JP3752309B2 - Barnacle third adhesion protein gene - Google Patents

Description

【００２１】

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to DNA used for producing a peptide as a raw material for an adhesive that can be used in water or in a wet environment by using recombinant DNA technology. The peptide obtained by culturing microorganisms or cultured cells containing recombinant DNA incorporating DNA encoding the adhesion protein in a culture solution and collecting the polypeptide accumulated in the culture It is expected to be used in a wide range of applications as an agent raw material and a cell culture substrate.
[0002]
[Prior art]
Various types of adhesives have been developed that exhibit strong adhesive strength under dry conditions. Many of them can maintain their strength once they are bonded under dry conditions, even in wet environments. However, there was no adhesive that could reach an effective strength when the adhesion was initiated under wet conditions or in water.
[0003]
Barnacles are secreted based on a protein called cement as a main component and can adhere strongly in seawater. The amino acid composition of this protein has been investigated and suggested to be different from general insoluble proteins (G. Walker, J. mar. Biol. Ass. UK (1972) 52, 429-7435).
[0004]
Regarding the first adhesive protein constituting the insoluble fraction of cement and the second adhesive protein contained in the soluble fraction of cement and having a molecular weight of about 60 kDa, the gene has already been cloned by the present inventor and the structure has also been determined. (Japanese Patent Application Laid-Open No. 7-265081, Japanese Patent Application No. 7-203250). However, as for proteins other than the first and second adhesion proteins contained in the cement, no examples of successful cloning of the genes have been known yet.
[0005]
[Problems to be solved by the invention]
As described above, a part of proteins contained in cement has been successfully cloned. However, the cement contains many proteins other than these, and it is considered that such proteins can be used as adhesives as well as the first adhesive protein and the second adhesive protein. It is an object of the present invention to provide a gene that is the basis for production in order to produce such proteins in large quantities using genetic engineering techniques.
[0006]
[Means for Solving the Problems]
As a result of intensive studies in order to solve the above problems, the present inventors have isolated a cDNA encoding a soluble protein having a molecular weight of about 20 kDa (hereinafter referred to as “third adhesion protein”) contained in cement. The present invention was completed successfully.
That is, the present invention is a barnacle third adhesion protein gene encoding an amino acid sequence represented by SEQ ID NO: 2 or an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2.
[0007]
Hereinafter, the present invention will be described in detail.
The barnacle third adhesion protein gene of the present invention encodes an amino acid sequence represented by SEQ ID NO: 2 or an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2. Here, the “amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 2” means that some amino acid residues of the amino acid sequence represented by SEQ ID NO: 2 are changed such as deletion, substitution, addition, etc. Is an amino acid sequence having the same adhesive properties as the above sequence. Making some amino acid residues change such as deletion, substitution, addition and the like can be achieved by techniques commonly used at the time of filing of the present application, such as site-directed mutagenesis (Nucleic Acid Research, Vol. 10, No. 20, p6487-6500 (1982)).
[0008]
The gene of the present invention can be obtained by the following procedure.
First, the third adhesion protein is isolated and its partial amino acid sequence is determined. The third adhesion protein can be isolated according to a method commonly used for isolating soluble proteins. For example, cement secreted from the barnacle bottom shell is collected and solubilized with 70% formic acid or the like. This fraction can be isolated as a supernatant by centrifugation, the solubilized fraction is separated by reverse-phase HPLC, and the major peak eluted first is collected. The partial amino acid sequence can be determined by a commercially available protein sequencer.
[0009]
Next, barnacle total RNA is isolated, and RNA having a polyadenylic acid chain (polyA-RNA) is prepared therefrom. Isolation of total RNA can be performed according to a conventional method. For example, whole barnacle tissue is solubilized with guanidine thiocyanate or the like, extracted with phenol / chloroform, and precipitated with isopropanol. The method for obtaining total RNA is not limited to this method, and can also be obtained by centrifuging in a LiCl precipitation method or cesium chloride solution. Poly A-RNA can be prepared from total RNA using, for example, an oligo dT cellulose column.
[0010]
Using the poly A-RNA obtained above as a template, prepare double-stranded cDNA using reverse transcriptase, insert it into a suitable vector, introduce this vector into a suitable host, amplify it, Make a rally. Double-stranded cDNA can be synthesized by the S1 nuclease method or the Okayama-berg method, but can also be synthesized using a commercially available cDNA synthesis kit. As the vector, various vectors derived from λ phage, such as λgt10 and λZapII, and plasmid vectors such as pBR322 can be used.
[0011]
Selection of a target clone from the library may be performed by synthesizing an oligonucleotide based on the partial amino acid sequence of the third adhesion protein determined above and selecting a clone that binds strongly to the oligonucleotide. The sequence can be determined by a general method such as the Sanger method or the Maxam-Gilbert method. By the above procedure, the full length of the third adhesion protein cDNA including the translation initiation codon to the termination codon and further the polyadenylate chain addition signal can be isolated.
[0012]
The isolated cDNA can be prepared in large quantities by inserting it into an appropriate expression vector and introducing it into a microorganism or cultured cell for expression. At this time, since the DNA contains a signal portion, the peptide can be secreted outside the host cell. It is also possible to produce intracellularly by removing the signal portion and incorporating it into an appropriate vector.
[0013]
DETAILED DESCRIPTION OF THE INVENTION
[0014]
【Example】
[Example 1] Determination of partial amino acid sequence of third adhesion protein of red barnacles Cement secreted from the bottom shell of red barnacles collected in Miyako Bay, Miyako City, Iwate Prefecture was collected and suspended in a 70% aqueous formic acid solution. After diluting the formic acid concentration to 10%, the mixture is centrifuged at 200,000 × g for 1 hour, and the resulting supernatant is separated by reverse-phase HPLC. The first major peak that is eluted is recovered and the third adhesion protein is isolated. did.
The partial amino acid sequence of the isolated third adhesion protein was determined by Protein Sequencer 473A (Upride Biosystems).
[0015]
[Example 2] Preparation of cDNA library of red barnacles Ten red barnacles with a diameter of 4-5cm in the bottom shell collected in Miyako Bay, Miyako City, Iwate Prefecture were obtained from guanidine thiocyanate, sodium citrate, sodium N-lauryl sarcosine, 2- The tissue is mechanically disrupted in a solution such as mercaptoethanol, extracted with phenol and chloroform to remove proteins, and then extracted with total alcohol by adding isopropanol and precipitating it on an oligo dT cellulose column. Conducted to prepare poly A-RNA. By this operation, about 2 μg of poly A-RNA was obtained.
[0016]
Next, double-stranded cDNA was prepared using reverse transcriptase with this poly A-RNA as a template. This operation was performed using an Amersham cDNA synthesis kit according to the attached protocol. An EcoRI-Notl-BamHI adapter was added to the resulting double-stranded DNA and inserted into the phage vector λZapII. This operation was performed using an Amersham cDNA synthesis system according to the attached protocol. After completion of insertion, the recombinant DNA was encapsulated in the phage using the in vitro packaging solution attached to the kit. Recombinant phage that had been completely encapsulated was infected with E. coli XL-I blue and amplified.
[0017]
Example 3 Selection of Recombinant Phage Containing Third Adhesive Protein cDNA The recombinant phage obtained in Example 2 was amplified, and 50,000 plaques obtained were immobilized on a nylon membrane high bond (Amersham). Next, oligonucleotide probes CA (A, G, T, C) AC (A, G, T, C) CC (A, G) TC (T, T) corresponding to the complementary strand of the partial amino acid sequence determined in Example 1 were used. C) TC (A, G) TG was synthesized with a milligen cyclone DNA synthesizer, labeled with α 32 P-ATP end label, and plaque hybridization was performed. As a result, more than 30 plaques that bind to the probe were obtained from 50,000 clones. Of these, 10 plaques are arbitrarily selected, the length of the inserted cDNA is examined by agarose electrophoresis, and the one with the longest inserted fragment is selected from the phage vector to the plasmid vector using the EXASSIST system (Stratagene). Was cut out. This plasmid vector was introduced into E. coli SF1-20K. Escherichia coli SF1-20K has been deposited as FERM P-15620 at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (1-3 Higashi 1-3, Tsukuba, Ibaraki, Japan) (Deposit date: May 15, 1996) Day).
[0018]
[Example 4] Sequence determination of the third adhesion protein gene The sequence of the insert fragment obtained in Example 3 was determined using a 373A-DNA sequencer and sequencing kit manufactured by Applied Biosystems. As a result, it was found that this inserted fragment was a sequence containing the full length of the mature body of the third adhesion protein. As shown in SEQ ID NO: 1, the obtained third adhesion protein gene is an 881 bp sequence encoding the 183 amino acid sequence from the N-terminal amino acid of the mature form to the translation termination codon, and the 19 residues from the most upstream are signal peptides. It hits. A polyadenylate chain addition signal AATAAA was present in the downstream untranslated region, and a polyadenylate chain was present further downstream.
[0019]
【The invention's effect】
The present invention provides a barnacle third adhesion protein gene. The protein produced from the gene of the present invention is extremely useful as a raw material for adhesives.
[0020]
[Sequence Listing]

[0021]

Claims (3)

A protein represented by the amino acid sequence represented by SEQ ID NO: 2 or a sequence in which some amino acid residues of the amino acid sequence represented by SEQ ID NO: 2 have been deleted, substituted, or added, and have adhesive properties A barnacle third adhesion protein gene encoding a protein. A barnacle third adhesion protein gene comprising the base sequence represented by SEQ ID NO: 1. A protein represented by the amino acid sequence represented by SEQ ID NO: 2 or a sequence in which some amino acid residues of the amino acid sequence represented by SEQ ID NO: 2 have been deleted, substituted or added, and have adhesive properties Barnacle third adhesion protein, which is a protein.