Membrane protein expression vector

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The present invention relates to a membrane protein expression vector comprising a fusion partner consisting of the envelope protein P9 of cystovirus phi 12, and to a membrane protein production method using the same. More specifically, the present invention concerns: a membrane protein expression vector comprising the envelope protein P9 gene of cystovirus phi 12, a multicloning site for inserting a target membrane protein, and a protease recognition site located between the P9 gene and the multicloning site; cells transformed by the expression vector; and a method for producing a membrane protein using the cells.

Compensation pursuant to Rule 26, 21.03.2012] Sisto viral membrane proteins and membrane using the same expression vector comprising an outer coat protein P9 as a fusion partner protein production method of phi12

The present invention relates to a film production method using a membrane protein and a protein expression vector which comprises an outer coat protein of P9 Sisto virus phi12 as a fusion partner. More particularly, the present invention is a film containing a protease recognition site is located between the multi-cloning site and the P9 gene and the multiple cloning site for insertion of the outer coat protein gene protein P9, it is an object of the Sisto virus phi12 membrane protein expression vector, the expression vector transfected in cells, and to methods for manufacturing and using the cellular membrane protein.

Membrane protein accounts for about 25-30% degree of proteome of an organism, such as photosynthesis, respiration and basic energy metabolism, cells and liver cells or liver cells and external communications, transportation, and material involved and lipid metabolism. In addition, approximately 50% of the commercially available drugs is a type of film and G- protein coupled receptor protein (G-protein coupled receptor, GPCR), and that the reports of the action point, 1/4 of the approximately 100 selling most GPCR has been reported that this action point.

However, membrane proteins, even though economically important group of proteins that study its function or structure is much behind, compared to water-soluble protein. The reason for this is that, unlike a membrane protein, in particular multiple times across the multilayer membrane protein and soluble protein (multipass transmembrane) was almost impossible to produce by recombinant DNA technology.

Thus, expression of membrane proteins using microorganisms other cases the water-soluble protein is extremely rare, and the protein expression level is very low . If you want to be particularly successful in neurotensin receptor and there are cases of E. coli cells obtained protein per 100 g of about 3 ㎎ through the expression of a fusion form of the maltose binding protein called.

However, when using E. coli to induce the expression of foreign membrane protein, the expression of the target protein is observed before the host ends up death. Membrane protein, and introducing the expression vector In order to solve this problem and may induce the expression of the membrane proteins do not die developed a mutant E. coli C41 and C43 , a membrane protein of the expression in E. coli C41 (DE3) and C43 (DE3) and was also used .

On the other hand, Mystic (Mistic) called Bacillus subtilis (Bacillus subtilus) The protein as a fusion partner, but the reports of eukaryotic cells capable of expressing the multi-membrane proteins, was not effective for the expression of membrane proteins .

In recent years, using the E. coli GlpF as a fusion partner Eau rudin (occludin), Chloe Dean 4 (claudin 4), cheolhwan original enzyme (ferric reductase) and human membrane proteins, such as potassium channel .This is the case sikyeoteuna the expression of a target protein with the amino terminus outside the cell membrane is not applied, and the expression level is also very small.

In addition, the development of expression systems in the membrane system is developed using the yeast cells have been tried. Green Fluorescent Protein (Green fluorescent protein, GFP) using a method reported body expressed protein to determine the presence or absence of expression weigh the presence of GFP fluorescence from the film and inserted in the yeast expression vector object membrane protein to form a fusion protein with GFP It has been developed in the recent years.In this case, while yeast-derived proteins are expressed high success rate, a very low success rate for the animal derived protein is expressed, including a human, the expression level is very low.

The present inventors are of prokaryotic or eukaryotic cell membrane proteins was derived from extensive studies in order to effectively express an object of membrane protein, the fusion partner Sisto et phi12 viral protein coat (major envelope protein) was coupled with the effective P9 confirmed that this can be expressed by the present invention has been completed.

The basic object of the invention is a membrane protein expression vector comprising a protease recognition site is located between the multi-cloning site and the P9 gene and the multiple cloning site for insertion of the outer coat protein gene protein P9, it is an object of film Sisto virus phi12 to provide a.

It is another object of the present invention to provide a membrane protein, a cell transformed with the expression vector of the present invention.

After a further object of the present invention is inserted the gene encoding the desired membrane protein, a multiple cloning site of a vector expressing the membrane protein of the present invention, it was transformed into cells, and then culturing said transformed cell The purpose is to provide a membrane protein for a method of manufacturing the same.

The primary purpose of the present invention described above is the film comprising a protease recognition site is located between the multi-cloning site and the P9 gene and the multiple cloning site for insertion of the outer coat protein gene protein P9, it is an object of the Sisto virus phi12 membrane protein It can be achieved by providing an expression vector.

An expression vector according to the invention is characterized by over-expressing the membrane protein of interest with an outer coating of proteins P9 Sisto virus phi12 as a fusion partner, the P9 5'- end of the gene encoding the protein, or 3 & apos; connection object just the gene encoding the protein of interest and express the protein at the end of a fusion protein fused to the N- terminal or C- terminal of the protein by incorporating the P9. Preferably, the pRphi12 may be used as the expression vector.

P9 get in the C- terminal of the protein was added to the protein of the transmembrane domain and the P9 extra protein by including connecting a gene encoding the target protein on the film 5'-end or 3'-end of the fusion protein N -terminal or a protein of interest and express the fusion protein fused to the C- terminus.

P9 is the outer coat protein may have an amino acid sequence of SEQ ID NO: 1 (P9) or SEQ ID NO: 2 (P9 + TM), the substitution of amino acids to the extent which does not affect the function of the protein in the amino acid sequence of the protein, addition or deletion can be made, depending on the purpose, only a portion of the protein is used or a specific domain may be used in duplicate. Such variations in the amino acid sequence is also included within the scope of the present invention. Accordingly, the present invention also can be used in the polypeptide and fragments thereof having substantially the same amino acid sequence of the protein, the polypeptide is substantially the same preferably at least 80%, more preferably at least 90%, and most preferably at least 95% It means those having a sequence identity.

Gene encoding the other protein P9 film comprises a base sequence deduced from the amino acid sequence of the protein P9 according to the genetic code (genetic code), and to optimize the codon to be well expressed in a selected host cell, such gene may be mentioned as a representative example of a nucleotide sequence of SEQ ID NO: 3 (P9) or 4 (P9 + TM). The nucleotide sequence of SEQ ID NO: 3 and 4 are each a coded amino acid sequence of the SEQ ID NOS: 1 and 2.

Expression vectors of the invention may include a protease recognition site and the appropriate linker DNA between the outer coat protein and P9, a portion to be inserted into the target protein. For example, the expression vectors of the invention in the direction of 5 'to 3' a promoter, Sisto virus gene encoding the outer coat protein of P9 phi12, a protease recognition site, the gene of the desired protein is inserted multiple cloning site (multicloning site and it may include the form of the associated MCS) and a histidine tag sequence fused to the translation (translational fusion). In addition, the antibiotic resistance gene or the like as needed may further include a.