KR100803094B1 - Perlucin protein from abalone coding gene and use thereof - Google Patents

Abstract

A perlucin protein derived from Haliotis discus discus is provided to precipitate effectively calcium carbonate and form aragonite crystals, thereby being usefully used for promoting the pearl formation. A perlucin protein consists of an amino acid sequence of SEQ ID : NO. 2 isolated from Haliotis discus discus, where the protein is coded by a nucleotide having a sequence of SEQ ID : NO. 1. A transformant is transformed by introducing a vector including a nucleic acid sequence which has a sequence of SEQ ID : NO. 1 and encodes a cDNA of the perlucin isolated from the Haliotis discus discus. An agent for promoting the pearl formation of the Haliotis discus discus comprises the perlucin protein as an effective ingredient.

Description

PELBLCCI protein derived from abalone, gene encoding the same, and use thereof {PERLUCIN PROTEIN FROM ABALONE, CODING GENE, AND USE THEREOF}

1 is abalone ( Haliotis discus The full-length cDNA and amino acid sequence of the perlucin gene isolated from the discus ) is shown.

FIG. 2 shows the homology analysis of the abalone perlucin amino acid sequence (cPLC) of the present invention and the perlucin (P82596) amino acid sequence derived from greenlip abalone. Dots indicate very important residues related to calcium binding, underlines indicate carbonate recognition sites, and two squares indicate important motifs of carbonate.

3 is a diagram of abalone perlucin protein induction and purification, where A represents the protein induced by IPTG (circular site) and B represents the purified protein, respectively.

4 is a graph showing calcium precipitation induced by abalone perlucin treatment, control 1 is a distilled water, control 2 is a protein elution buffer solution, Perlucin is a group treated with Perlucin, respectively.

5 is a view showing the change of the structure of the crystal over time after the abalone perlucin treatment, the control group is a single and multiple crystals of calcium carbonate without perlucin, PLC is a single and multiple crystals of calcium carbonate added perlucin, respectively Pointing.

1 is abalone ( Haliotis discus The full-length cDNA and amino acid sequence of the perlucin gene isolated from the discus ) is shown.

FIG. 2 shows the homology analysis of the abalone perlucin amino acid sequence (cPLC) of the present invention and the perlucin (P82596) amino acid sequence derived from greenlip abalone. Dots indicate very important residues related to calcium binding, underlines indicate carbonate recognition sites, and two squares indicate important motifs of carbonate.

3 is a diagram of abalone perlucin protein induction and purification, where A represents the protein induced by IPTG (circular site) and B represents the purified protein, respectively.

4 is a graph showing calcium precipitation induced by abalone perlucin treatment, control 1 is a distilled water, control 2 is a protein elution buffer solution, Perlucin is a group treated with Perlucin, respectively.

5 is a view showing the change of the structure of the crystal over time after the abalone perlucin treatment, the control group is a single and multiple crystals of calcium carbonate without perlucin, PLC is a single and multiple crystals of calcium carbonate added perlucin, respectively Pointing.

The invention abalone (Abalone, Haliotis discus perlucin gene isolated from the discus ), the expression vector containing the gene, the transformant into which the expression vector is introduced, the perlucin protein produced therefrom and the usefulness of the protein.

The shell of a mollusk is the main barrier to protect itself from the outside and is the most discriminated feature. In understanding the mechanical strength of the skin and the nanoscale microstructure, the skin formation mechanism can be very strange and interesting. The nacreous layer of the sheath is involved in forming pearls of expensive ornaments. In addition, in the medical field, the pearl shell may contain any signal substance having bone formation ability and thus may be used as a bone regeneration material. This feature of the skin is mainly due to the inclusion of an organic substrate between the calcium carbonate layers of the skin. Although these proteins and chitin are limited to only 5% of the envelope, they play a very important role in regulating the growth of the shell structure, nucleation aragonite crystals and molluscs. Coral, for example, is an aragonite form of calcium carbonite, which can grow without the aid of any protein, but its robustness is much lower than that of mollusk shells. In addition, scientists have isolated and identified interesting proteins derived from the shells of mollusks to reveal the secrets of their formation. (Cen Zhang & Rongqing ZhangBiotechnology, 0: 1-15, 2006).

Perlucin is one of the most promising soluble envelope matrix proteins. Many previous studies have shown that perlucin can promote nucleated calcium precipitation and aragonite crystal formation. Therefore, the gene constituting the protein can be said to be a very important gene associated with pearling (J, Gomez-Morales et al., Journal of Crystal Growth, 169: 331-338, 1996). More interestingly, through amino acid sequencing, perlucin is called a functional C-type lectin that specifically binds to D-galactose or D-mannose / D-glucose. Known (Marion, M Bradford, Analytical. Biochemistry, 72: 248-254, 1976). Similar C-type lectins in fish and shrimp have been found to have blood cell function and bacterial deadlocks by themselves. This means that perlucin can play a very important role in the innate immune response of mollusks as well as promoting pearling.

In order to achieve the above object, the present invention provides a polynucleotide having a nucleotide sequence of SEQ ID NO: 1, encoding a perlucin cDNA isolated from the abalone.

The present invention also provides a vector and a transformant comprising the nucleic acid sequence of the gene.

In addition, the present invention is abalone ( Haliotis discus provide perlucin protein isolated from the discus ). Preferably the present invention is a peptide having an amino acid sequence of SEQ ID NO: 2, more preferably encoded by the nucleotide sequence of SEQ ID NO: 1.

In addition, the present invention provides a pearl formation promoter comprising the perlucin protein as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention is abalone ( Haliotis discus provide a perlucin gene isolated from the discus ).

Abalone ( Haliotis discus In order to isolate the perlucin gene related to pearl formation from the discus ), six candidate clones were selected from the EST cDNA library, and the sequence was analyzed to finally select one single clone (clone 1). At this time, the forward and reverse primers used are not particularly limited, but preferably SEQ ID NO: 3 and 4 in clone 1, SEQ ID NO: 5 and 6 in clone 2, SEQ ID NO: 7 and 8 in clone 3, sequence in clone 4, respectively Primers 9 and 10, SEQ ID NOs: 11 and 12 in clone 5, and SEQ ID NOs: 13 and 14 in clone 6 can be used. As a result of sequencing, clone 1 of the clone had 36% sequence homology with the amino acid sequence of the perlucin gene derived from greenlip abalone (NCBI accession no. It was confirmed that it has a 648 bp cDNA including the bp open reading frame (ORF, SEQ ID NO: 1) (see Figure 1).

The present invention also provides a vector and a transformant comprising the nucleic acid sequence of the gene.

The vector system of the present invention can be constructed through various methods known in the art, and specific methods thereof are described in Sambrook et al., Molecular . Cloning , A Laboratory Manual , Cold Spring Harbor Laboratory Press, 2001, which is incorporated herein by reference.

Vectors of the present invention can typically be constructed as vectors for cloning or vectors for expression. In addition, the vector of the present invention can be constructed using prokaryotic or eukaryotic cells as hosts. It is preferable that the nucleic acid molecule of the present invention is derived from a prokaryotic cell, and the prokaryotic cell is used as a host in consideration of the convenience of culture. Vectors of the present invention can typically be constructed as vectors for cloning or vectors for expression.

In a specific embodiment of the present invention, in order to prepare an expression vector containing the coding sequence of the abalone-derived perlucin gene, the perlucin cDNA isolated from the clone is cloned and transformed into Rosetta gammi (DE3) strain. In addition, the expression vector may be selected from the group consisting of plasmids, cosmids and phages, and preferably, a pMAL-c2X vector may be used. The host cell capable of transforming the expression vector may be selected from the group consisting of E. coli, prokaryote, fungus, plant and animal cells, preferably Roseetta (DE3) E. coli.

The perlucin protein according to the present invention has a molecular weight range of 50 to 75 kDa and has an activity of promoting calcium carbonate precipitation and aragonite crystal formation. This suggests that perlucin protein derived from abalone can be usefully used as a pearling accelerator as mentioned above (J, Gomez-Morales et al., Journal of Crystal Growth, 169: 331-338, 1996).

In addition, as a result of comparing and analyzing the homology of proteins of species having a molecular structure similar to the perlucin protein of the present invention, the abalone perlucin protein has a homology of 30% with protein protein of Zebrafish (Proteoglycan) It has 29% homology with its Asianloglycoprotein receptor, and has 30% homology with the protein protein protein of chicken. It is ayu fish, eel and Carp C-type lectin (Cartin) it was confirmed that it has a sequence homology of 30%, 32% and 29%, respectively (Table 2).

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the invention, the present invention is not limited by the following examples.

< Example  1> Abalone perlucin  Characterization of genes and proteins

<1-1> Clonal Selection and Sequence Analysis

Using short gun sequencing, the original prototype sequence of the overturned EST cDNA library was obtained, and the cap3 Sequence Assembly Program ( http://pbil.univlyon1.fr/cap3.php ) was used to maintain proper clustering and unification. Based on this, in this example six candidate clones with perlucin were selected and their sequences analyzed respectively. Their ORF sequences were detected using DNAssit 2.0 and applied to blast X for comparison with known sequences. In addition, attempts were made to detect both single peptides and N- and O-glycosylation sites using a web page server ( http://www.cbs.dtu.dk/services/ ).

<1-2> perlucin  gene Cloning , Protein purification and expression analysis

In order to clone the perlucin gene from the six clones selected in Example <1-1>, a primer encoding the perlucin gene of each of the six clones was first designed, and then the coding region was amplified by PCR. At this time, the primers used are described in Table 1, respectively, and the EcoR I and Hind III restriction sites were inserted at the primer ends for cloning into the vector.

Clone order SEQ ID NO: Clone 1 Forward primer 5'-gagagaGAATTCATGCTTCTGTTTCTCGCGAT-3 ' 3 Reverse primer 5'-gagagaAAGCTTTTAGCGTCCAACACCAGGA-3 ' 4 Clone 2 Forward primer 5'-gagagaGAATTCATGAAAACGTTCTGCTGTACCCT-3 ' 5 Reverse primer 5'-gagagaAAGCTTTTACATTTTACATATGAAATTGGCGA-3 ' 6 Clone 3 Forward primer 5'-gagagaGAATTCATGAGACTCGAAGTCCTATCATTCC-3 ' 7 Reverse primer 5'-gagagaAAGCTTTTAAAGGTTCTTCTCACAGATAAAGTTTAAG-3 ' 8 Clone 4 Forward primer 5'-gagagaGAATTCATGGAGAAGGTACTCAAGTATTTGTC-3 ' 9 Reverse primer 5'-gagagaAAGCTTCTACCCAATAGGATTTGAATCCA-3 ' 10 Clone 5 Forward primer 5'-gagagaGAATTCATGTCTCCATTGTCCGTGGT-3 ' 11 Reverse primer 5'-gagagaAAGCTTTCATCCCACGAGTATGCTTCC-3 ' 12 Clone 6 Forward primer 5'-gagagaGAATTCATGTTGATCCCGCGCGT-3 ' 13 Reverse primer 5'-gagagaAAGCTTTTATTTTCCTGTTATGTTAGGCGC-3 ' 14

All PCR products were purified using a Bioneer PCR purification kit, inserted into a pMAL-c2X expression vector (New England Biolabs, USA) and transformed into 10G cells to obtain plasmid DNA. As a result, one clone 1 having 36% homology with the perlucin sequence derived from greenlip abalone (NCBI accession number: P82596) was finally selected among the six clones (FIG. 2). Through sequencing, the full-length cDNA sequence of the clone consists of 648 bp of nucleotides and contains a 495 bp open reading frame (ORF, SEQ ID NO: 1) encoding a total of 165 amino acid residues (SEQ ID NO: 2) It was confirmed that the presence (Fig. 1).

For protein expression, the isolated plasmids were transformed into Rosetta gammi (DE3) strains, and then the initial cultured 5 ml of the culture solution was added with 100 ul empicillin (100 mg / ml) and 10 mM glucose (2% final concentration). Inoculated in the added 100ml Luria broth medium was incubated at 180 rpm, 37 ℃ until the OD ₆₀₀ value is 0.5 ~ 0.8. Subsequently, 1.0 mM IPTG was added to induce protein expression for 6 hours at 37 ° C., and then the cells were centrifuged at 4 ° C. and 3500 rpm for 30 minutes, and then all the cells were suspended in a 5 ml column solution at −70 ° C. Stored for one day. After thawing, the cells were crushed in an ice-water bath, centrifuged at 9000 g for 30 minutes at 4 ° C, and the supernatant obtained therefrom was passed through a maltose binding resin column. The protein passing through it was prepared for purification and protein activity analysis. In addition, to confirm the expression and purity of the protein, SDS-PAGE was used, and the protein concentration was quantitated based on the Bradford method (Marion. M Bradford, Analytical. Biochemistry, 72: 248-254, 1976). Calcium carbonate precipitation and crystal formation assays used proteins expressed at the appropriate concentrations and purity prepared above. As a result, the inventors confirmed strong protein expression (FIGS. 3A and 3B), and on average, 2 mg of purified soluble protein was obtained in 100 ml of LB broth medium.

<1-3> Perlucin  Signal peptides of proteins and Saccharification  Site measurement

Because perlucin is a glycoprotein or not glycosylated, only one candidate N glycosylation site is known for the greenlip abalone protein (Kalheinz Mann, et al., Journal of Biochemistry, 267: 5257-5264, 2000). In the perlucin cDNA sequencing the candidate glycosylation site was measured, it was confirmed that only one glycosylation site was not detected (Fig. 1). This means that the perlucin protein can be successfully expressed in the prokaryotic protein expression system. In addition, the tenth amino acid residue (serine) was identified as a single peptide, and the perlucin protein of the present invention was similar to the extracellular matrix protein. Therefore, it can be seen that the perlucin protein of the present invention contains 144 amino acid residues and is shorter than the greenlip abalone perlucin protein.

<1-4> Perlucin  Amino Acid Substitution Analysis of Proteins

The most amino acids in the amino acid sequence of the perlucin protein are aspartic acid (8%) and glycine (7%). These two amino acid residues are known to directly affect carbonate crystal formation because they have a C-terminal group that can modify the thermodynamic equilibrium of the crystal forming surface (H. Henry Teng, et al., 282 : SCIENCE, 1998). In other words, abundance of substrate proteins such as aspartic acid or glycine can quickly promote the overcoat membrane growth of the abalone. Arginine corresponds to 3% of the total amino acid residues but is another important amino acid residue. Given the difference in translation codon frequencies in eukaryotic and prokaryotic cells, the presence of five arginines allowed the inventor to select the Rosetta gammi (DE3) strain as the strain for protein expression.

<1-5> Perlucin  Functional Domain Analysis of Proteins

Although the perlucin protein of the present invention has a shorter sequence length than the perlucin protein derived from greenlip abalone, it was confirmed that the two perlucin proteins contained almost identical functional domains (FIG. 2). That is, it has all the functional components (round point) associated with calcium binding, six highly conserved cysteines (star shape), and one single carbonate cognitive domain (underlined). In addition, the two domains in the rectangle are known to be very important for binding to carbonates and Ca ²⁺ , and to polysaccharides with specific structures (Drickamer, K., J. Biol. Chem., 263: 9557). -9560, 1988 and Drickamer, K., Nucleic Acid Research and Molecular Biology, 45: 207-232, 1993). That is, if the domain is present near the carbonate recognition domain it is a "QPD" protein that specifically binds to galactose. On the other hand, if the domain is "EPN" the protein is able to bind specifically to most mannose. In addition, a domain near the N-terminal portion of the carbonate recognition domain can determine calcium binding capacity.

<1-6> with other proteins Homology  analysis

In order to investigate what function perlucin performs in vivo, in this example, homology between several types of proteins having a molecular structure similar to the abalone perlucin protein of the present invention was analyzed. Table 1 below shows the six most similar proteins obtained through the results of blust X of the NCBI webpage server.

Six proteins of the species most similar to abalone perlucin Animal species Protein name Blast score Sequence identity (%) Zebrafish Proteoglycan precursor 79.7 30 Ayu fish C-tpye lectin 79.7 30 Human Asialoglycoprotein receptor 75.3 29 Eel C-tpye lectin 74.7 32 Chicken Proteoglycan core protein 74.3 30 Carp C-tpye lectin 73.9 29

As a result, the abalone perlucin protein is 30% homologous to the protein of Proteoglycan in Zebrafish, 29% homologous to the human Asian glycoprotein receptor, and the protein of chicken 30% homology with Proteoglycan core protein, 30%, 32% and 29%, respectively, with C-type lectins of Ayu fish, Eel and Carp It was confirmed that the homology of (Table 2).

< Example  2> perlucin  Protein Activity Analysis

<2-1> calcium precipitation analysis

Calcium precipitation analysis has been previously reported (Weiss, IM, et al., Biochem. Biophys. Res. Commun., 267: 17-21, 2000 and Yong Zhang, et al., Pinctada fucata Comparative Biochemistry and Physiology Part B 135 : 565-573, 2003). Specifically, 1N NaOH was added to bring the pH of 0.02 mM NaHCO 3 to 8.7, and 0.5 mM KCL was added to maintain overall ionic strength similar to seawater. 6 ml of NaHCO solution prepared above and 0.02 mM CaCl 2 were rapidly mixed in a 50 ml tube. 50 μl of 1 mg / ml Perlucin was added to the calcium carbonate solution. As a control, distilled water or protein buffer (1.0 M Tris-HCL, PH 7.4; 200 mM NaCl) without 1 mg / ml perlucin was added to the saturated calcium carbonate solution. Then, an istek (720P model) pH meter was used to measure the pH value of the solution, and data was recorded every 10 seconds.

As a result, the pH of the saturated calcium carbonate solution was significantly reduced compared to the control (sterile water and perlucin elution buffer) by the addition of perlucin of the present invention as shown in FIG. This is because the ionization equilibrium of calcium carbonate solution is broken by perlucin protein and the pH value is rapidly decreased.

Therefore, the above results indicate that the perlucin protein of the present invention can bind to calcium and can promote crystal formation with high activity.

<2-2> calcium Carbonate crystal  Formation analysis

Crystal formation assays were based on previously known methods (S. Black, et al., Journal of Microbiology, 212: 280-291, 2003), about 30 ml of 100 mM NaHCO3 and 120 ml of 40 ml of 53 mM MgCl2. Dropwise addition was added whilst continuously swirling to mmol CaCl2. When the solution was suspended, the reaction was stopped by adding CaCl 2 and adjusted to pH 8.2 by addition of 1N NaOH. Since the pH of the crystal forming solution may be too low (less than 7.7) or too high, resulting in pH drift during culture (Deron A. Walters, et al., Biopysical Journal., 72: 1425-1433, 1997). In the example, 8.2 was chosen as the optimal pH for crystal formation. The saturated calcium carbonate solution was then filtered through a 0.22 um filter. To promote calcium carbonate crystal formation using a 24-well plate, 50 μl of 1 mg / ml perlucin was added to the 300 μl calcium carbonate solution prepared above. As a control, the same volume of protein buffer without perlucin prepared above was added. After culturing for 1 week in a 4 ° C. refrigerator, crystals induced by perlucin protein were observed with a stereo invert microscope.

As a result, as shown in Figure 5, the control group without the addition of perlucin forms a pointed crystal, while the experimental group to which perlucin was added could be observed that the pointed part of the crystal was circular with time. This suggests that perlucin plays a very important role in pearl structure formation.

As discussed above, the abalone-derived perlucin protein according to the present invention can be effectively used to promote pearling because it effectively acts on calcium carbonate precipitation and aragonite crystal formation.

Attach sequence list electronic file  

Claims (7)

A perlucin protein consisting of the amino acid sequence of SEQ ID NO: 2 isolated from Haliotis discus discus . delete The perlucin protein of claim 1, wherein the protein is encoded by a nucleotide having a nucleotide sequence of SEQ ID NO: 1. A polynucleotide having the nucleotide sequence of SEQ ID NO: 1 and encoding the cDNA of perlucin isolated from abalone. A vector comprising the nucleic acid sequence of claim 4. A transformant transformed by introducing the vector of claim 5. An abalone pearling accelerator comprising the perlucin protein of claim 1 or 3 as an active ingredient.

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