Example 5

Analysis of Suitability of the Expression System of Frog Eggs According to the Present Invention

(1) Pulse and Chase Experiment

Frog eggs selected previously were injected with a total volume of 50 nl of reaction containing ²⁵ ng IGFBP-3 variant mRNA and / or 25 ng ALS mRNA and 0.5 μCi [ ³⁵ S] methionine / cysteine using an Ependorf Micromanipulation System. The eggs so injected were further incubated at 14 ° C. with 100 μl MOR-2 medium. After 1 hour of pulse-labeling translation of ALS mRNA and / or IGFBP-3 mRNA according to this culture, unlabeled Met / Cys in MOR-2 medium was brought to 9 mM concentration ( ³⁵ S-labeled Met). 100 times greater than / Cys). Radioactivity of ³² S-labeled ALS or IGFBP-3 accumulated in MOR-2 medium was measured at 0, 1, 3, 6 and 12 hours from the start of wave-labeling and the results are shown in FIG. 1.

According to FIG. 1 It can be seen that IGFBP-3 variant or ALS accumulates in the medium. No expression products were identified in frog eggs that were not injected with external messages (mRNA) such as IGFBP-3 or ALS. These results indicate that endogenous mRNA detoxification does not occur in the developmental stage of the frog eggs used in the present invention. The frog egg culture system according to the present invention studies the expression and secretion of IGFBP-3 and ALS. It was found to be suitable.

In addition, IGFBP-3 and ALS were able to detect radiolabeled IGFBP-3 or ALS for up to 12 hours in co-injected eggs. Since secretion of protein was detected in the medium, IGFBP-3 or ALS accumulated at a constant rate in the medium. Frog eggs synthesized and secreted proteins in a straight line for at least 6 hours. On the other hand, the reaction by addition of unlabeled Met / Cys was confirmed by measuring the amount of radioactivity inserted into IGFBP-3 or ALS between the pulse and the chase period. However, the insertion of IGFBP-3 or ALS labeled Met / Cys in the presence of unlabeled Met / Cys rapidly decreased within 30 minutes of the chase and substantially disappeared within 1 hour. This result means that the frog eggs according to the present invention synthesize and secrete the injected message in a straight and highly sensitive manner.

(2) Comparison of expression amount by single injection and simultaneous injection

Cultures of eggs injected with mRNA encoding Δ11-40, Δ11-60, or Δ11-88 were immunoprecipitated using IGFBP-3 antiserum and visualized by SDS-PAGE and radiographs. 39-, 36- and 33-kDa bands respectively appeared (lane 1 in FIG. 2). In addition, the culture medium of the egg injected with the mRNA encoding ALS alone was immunoprecipitated using ALS antiserum, and visualized by SDS-PAGE and radiograph. As a result, it was confirmed that the expected 85kDa ALS protein was secreted ( Lane 3 in FIG. 2). A culture of frog eggs injected with both ALS-encoding mRNA and native type IGFBP-3 was simultaneously immunoprecipitated with IGFBP-3 antiserum, followed by immunoprecipitation with ALS antiserum, followed by SDS-PAGE and radioactivity. Visualization by automated photography revealed the expected 85 kDa ALS and 43 kDa IGFBP-3 bands (lane 2 in FIG. 2).

From these results, the intensity of co-injected (lane 2) ALS bands can be compared with single-injected ALS in neighboring lanes (lane 3), where the amount of ALS expressed when co-injected and when injected alone is similar. And it was found. In the same vein, co-expression of three IGFBP-3 deletion variants (lane 1) did not reduce the amount of expression of each of the variants (lane 2). Therefore, it can be seen that the frog egg expression system according to the present invention is suitable for the screening method including the step of injecting, incubating and expressing IGFBP-3 and ALS into the frog egg simultaneously.

<Example 6>

Detection of complexes formed in egg culture medium after co-expression of ALS and double IGFBP-3 variants

A total volume of 50nl reactants containing mRNA encoding 25 ng ALS and 25 ng double IGFBP-3 variant and 0.5 μCi [ ³⁵ S] methionine / cysteine were injected into frog eggs, and 200 μl of MOR2 medium (2 eggs Incubated with sugar). Negative control eggs were injected and cultured with mRNA encoding Ovalbumin and mRNA encoding ALS. After incubation, the culture medium was cross-linked with 0.25 mM DSS (dissucinimidyl suberate) at room temperature for 30 minutes and then the ALS / IGFBP complex was immunoprecipitated with IGFBP-3 antiserum (diluted at 1: 500). The immunoprecipitation was performed for 4 hours with BIPIP buffer (150 mM NaCl, 1 mM PMSF, 1 mM EDTA, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, 10 mM Tris-HCl, pH 7.4). It was performed at 15 ° C. Ovalbumin was immunoprecipitated using polyclonal antibodies against ovalbumin. The precipitate formed therefrom was separated under reducing conditions on 5% polyacrylamide-SDS-gel and visualized by radiograph, the results of which are shown in FIG. 3.

According to Figure 3 (A), in the case of Δ11-40, it was able to complex with ALS to a degree similar to the C-terminal substituted IGFBP-3 variant. However, in the case of Δ11-60 and Δ11-88, there was almost no complex with ALS. Through this, it was confirmed that the 60th to 88th amino acid residue in IGFBP-3 is a residue that plays an important role in binding to ALS. According to (B) of FIG. 3, I56S + Y57A and L80S + L81S did not form a complex with ALS, whereas D72N + E73G and C87W + V88A formed a complex with ALS. As a result, it was found that 56th and 57th amino acid residues and 80th and 81th amino acid residues of IGFBP-3 are amino acid residues that play an important role in binding to ALS.

<Example 7>

Detection of the Stability of Complexes Formed in Egg Culture Media After Simultaneous Expression of ALS and Single IGFBP-3 Variants

A total volume of 50nl reactants containing ²⁵ ng ALS and 25 ng single IGFBP-3 variant and 0.5 μCi [ ³⁵ S] methionine / cysteine were injected into the frog eggs and 200 μl of MOR2 medium (2 eggs) Incubated with sugar). After 3 hours of incubation, the culture medium was cross-linked with 0.25 mM DSS for 30 minutes at room temperature and then the ALS / IGFBP-3 complex was immunoprecipitated with IGFBP-3 antiserum (diluted at 1: 500). The immunoprecipitation was performed for 4 hours with BIPIP buffer (150 mM NaCl, 1 mM PMSF, 1 mM EDTA, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, 10 mM Tris-HCl, pH 7.4). It was performed at 15 ° C. The precipitate formed therefrom was separated on reducing 5% polyacrylamide-SDS-gel under reducing conditions and visualized by radiograph. After another 3 hours of further incubation, cross-linking, immunoprecipitation followed by SDS-PAGE followed by visualizing by radiograph, and the results are all shown in FIG.

After 3 hours of incubation of eggs containing ALS and a single IGFBP-3 variant mRNA injection, it was confirmed that all media contained the ALS / IGFBP-3 complex. The amount of complex that could be crosslinked in the medium did not visually decrease after 3 hours of incubation at room temperature. However, during subsequent 3 h incubation, little complex was detected for Δ11-60 and for Δ11-88, whereas for the native and Δ11-40 the complex remained in the medium. In addition, variants of 87/88 (ie, D72N + E73G and C87W + V88A) containing 72/73 and twelfth conserved Cys, the “variable loop” regions of IGF-binding patches, Except for the known IGF-binding residues 56/57 and 80/81, the variants (ie, I56S + Y57A and L80S + L80S) reduced the amount of complexes in the medium during the second 3 hours of incubation. As a result, it was confirmed that IGF-binding residues of IGFBP-3 as well as amino acid residues corresponding to 41-88th of IGFBP-3 play an important role in stabilizing the ALS / IGFBP-3 complex in the medium.

The screening method according to the invention can be used to easily screen amino acid residues involved in binding to ALS in IGFBP-3.

<110> Jinju National University <120> A method for screening amino acid residues of IGFBP-3 which are necessary for binding ALS <160> 23 <170> KopatentIn 1.71 <210> 1 <211> 291 <212> PRT <213> Artificial Sequence <220> <223> variant <400> 1 Met Gln Arg Ala Arg Pro Thr Leu Trp Ala Ala Ala Leu Thr Leu Leu 1 5 10 15 Val Leu Leu Arg Gly Pro Pro Val Ala Arg Ala Gly Ala Ser Ser Gly 20 25 30 Gly Leu Gly Pro Val Val Arg Cys Glu Pro Cys Asp Ala Arg Ala Leu 35 40 45 Ala Gln Cys Ala Pro Pro Ala Val Cys Ala Glu Leu Val Arg Glu 50 55 60 Pro Gly Cys Gly Cys Cys Leu Thr Cys Ala Leu Ser Glu Gly Gln Pro 65 70 75 80 Cys Gly Ile Tyr Thr Glu Arg Cys Gly Ser Gly Leu Arg Cys Gln Pro 85 90 95 Ser Pro Asp Glu Ala Arg Pro Leu Gln Ala Leu Leu Asp Gly Arg Gly 100 105 110 Leu Cys Val Asn Ala Ser Ala Val Ser Arg Leu Arg Ala Tyr Leu Leu 115 120 125 Pro Ala Pro Pro Ala P ro Gly Asn Ala Ser Glu Ser Glu Glu Asp Arg 130 135 140 Ser Ala Gly Ser Val Glu Ser Pro Ser Val Ser Ser Thr His Arg Val 145 150 155 160 Ser Asp Pro Lys Phe His Pro Leu His Ser Lys Ile Ile Ile Ile Lys 165 170 175 Lys Gly His Ala Lys Asp Ser Gln Arg Tyr Lys Val Asp Tyr Glu Ser 180 185 190 Gln Ser Thr Asp Thr Gln Asn Phe Ser Ser Glu Ser Lys Arg Glu Thr 195 200 205 Glu Tyr Gly Pro Cys Arg Arg Glu Met Glu Asp Thr Leu Asn His Leu 210 215 220 Lys Phe Leu Asn Val Leu Ser Pro Arg Gly Val His Ile Pro Asn Cys 225 230 235 240 Asp Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Met Asp 245 250 255 Glu Gly Ala Gly Phe Cys Trp Cys Val Asp Lys Tyr G ly Gln Pro Leu 260 265 270 Pro Gly Tyr Thr Thr Lys Gly Lys Glu Asp Val His Cys Tyr Ser Met 275 280 285 Gln Ser Lys 290 <210> 2 <211> 250 <212> PRT <213> Artificial Sequence <220> <223> variant <400> 2 Met Gln Arg Ala Arg Pro Thr Leu Trp Ala Ala Ala Leu Thr Leu Leu 1 5 10 15 Val Leu Leu Arg Gly Pro Pro Val Ala Arg Gly Cys Cys Leu Thr Cys 20 25 30 Ala Leu Ser Glu Gly Gln Pro Cys Gly Ile Tyr Thr Glu Arg Cys Gly 35 40 45 Ser Gly Leu Arg Cys Gln Pro Ser Pro Asp Glu Ala Arg Pro Leu Gln 50 55 60 Ala Leu Leu Asp Gly Arg Gly Leu Cys Val Asn Ala Ser Ala Val Ser 65 70 75 80 Arg Leu Arg Ala Tyr Leu Leu Pro Ala Pro Pro Ala Pro Gly Asn Ala 85 90 95 Ser Glu Ser Glu Glu Asp Arg Ser Ala Gly Ser Val Glu Ser Pro Ser 100 105 110 Val Ser Ser Thr His Arg Val Ser Asp Pro Lys Phe His Pro Leu His 115 120 125 Ser Lys Ile Ile Ile I le Lys Lys Gly His Ala Lys Asp Ser Gln Arg 130 135 140 Tyr Lys Val Asp Tyr Glu Ser Gln Ser Thr Asp Thr Gln Asn Phe Ser 145 150 155 160 Ser Glu Ser Lys Arg Glu Thr Glu Tyr Gly Pro Cys Arg Arg Glu Met 165 170 175 Glu Asp Thr Leu Asn His Leu Lys Phe Leu Asn Val Leu Ser Pro Arg 180 185 190 Gly Val His Ile Pro Asn Cys Asp Lys Lys Gly Phe Tyr Lys Lys Lys 195 200 205 Gln Cys Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys Trp Cys Val 210 215 220 Asp Lys Tyr Gly Gln Pro Leu Pro Gly Tyr Thr Thr Lys Gly Lys Glu 225 230 235 240 Asp Val His Cys Tyr Ser Met Gln Ser Lys 245 250 <210> 3 <211> 230 <212> PRT <213> Artificial Sequence <220> <223> variant <400> 3 Met Gln Arg Ala Arg Pro Thr Leu Trp Ala Ala Ala Leu Thr Leu Leu 1 5 10 15 Val Leu Leu Arg Gly Pro Pro Val Ala Arg Cys Gly Ser Gly Leu Arg 20 25 30 Cys Gln Pro Ser Pro Asp Glu Ala Arg Pro Leu Gln Ala Leu Leu Asp 35 40 45 Gly Arg Gly Leu Cys Val Asn Ala Ser Ala Val Ser Arg Leu Arg Ala 50 55 60 Tyr Leu Leu Pro Ala Pro Pro Ala Pro Gly Asn Ala Ser Glu Ser Glu 65 70 75 80 Glu Asp Arg Ser Ala Gly Ser Val Glu Ser Pro Ser Val Ser Ser Thr 85 90 95 His Arg Val Ser Asp Pro Lys Phe His Pro Leu His Ser Lys Ile Ile 100 105 110 Ile Ile Lys Lys Gly His Ala Lys Asp Ser Gln Arg Tyr Lys Val Asp 115 120 125 Tyr Glu Ser Gln Ser T hr Asp Thr Gln Asn Phe Ser Ser Glu Ser Lys 130 135 140 Arg Glu Thr Glu Tyr Gly Pro Cys Arg Arg Glu Met Glu Asp Thr Leu 145 150 155 160 Asn His Leu Lys Phe Leu Asn Val Leu Ser Pro Arg Gly Val His Ile 165 170 175 Pro Asn Cys Asp Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro 180 185 190 Ser Lys Gly Arg Lys Arg Gly Phe Cys Trp Cys Val Asp Lys Tyr Gly 195 200 205 Gln Pro Leu Pro Gly Tyr Thr Thr Lys Gly Lys Glu Asp Val His Cys 210 215 220 Tyr Ser Met Gln Ser Lys 225 230 <210> 4 <211> 201 <212> PRT <213> Artificial Sequence <220> <223> variant <400> 4 Met Gln Arg Ala Arg Pro Thr Leu Trp Ala Ala Ala Leu Thr Leu Leu 1 5 10 15 Val Leu Leu Arg Gly Pro Pro Val Ala Asn Ala Ser Ala Val Ser Arg 20 25 30 Leu Arg Ala Tyr Leu Leu Pro Ala Pro Pro Ala Pro Gly Asn Ala Ser 35 40 45 Glu Ser Glu Glu Asp Arg Ser Ala Gly Ser Val Glu Ser Pro Ser Val 50 55 60 Ser Ser Thr His Arg Val Ser Asp Pro Lys Phe His Pro Leu His Ser 65 70 75 80 Lys Ile Ile Ile Ile Lys Lys Gly His Ala Lys Asp Ser Gln Arg Tyr 85 90 95 Lys Val Asp Tyr Glu Ser Gln Ser Thr Asp Thr Gln Asn Phe Ser Ser 100 105 110 Glu Ser Lys Arg Glu Thr Glu Tyr Gly Pro Cys Arg Arg Glu Met Glu 115 120 125 Asp Thr Leu Asn His L eu Lys Phe Leu Asn Val Leu Ser Pro Arg Gly 130 135 140 Val His Ile Pro Asn Cys Asp Lys Lys Gly Phe Tyr Lys Lys Lys Gln 145 150 155 160 Cys Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys Trp Cys Val Asp 165 170 175 Lys Tyr Gly Gln Pro Leu Pro Gly Tyr Thr Thr Lys Gly Lys Glu Asp 180 185 190 Val His Cys Tyr Ser Met Gln Ser Lys 195 200 <210> 5 <211> 291 <212> PRT <213> Artificial Sequence <220> <223> variant <400> 5 Met Gln Arg Ala Arg Pro Thr Leu Trp Ala Ala Ala Leu Thr Leu Leu 1 5 10 15 Val Leu Leu Arg Gly Pro Pro Val Ala Arg Ala Gly Ala Ser Ser Gly 20 25 30 Gly Leu Gly Pro Val Val Arg Cys Glu Pro Cys Asp Ala Arg Ala Leu 35 40 45 Ala Gln Cys Ala Pro Pro Ala Val Cys Ala Glu Leu Val Arg Glu 50 55 60 Pro Gly Cys Gly Cys Cys Leu Thr Cys Ala Leu Ser Glu Gly Gln Pro 65 70 75 80 Cys Gly Ser Ala Thr Glu Arg Cys Gly Ser Gly Leu Arg Cys Gln Pro 85 90 95 Ser Pro Asp Glu Ala Arg Pro Leu Gln Ala Leu Leu Asp Gly Arg Gly 100 105 110 Leu Cys Val Asn Ala Ser Ala Val Ser Arg Leu Arg Ala Tyr Leu Leu 115 120 125 Pro Ala Pro Pro Ala P ro Gly Asn Ala Ser Glu Ser Glu Glu Asp Arg 130 135 140 Ser Ala Gly Ser Val Glu Ser Pro Ser Val Ser Ser Thr His Arg Val 145 150 155 160 Ser Asp Pro Lys Phe His Pro Leu His Ser Lys Ile Ile Ile Ile Lys 165 170 175 Lys Gly His Ala Lys Asp Ser Gln Arg Tyr Lys Val Asp Tyr Glu Ser 180 185 190 Gln Ser Thr Asp Thr Gln Asn Phe Ser Ser Glu Ser Lys Arg Glu Thr 195 200 205 Glu Tyr Gly Pro Cys Arg Arg Glu Met Glu Asp Thr Leu Asn His Leu 210 215 220 Lys Phe Leu Asn Val Leu Ser Pro Arg Gly Val His Ile Pro Asn Cys 225 230 235 240 Asp Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly 245 250 255 Arg Lys Arg Gly Phe Cys Trp Cys Val Asp Lys Tyr G ly Gln Pro Leu 260 265 270 Pro Gly Tyr Thr Thr Lys Gly Lys Glu Asp Val His Cys Tyr Ser Met 275 280 285 Gln Ser Lys 290 <210> 6 <211> 291 <212> PRT <213> Artificial Sequence <220> <223> variant <400> 6 Met Gln Arg Ala Arg Pro Thr Leu Trp Ala Ala Ala Leu Thr Leu Leu 1 5 10 15 Val Leu Leu Arg Gly Pro Pro Val Ala Arg Ala Gly Ala Ser Ser Gly 20 25 30 Gly Leu Gly Pro Val Val Arg Cys Glu Pro Cys Asp Ala Arg Ala Leu 35 40 45 Ala Gln Cys Ala Pro Pro Ala Val Cys Ala Glu Leu Val Arg Glu 50 55 60 Pro Gly Cys Gly Cys Cys Leu Thr Cys Ala Leu Ser Glu Gly Gln Pro 65 70 75 80 Cys Gly Ile Tyr Thr Glu Arg Cys Gly Ser Gly Leu Arg Cys Gln Pro 85 90 95 Ser Pro Asn Gly Ala Arg Pro Leu Gln Ala Leu Leu Asp Gly Arg Gly 100 105 110 Leu Cys Val Asn Ala Ser Ala Val Ser Arg Leu Arg Ala Tyr Leu Leu 115 120 125 Pro Ala Pro Pro Ala P ro Gly Asn Ala Ser Glu Ser Glu Glu Asp Arg 130 135 140 Ser Ala Gly Ser Val Glu Ser Pro Ser Val Ser Ser Thr His Arg Val 145 150 155 160 Ser Asp Pro Lys Phe His Pro Leu His Ser Lys Ile Ile Ile Ile Lys 165 170 175 Lys Gly His Ala Lys Asp Ser Gln Arg Tyr Lys Val Asp Tyr Glu Ser 180 185 190 Gln Ser Thr Asp Thr Gln Asn Phe Ser Ser Glu Ser Lys Arg Glu Thr 195 200 205 Glu Tyr Gly Pro Cys Arg Arg Glu Met Glu Asp Thr Leu Asn His Leu 210 215 220 Lys Phe Leu Asn Val Leu Ser Pro Arg Gly Val His Ile Pro Asn Cys 225 230 235 240 Asp Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly 245 250 255 Arg Lys Arg Gly Phe Cys Trp Cys Val Asp Lys Tyr G ly Gln Pro Leu 260 265 270 Pro Gly Tyr Thr Thr Lys Gly Lys Glu Asp Val His Cys Tyr Ser Met 275 280 285 Gln Ser Lys 290 <210> 7 <211> 291 <212> PRT <213> Artificial Sequence <220> <223> variant <400> 7 Met Gln Arg Ala Arg Pro Thr Leu Trp Ala Ala Ala Leu Thr Leu Leu 1 5 10 15 Val Leu Leu Arg Gly Pro Pro Val Ala Arg Ala Gly Ala Ser Ser Gly 20 25 30 Gly Leu Gly Pro Val Val Arg Cys Glu Pro Cys Asp Ala Arg Ala Leu 35 40 45 Ala Gln Cys Ala Pro Pro Ala Val Cys Ala Glu Leu Val Arg Glu 50 55 60 Pro Gly Cys Gly Cys Cys Leu Thr Cys Ala Leu Ser Glu Gly Gln Pro 65 70 75 80 Cys Gly Ile Tyr Thr Glu Arg Cys Gly Ser Gly Leu Arg Cys Gln Pro 85 90 95 Ser Pro Asp Glu Ala Arg Pro Leu Gln Ala Ser Ser Asp Gly Arg Gly 100 105 110 Leu Cys Val Asn Ala Ser Ala Val Ser Arg Leu Arg Ala Tyr Leu Leu 115 120 125 Pro Ala Pro Pro Ala P ro Gly Asn Ala Ser Glu Ser Glu Glu Asp Arg 130 135 140 Ser Ala Gly Ser Val Glu Ser Pro Ser Val Ser Ser Thr His Arg Val 145 150 155 160 Ser Asp Pro Lys Phe His Pro Leu His Ser Lys Ile Ile Ile Ile Lys 165 170 175 Lys Gly His Ala Lys Asp Ser Gln Arg Tyr Lys Val Asp Tyr Glu Ser 180 185 190 Gln Ser Thr Asp Thr Gln Asn Phe Ser Ser Glu Ser Lys Arg Glu Thr 195 200 205 Glu Tyr Gly Pro Cys Arg Arg Glu Met Glu Asp Thr Leu Asn His Leu 210 215 220 Lys Phe Leu Asn Val Leu Ser Pro Arg Gly Val His Ile Pro Asn Cys 225 230 235 240 Asp Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly 245 250 255 Arg Lys Arg Gly Phe Cys Trp Cys Val Asp Lys Tyr G ly Gln Pro Leu 260 265 270 Pro Gly Tyr Thr Thr Lys Gly Lys Glu Asp Val His Cys Tyr Ser Met 275 280 285 Gln Ser Lys 290 <210> 8 <211> 291 <212> PRT <213> Artificial Sequence <220> <223> variant <400> 8 Met Gln Arg Ala Arg Pro Thr Leu Trp Ala Ala Ala Leu Thr Leu Leu 1 5 10 15 Val Leu Leu Arg Gly Pro Pro Val Ala Arg Ala Gly Ala Ser Ser Gly 20 25 30 Gly Leu Gly Pro Val Val Arg Cys Glu Pro Cys Asp Ala Arg Ala Leu 35 40 45 Ala Gln Cys Ala Pro Pro Ala Val Cys Ala Glu Leu Val Arg Glu 50 55 60 Pro Gly Cys Gly Cys Cys Leu Thr Cys Ala Leu Ser Glu Gly Gln Pro 65 70 75 80 Cys Gly Ile Tyr Thr Glu Arg Cys Gly Ser Gly Leu Arg Cys Gln Pro 85 90 95 Ser Pro Asp Glu Ala Arg Pro Leu Gln Ala Leu Leu Asp Gly Arg Gly 100 105 110 Leu Trp Ala Asn Ala Ser Ala Val Ser Arg Leu Arg Ala Tyr Leu Leu 115 120 125 Pro Ala Pro Pro Ala P ro Gly Asn Ala Ser Glu Ser Glu Glu Asp Arg 130 135 140 Ser Ala Gly Ser Val Glu Ser Pro Ser Val Ser Ser Thr His Arg Val 145 150 155 160 Ser Asp Pro Lys Phe His Pro Leu His Ser Lys Ile Ile Ile Ile Lys 165 170 175 Lys Gly His Ala Lys Asp Ser Gln Arg Tyr Lys Val Asp Tyr Glu Ser 180 185 190 Gln Ser Thr Asp Thr Gln Asn Phe Ser Ser Glu Ser Lys Arg Glu Thr 195 200 205 Glu Tyr Gly Pro Cys Arg Arg Glu Met Glu Asp Thr Leu Asn His Leu 210 215 220 Lys Phe Leu Asn Val Leu Ser Pro Arg Gly Val His Ile Pro Asn Cys 225 230 235 240 Asp Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly 245 250 255 Arg Lys Arg Gly Phe Cys Trp Cys Val Asp Lys Tyr G ly Gln Pro Leu 260 265 270 Pro Gly Tyr Thr Thr Lys Gly Lys Glu Asp Val His Cys Tyr Ser Met 275 280 285 Gln Ser Lys 290 <210> 9 <211> 250 <212> PRT <213> Artificial Sequence <220> <223> variant <400> 9 Met Gln Arg Ala Arg Pro Thr Leu Trp Ala Ala Ala Leu Thr Leu Leu 1 5 10 15 Val Leu Leu Arg Gly Pro Pro Val Ala Arg Gly Cys Cys Leu Thr Cys 20 25 30 Ala Leu Ser Glu Gly Gln Pro Cys Gly Ile Tyr Thr Glu Arg Cys Gly 35 40 45 Ser Gly Leu Arg Cys Gln Pro Ser Pro Asp Glu Ala Arg Pro Leu Gln 50 55 60 Ala Leu Leu Asp Gly Arg Gly Leu Cys Val Asn Ala Ser Ala Val Ser 65 70 75 80 Arg Leu Arg Ala Tyr Leu Leu Pro Ala Pro Pro Ala Pro Gly Asn Ala 85 90 95 Ser Glu Ser Glu Glu Asp Arg Ser Ala Gly Ser Val Glu Ser Pro Ser 100 105 110 Val Ser Ser Thr His Arg Val Ser Asp Pro Lys Phe His Pro Leu His 115 120 125 Ser Lys Ile Ile Ile I le Lys Lys Gly His Ala Lys Asp Ser Gln Arg 130 135 140 Tyr Lys Val Asp Tyr Glu Ser Gln Ser Thr Asp Thr Gln Asn Phe Ser 145 150 155 160 Ser Glu Ser Lys Arg Glu Thr Glu Tyr Gly Pro Cys Arg Arg Glu Met 165 170 175 Glu Asp Thr Leu Asn His Leu Lys Phe Leu Asn Val Leu Ser Pro Arg 180 185 190 Gly Val His Ile Pro Asn Cys Asp Lys Lys Gly Phe Tyr Lys Lys Lys 195 200 205 Gln Cys Arg Pro Ser Met Asp Gly Glu Ala Gly Phe Cys Trp Cys Val 210 215 220 Asp Lys Tyr Gly Gln Pro Leu Pro Gly Tyr Thr Thr Lys Gly Lys Glu 225 230 235 240 Asp Val His Cys Tyr Ser Met Gln Ser Lys 245 250 <210> 10 <211> 230 <212> PRT <213> Artificial Sequence <220> <223> variant <400> 10 Met Gln Arg Ala Arg Pro Thr Leu Trp Ala Ala Ala Leu Thr Leu Leu 1 5 10 15 Val Leu Leu Arg Gly Pro Pro Val Ala Arg Cys Gly Ser Gly Leu Arg 20 25 30 Cys Gln Pro Ser Pro Asp Glu Ala Arg Pro Leu Gln Ala Leu Leu Asp 35 40 45 Gly Arg Gly Leu Cys Val Asn Ala Ser Ala Val Ser Arg Leu Arg Ala 50 55 60 Tyr Leu Leu Pro Ala Pro Pro Ala Pro Gly Asn Ala Ser Glu Ser Glu 65 70 75 80 Glu Asp Arg Ser Ala Gly Ser Val Glu Ser Pro Ser Val Ser Ser Thr 85 90 95 His Arg Val Ser Asp Pro Lys Phe His Pro Leu His Ser Lys Ile Ile 100 105 110 Ile Ile Lys Lys Gly His Ala Lys Asp Ser Gln Arg Tyr Lys Val Asp 115 120 125 Tyr Glu Ser Gln Ser Thr Asp Thr Gln Asn Phe Ser Ser Glu Ser Lys 130 135 140 Arg Glu Thr Glu Tyr Gly Pro Cys Arg Arg Glu Met Glu Asp Thr Leu 145 150 155 160 Asn His Leu Lys Phe Leu Asn Val Leu Ser Pro Arg Gly Val His Ile 165 170 175 Pro Asn Cys Asp Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro 180 185 190 Ser Met Asp Glu Gly Ala Gly Phe Cys Trp Cys Val Asp Lys Tyr Gly 195 200 205 Gln Pro Leu Pro Gly Tyr Thr Thr Lys Gly Lys Glu Asp Val His Cys 210 215 220 Tyr Ser Met Gln Ser Lys 225 230 <210> 11 <211> 202 <212> PRT <213> Artificial Sequence <220> <223> variant <400> 11 Met Gln Arg Ala Arg Pro Thr Leu Trp Ala Ala Ala Leu Thr Leu Leu 1 5 10 15 Val Leu Leu Arg Gly Pro Pro Val Ala Asn Ala Ser Ala Val Ser Arg 20 25 30 Leu Arg Ala Tyr Leu Leu Pro Ala Pro Pro Ala Pro Gly Asn Ala Ser 35 40 45 Glu Ser Glu Glu Asp Arg Ser Ala Gly Ser Val Glu Ser Pro Ser Val 50 55 60 Ser Ser Thr His Arg Val Ser Asp Pro Lys Phe His Pro Leu His Ser 65 70 75 80 Lys Ile Ile Ile Ile Lys Lys Gly His Ala Lys Asp Ser Gln Arg Tyr 85 90 95 Lys Val Asp Tyr Glu Ser Gln Ser Thr Asp Thr Gln Asn Phe Ser Ser 100 105 110 Glu Ser Lys Arg Glu Thr Glu Tyr Gly Pro Cys Arg Arg Glu Met Glu 115 120 125 Asp Thr Leu Asn His Leu Lys Phe Leu Asn Val Leu Ser Pro Arg Gly 130 135 140 Val His Ile Pro Asn Cys Asp Lys Lys Gly Phe Tyr Lys Lys Lys Gln 145 150 155 160 Cys Arg Pro Ser Met Asp Glu Gly Ala Arg Gly Phe Cys Trp Cys Val 165 170 175 Asp Lys Tyr Gly Gln Pro Leu Pro Gly Tyr Thr Thr Lys Gly Lys Glu 180 185 190 Asp Val His Cys Tyr Ser Met Gln Ser Lys 195 200 <210> 12 <211> 291 <212> PRT <213> Artificial Sequence <220> <223> variant <400> 12 Met Gln Arg Ala Arg Pro Thr Leu Trp Ala Ala Ala Leu Thr Leu Leu 1 5 10 15 Val Leu Leu Arg Gly Pro Pro Val Ala Arg Ala Gly Ala Ser Ser Gly 20 25 30 Gly Leu Gly Pro Val Val Arg Cys Glu Pro Cys Asp Ala Arg Ala Leu 35 40 45 Ala Gln Cys Ala Pro Pro Ala Val Cys Ala Glu Leu Val Arg Glu 50 55 60 Pro Gly Cys Gly Cys Cys Leu Thr Cys Ala Leu Ser Glu Gly Gln Pro 65 70 75 80 Cys Gly Ser Ala Thr Glu Arg Cys Gly Ser Gly Leu Arg Cys Gln Pro 85 90 95 Ser Pro Asp Glu Ala Arg Pro Leu Gln Ala Leu Leu Asp Gly Arg Gly 100 105 110 Leu Cys Val Asn Ala Ser Ala Val Ser Arg Leu Arg Ala Tyr Leu Leu 115 120 125 Pro Ala Pro Pro Ala Pro Gly Asn Ala Ser Glu Ser Glu Glu Asp Arg 130 135 140 Ser Ala Gly Ser Val Glu Ser Pro Ser Val Ser Ser Thr His Arg Val 145 150 155 160 Ser Asp Pro Lys Phe His Pro Leu His Ser Lys Ile Ile Ile Ile Lys 165 170 175 Lys Gly His Ala Lys Asp Ser Gln Arg Tyr Lys Val Asp Tyr Glu Ser 180 185 190 Gln Ser Thr Asp Thr Gln Asn Phe Ser Ser Glu Ser Lys Arg Glu Thr 195 200 205 Glu Tyr Gly Pro Cys Arg Arg Glu Met Glu Asp Thr Leu Asn His Leu 210 215 220 Lys Phe Leu Asn Val Leu Ser Pro Arg Gly Val His Ile Pro Asn Cys 225 230 235 240 Asp Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Met Asp 245 250 255 Glu Gly Ala Gly Phe Cys Trp Cys Val Asp Lys Tyr Gly Gln Pro Leu 260 265 270 Pro Gly Tyr Thr Thr Lys Gly Lys Glu Asp Val His Cys Tyr Ser Met 275 280 285 Gln Ser Lys 290 <210> 13 <211> 291 <212> PRT <213> Artificial Sequence <220> <223> variant <400> 13 Met Gln Arg Ala Arg Pro Thr Leu Trp Ala Ala Ala Leu Thr Leu Leu 1 5 10 15 Val Leu Leu Arg Gly Pro Pro Val Ala Arg Ala Gly Ala Ser Ser Gly 20 25 30 Gly Leu Gly Pro Val Val Arg Cys Glu Pro Cys Asp Ala Arg Ala Leu 35 40 45 Ala Gln Cys Ala Pro Pro Ala Val Cys Ala Glu Leu Val Arg Glu 50 55 60 Pro Gly Cys Gly Cys Cys Leu Thr Cys Ala Leu Ser Glu Gly Gln Pro 65 70 75 80 Cys Gly Ile Tyr Thr Glu Arg Cys Gly Ser Gly Leu Arg Cys Gln Pro 85 90 95 Ser Pro Asn Gly Ala Arg Pro Leu Gln Ala Leu Leu Asp Gly Arg Gly 100 105 110 Leu Cys Val Asn Ala Ser Ala Val Ser Arg Leu Arg Ala Tyr Leu Leu 115 120 125 Pro Ala Pro Pro Ala Pro Gly Asn Ala Ser Glu Ser Glu Glu Asp Arg 130 135 140 Ser Ala Gly Ser Val Glu Ser Pro Ser Val Ser Ser Thr His Arg Val 145 150 155 160 Ser Asp Pro Lys Phe His Pro Leu His Ser Lys Ile Ile Ile Ile Lys 165 170 175 Lys Gly His Ala Lys Asp Ser Gln Arg Tyr Lys Val Asp Tyr Glu Ser 180 185 190 Gln Ser Thr Asp Thr Gln Asn Phe Ser Ser Glu Ser Lys Arg Glu Thr 195 200 205 Glu Tyr Gly Pro Cys Arg Arg Glu Met Glu Asp Thr Leu Asn His Leu 210 215 220 Lys Phe Leu Asn Val Leu Ser Pro Arg Gly Val His Ile Pro Asn Cys 225 230 235 240 Asp Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Met Asp 245 250 255 Gly Glu Ala Gly Phe Cys Trp Cys Val Asp Lys Tyr Gly Gln Pro Leu 260 265 270 Pro Gly Tyr Thr Thr Lys Gly Lys Glu Asp Val His Cys Tyr Ser Met 275 280 285 Gln Ser Lys 290 <210> 14 <211> 291 <212> PRT <213> Artificial Sequence <220> <223> variant <400> 14 Met Gln Arg Ala Arg Pro Thr Leu Trp Ala Ala Ala Leu Thr Leu Leu 1 5 10 15 Val Leu Leu Arg Gly Pro Pro Val Ala Arg Ala Gly Ala Ser Ser Gly 20 25 30 Gly Leu Gly Pro Val Val Arg Cys Glu Pro Cys Asp Ala Arg Ala Leu 35 40 45 Ala Gln Cys Ala Pro Pro Ala Val Cys Ala Glu Leu Val Arg Glu 50 55 60 Pro Gly Cys Gly Cys Cys Leu Thr Cys Ala Leu Ser Glu Gly Gln Pro 65 70 75 80 Cys Gly Ile Tyr Thr Glu Arg Cys Gly Ser Gly Leu Arg Cys Gln Pro 85 90 95 Ser Pro Asp Glu Ala Arg Pro Leu Gln Ala Ser Ser Asp Gly Arg Gly 100 105 110 Leu Cys Val Asn Ala Ser Ala Val Ser Arg Leu Arg Ala Tyr Leu Leu 115 120 125 Pro Ala Pro Pro Ala Pro Gly Asn Ala Ser Glu Ser Glu Glu Asp Arg 130 135 140 Ser Ala Gly Ser Val Glu Ser Pro Ser Val Ser Ser Thr His Arg Val 145 150 155 160 Ser Asp Pro Lys Phe His Pro Leu His Ser Lys Ile Ile Ile Ile Lys 165 170 175 Lys Gly His Ala Lys Asp Ser Gln Arg Tyr Lys Val Asp Tyr Glu Ser 180 185 190 Gln Ser Thr Asp Thr Gln Asn Phe Ser Ser Glu Ser Lys Arg Glu Thr 195 200 205 Glu Tyr Gly Pro Cys Arg Arg Glu Met Glu Asp Thr Leu Asn His Leu 210 215 220 Lys Phe Leu Asn Val Leu Ser Pro Arg Gly Val His Ile Pro Asn Cys 225 230 235 240 Asp Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Met Asp 245 250 255 Gly Glu Ala Gly Phe Cys Trp Cys Val Asp Lys Tyr Gly Gln Pro Leu 260 265 270 Pro Gly Tyr Thr Thr Lys Gly Lys Glu Asp Val His Cys Tyr Ser Met 275 280 285 Gln Ser Lys 290 <210> 15 <211> 291 <212> PRT <213> Artificial Sequence <220> <223> variant <400> 15 Met Gln Arg Ala Arg Pro Thr Leu Trp Ala Ala Ala Leu Thr Leu Leu 1 5 10 15 Val Leu Leu Arg Gly Pro Pro Val Ala Arg Ala Gly Ala Ser Ser Gly 20 25 30 Gly Leu Gly Pro Val Val Arg Cys Glu Pro Cys Asp Ala Arg Ala Leu 35 40 45 Ala Gln Cys Ala Pro Pro Ala Val Cys Ala Glu Leu Val Arg Glu 50 55 60 Pro Gly Cys Gly Cys Cys Leu Thr Cys Ala Leu Ser Glu Gly Gln Pro 65 70 75 80 Cys Gly Ile Tyr Thr Glu Arg Cys Gly Ser Gly Leu Arg Cys Gln Pro 85 90 95 Ser Pro Asp Glu Ala Arg Pro Leu Gln Ala Leu Leu Asp Gly Arg Gly 100 105 110 Leu Trp Ala Asn Ala Ser Ala Val Ser Arg Leu Arg Ala Tyr Leu Leu 115 120 125 Pro Ala Pro Pro Ala Pro Gly Asn Ala Ser Glu Ser Glu Glu Asp Arg 130 135 140 Ser Ala Gly Ser Val Glu Ser Pro Ser Val Ser Ser Thr His Arg Val 145 150 155 160 Ser Asp Pro Lys Phe His Pro Leu His Ser Lys Ile Ile Ile Ile Lys 165 170 175 Lys Gly His Ala Lys Asp Ser Gln Arg Tyr Lys Val Asp Tyr Glu Ser 180 185 190 Gln Ser Thr Asp Thr Gln Asn Phe Ser Ser Glu Ser Lys Arg Glu Thr 195 200 205 Glu Tyr Gly Pro Cys Arg Arg Glu Met Glu Asp Thr Leu Asn His Leu 210 215 220 Lys Phe Leu Asn Val Leu Ser Pro Arg Gly Val His Ile Pro Asn Cys 225 230 235 240 Asp Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Met Asp 245 250 255 Glu Gly Ala Gly Phe Cys Trp Cys Val Asp Lys Tyr Gly Gln Pro Leu 260 265 270 Pro Gly Tyr Thr Thr Lys Gly Lys Glu Asp Val His Cys Tyr Ser Met 275 280 285 Gln Ser Lys 290 <210> 16 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 atggacgggg aggcg 15 <210> 17 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 acccagaact tctcc 15 <210> 18 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 aagaagggat tttat 15 <210> 19 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 gacgtgcact gcta 14 <210> 20 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 gacaggcagg catac 15 <210> 21 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 ggcacaatta tatca 15 <210> 22 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 22 ataggcctag catatc 16 <210> 23 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 23 catgatccga tacgac 16

Claims (13)

(1) preparing an mRNA encoding an IGFBP-3 variant having the amino acid sequence of any one of SEQ ID NO: 2 to SEQ ID NO: 15 and mRNA encoding a native type ALS (Acid Labile Subunit), (2) prepared above Injecting both mRNA and radiolabeled amino acid into the Xenopus Oocyte, (3) culturing the frog egg to form a complex of ALS / IGFBP-3 variant, and (4) separating the complex and A method for screening amino acid residues involved in binding to ALS in IGFBP-3 comprising detecting the amount thereof. delete delete delete delete delete delete delete delete An IGFBP-3 variant comprising the amino acid sequence of any one of SEQ ID NOs: 2-15. A nucleic acid sequence encoding the IGFBP-3 variant according to claim 10. The nucleic acid sequence of claim 11 wherein said nucleic acid sequence is mRNA or DNA. A primer having a nucleotide sequence of any one of SEQ ID NOs: 17 to 23 used to prepare a DNA encoding an IGFBP-3 variant having an amino acid sequence of any one of SEQ ID NOs: 2 to 15.