Method for cell surface displaying of target proteins using
The present invention relates to a method for expressing a target protein on the surface of a microorganism using Bacillus anthracis exosporium protein. More particularly, to an expression vector constructed such that it comprises bclA gene encoding Bacillus anthracis exosporium protein BclA or fragments thereof as a cell surface anchoring motif and the target protein can be expressed on the surface of a cell in a form fused with BclA or a fragment thereof when the gene encoding the target protein is expressed in a host cell, as well as, a method for expressing a target protein on the surface of a microorganism using the vector. The expression vector according to the present invention is capable of effectively expressing a target protein or a peptide on the cell surface using BclA, Bacillus anthracis exosporium protein as a cell surface anchoring motif, and since a target protein can be stably expressed on the cell surface in large amounts by culturing a microorganism transformed with the expression vector, thus making it possible to effectively use for the various purposes of recombinant live vaccines, whole cells absorbents, whole cell bioconversion and the like.
1. A surface expression vector comprising a fragment of bclA gene encoding a fragment of Bacillus anthracis exosporium protein BclA as a cell surface anchoring motif, wherein the fragment of the bclA gene is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and a gene encoding a promoter and a target protein, wherein the expression vector is constructed such that the target protein can be expressed in a form fused with the fragment of protein BclA on the cell surface when the gene encoding the target protein is expressed in a host cell.
2. The surface expression vector according to claim 1 , wherein the promoter is tac promoter or any other inducible promoter.
3. The surface expression vector according to claim 1 , wherein the target protein is a protein prepared by deleting a part of amino acid sequences of the target protein or subjecting to site-specific mutation in order to facilitate surface expression.
4. A transformed microorganism obtained by introducing the expression vector of claim 1 into a cell selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria, Actinomyces , yeast and fungus.
5. The transformed microorganism according to claim 4 , wherein the microorganism is mutated such that an intracellular or extracellular protease that degrades the expressed target protein cannot be produced, in order to facilitate surface expression of the target protein.
6. The transformed microorganism according to claim 5 , wherein the microorganism is Escherichia coli.
7. A method for expressing a target protein on the surface of a cell, the method comprising the steps of: expressing the target protein on the cell surface by culturing the transformed microorganism of claim 4 ; and recovering the cell having the target protein expressed on the surface thereof.
8. The method according to claim 7 , wherein the target protein is any one selected from the group consisting of hormones, hormone analogues, enzymes, enzyme inhibitors, signal transduction proteins or their fragments, antibodies or their fragments, single chain antibodies, binding proteins, binding domains, peptides, antigens, adherent proteins, structural proteins, regulatory proteins, toxin proteins, cytokines, transcription factors, blood coagulation factors, and plant defense-inducing proteins.
9. The method according to claim 8 , wherein the target protein is streptavidin.
10. The method according to claim 8 , wherein the enzyme is lipase.
11. A bioconversion method, the method is characterized by using the cells, prepared by the method of claim 8 and expressing the target protein having enzyme activity on the surface thereof.
12. A method for manufacturing a protein array, the method is characterized by attaching the cells, prepared by the method of claim 8 and expressing the target protein having enzyme activity on the surface thereof, on a substrate.
13. A method for producing an antibody in vertebrates, the method comprises:
inducing an immune response by administering the cells, prepared by the method of claim 8 and expressing antigen on the surface thereof, to a vertebrate except a human being, and recovering the antibody produced by the immune response.
14. A method for preparing a chiral compound by carrying out optical resolution of racemic ester compounds using lipase, the method is characterized by using the lipase expressed on the surface of the cells prepared by the method of claim 10 .