INTACT SURFACE DISPLAY OF SUBSTANCES OF INTEREST

BACKGROUND OF THE INVENTION FIELD OF THE INVENTION The present invention relates to a method for the surface display and the application thereof.

DESCRIPTION OF THE RELATED ART

The technology of surface display in which organism displays on its surface the desired proteinaceous substance such as peptide and polypeptide has wider application fields depending on the types of protein displayed or host organism (Georgiou et al., 1993, 1997; Fischetti et al., 1993; and Schreuder et al., 1996). Such conventional surface display technology has been developed by use of several unicellular organisms such as bacteriophage, bacteria, yeast and mammalian cell. The gene of protein to be displayed is contained in host organism and thus the host can be selectively screened using the characteristics of the protein displayed, thereby obtaining the desired gene from the selected host with easiness. Therefore, such surface display technology can guarantee a powerful tool on molecular evolution of protein (see WO 9849286; and U.S. Pat. No. 5,837,500).

High-Throughput Screening

For instance, phage displaying on its surface antibody having desired binding affinity is bound to immobilized antigen and then eluted, followed by propagating the eluted phage, thereby yielding the gene coding for target antibody from phage (U.S. Pat. No. 5,837,500). The bio panning method described above can provide a tool to select target antibody by surface displaying antibody library on phage surface in large amount and comprises the consecutive steps as follows: (1) constructing

library; (2) surface displaying the library; (3) binding to immobilized antigen; (4) eluting the bound phage; finally (5) propagating selected clones.

The technology of phage surface display has been found to be useful in obtaining the desired monoclonal variant form enormous library (e.g., 10 ⁵ -10 ⁹ variants) and thus applied to the field of high-throughput screening of antibody. Antibody has been used in various fields such as therapy, diagnosis, analysis, etc. and thus its demand has been largely increased. In this context, there has been a need for novel antibody to have binding affinity to new substance or catalyze biochemical reaction. The hybridoma technology to produce monoclonal antibody has been conventionally used so as to satisfy the need. However, the conventional method needs high expenditure and long time for performance whereas the yield of antibody is very low. In addition to this, to screen novel antibody, more than 10 ¹⁰ antibody libraries is generally used, as a result, the hybridoma technology has been thought to be inadequate in finding antibody exhibiting new binding property. Many researches has focused on novel methods which is easier and more effective that the bio panning method described above and then developed novel technologies performed in such a manner that libraries are displayed on surface of bacteria or yeast and then cells displaying target protein is sorted with flow cytometry in a high-throughput manner. According to the technology, antigen labeled with fluorescent dye is bound to surface-displaying cell and the antibody having the desired binding affinity is isolated with flow cytometry capable of analyzing more than 10 ⁸ cells a hr. Francisco, et al., have demonstrated the usefulness of microbial display technology by revealing that surface-displayed monoclonal antibody could be concentrated with flow cytometry at rate of more than 10 ⁵ , finally more than 79% have been proved to be the desired cells (Daugherty et al., 1998).

Live Vaccine

The surface display technology mentioned above can display antigen or fragment thereof and hence provide a delivery system for recombinant live vaccine. Up to now, attenuated pathogens or viruses have been predominantly employed as vaccine. Particularly, the bacteria have been found to express antigen intracellular^ or extracellularly or on its cell membrane, thereby delivering antigen to host cell. The surface-displayed live vaccine induces a potential immune reaction and expresses continuously antigen during propagation in host cell; therefore, it has been highlighted as novel delivery system for vaccine. In particular, pathogen-derived antigenic epitope displayed on surface of nonpathogenic E coli or Salmonella is administered orally in viable form and then exhibits to induce immune reaction in more continuous and powerful manner (Georgiou eta/., 1997; and Lee eta/., 2000).

Whole Cell Bioconversion

Whole cell as biocatalyst displaying on its surface enzyme capable of catalyzing chemical reaction can avoid necessities for direct expression, isolation and stabilization of enzyme. In case of expressing enzyme in cell for bioconversion, the cell is compelled to recovery and chemical (e.g., toluene) treatment to ensure impermeability of substrate. In addition, the lasting use renders the enzyme inactive or gives a problem on transference of substrate and product, thus dropping the productivity of overall process.

The above-mentioned shortcomings can be removed using enzyme displayed on cell surface (Jung et al, 1998a: 1998b). With whole cell displaying on its surface phosphodiesterase, organophosphorous-typed parathion and paraoxon with higher toxicity can be degraded, which is a typical example representing the applicability of cells displaying enzyme to environmental purification process (Richins et al., 1997).

Antipeptide Antibody

Martineau et al. have reported a highly simple method for production of antipeptide antibody using surface display technology of E coll (Martineau et al., 1991). As described, the desired peptide is displayed on the protruding region of MaIE and outer membrane protein, LamB and then whole cell or fragmented cell is administered to animal so as to generate antipeptide antibody. The method makes it possible to produce antibody with avoiding chemical synthesis of peptide and its linkage to carrier protein.

Whole Cell Adsorber To immobilize antibody or polypeptide on suitable carrier, which is useful in absorption chromatography, several subsequent steps must be performed, for example, protein production by fermentation, isolation of protein in pure form, and immobilization on a carrier. Generally, it is difficult to prepare the bioadsorber.

As adsorber, a whole cell displaying absorption protein has been developed. The whole cell adsorber known mostly is Staphylococcus aureus displaying on its surface protein A naturally, which has a high binding affinity to Fc domain of mammalian antibody. Currently, novel method has been proposed to remove and recover heavy metals, which employs metallothionein or metal-absorption protein displayed on microbial cell surface in large amount (Sousa et al., 1996, 1998; and Samuelson et al., 2000). The method is more effective in removing and recovering heavy metals from contamination source in comparison with the conventional method using metal-absorption microbes.

As understood based on the matters described above, in order to display foreign protein on cell surface, a suitable surface protein and foreign protein must be linked each other in gene level to express fusion protein, and the fusion protein should pass stably across inner membrane of cell to be attached to cell surface.

Preferably, the surface protein having the following characteristics is recommended

as surface display motif: 1) existence of secretory signal enabling passage across inner membrane of cell, 2) existence of target signal enabling stable attachment to cell surface, 3) high expression level on cell surface, and 4) stable expression regardless of protein size (Georgiou et al., 1993). Meanwhile, according to the existing surface display methods described above, the motif for surface display is required to genetically modified in order to incorporate a protein of interest to N- or C-terminal, or central region of surface protein. All the proteins surface-displayed is expressed in a fusion form with surface display motif. Therefore, the resulting protein surface-displayed is a modified protein rather than wild type protein.

Up to date, the developed surface display systems are as follows: phage surface display system (Chiswell and McCarferty, 1992), bacterial surface display system (Georgiou et al., 1993; Little et al., 1993; and Georgiou et al., 1997), surface display system of Gram negative bacteria (Francisco et al., 1992; Fuchs et al., 1991; Klauser et al., 1990, 1992; and Hedegaard et al., 1989), surface display system of Gram positive bacteria (Samuelson et al., 1995; Palva et al., 1994; and Sleytr and Sara, 1997), and surface display system of yeast (Ferguson, 1988; and Schreuder et al., 1996). Furthermore, it has been developed that a protein of interest fused to spore coat protein is displayed on spore surface. For example, U.S. Pat. No. 5,766,914 discloses a method of producing and purifying enzymes using fusion protein between cotC or cotD among spore coat proteins of Bacillus subtilis and lacZ as reporter. U.S. Pat. Nos. 5,837,500 and 5,800,821 also indicate cotC and cotD as a preferable surface display motif, but its experimental demonstrations are not described. According to a surface display system of Gram negative bacteria, the incorporation of foreign polypeptide into surface structure results in not only its steric limitation which makes it impossible to have stable membrane protein (Charbit et al., J. Immunol, 139:1644-1658(1987); and Agterberg et al., Gene, 88:37-

45(1990)) but also drop of the stability of cell outer membrane and its viability. £. coli as display host, which has been intensively studied, uses generally cell outer membrane protein as surface display motif. However, the over-expression of cell outer membrane protein fused to foreign protein is likely to bring about structural instability of cell outer membrane, consequently, diving the viability of host cell (Georgiou et al., 1996).

The problems in the conventional display methods described above, is due to preparation of fusion protein between a protein of interest and surface display motif for display. Where the fusion protein is expressed in small amount, the reaction efficiency in whole cell bioconversion, protein array and antibody production is decreased; if overexpressed, it is very likely to lead to the shortcomings mentioned above. In addition, the surface display methods using the fusion protein are depended on the extent of incorporation of surface display motif into cell, spore or phage surface, giving rise to limitation of the amount of protein displayed. As described above, the conventional surface display technology is fundamentally dependent on formation of fusion protein between a protein of interest and surface display motif. Consequently, there occur several shortcomings in conventional surface display systems: (1) necessity of getting knowledge of a gene sequence of surface display motif; (2) necessity of cloning a gene of surface display motif; (3) being very likely to affect the tertiary structure of a protein of interest by surface display motif; (4) rendering a protein of interest inactive when a protein of interest is active only in multimeric form and a fusion protein is independently surface-displayed; (5) limitation of the amount of protein displayed since the surface display methods using the fusion protein are depended on the extent of incorporation of surface display motif into host cell surface; (6) inducing a structural instability of host cell surface when a protein of interest is surface displayed in excess, thereby dropping resistance to environment and viability of host cell.

Consequently, for developing novel surface display system, which is capable of overcoming the shortcomings in conventional methods, the following characteristics should be accomplished: (1) being capable of constructing system without knowledge on a gene sequence of surface display motif; (2) being capable of constructing system without cloning a gene of surface display motif; (3) being capable of displaying a protein of interest on host cell surface after forming a inherent structure thereof; (4) being capable of increasing an amount of protein surface-displayed by means of nonselective linkages; and/or (6) not reducing a resistance to environment and a viability of host cell even when a protein of interest is surface displayed in excess.

Throughout this application, various patents and publications are referenced and citations are provided in parentheses. The disclosure of these patents and publications in their entities are hereby incorporated by references into this application in order to more fully describe this invention and the state of the art to which this invention pertains.

DETAILED DESCRIPTION OF THIS INVETNION

The present inventors have made intensive researches to develop novel methods for surface display of proteins, sugars or nucleic acid molecules, particularly proteins in more convenient and effective manner. As a result, we have discovered that contacting substances of interest to suitable surface donors permits to display substances of interest on the surface of surface donors in much higher efficiency.

Accordingly, it is an object of this invention to provide a method for an intact surface display.

It is another object of this invention to provide a method of a bioconversion using a biocatalyst.

It is still another object of this invention to provide a method for producing an antibody binding specifically to an antigen in vertebrates.

It is further object of this invention to provide a method for preparing a whole cell adsorber. It is still further object of this invention to provide a method for preparing a protein microarray.

Other objects and advantages of the present invention will become apparent from the detailed description to follow taken in conjugation with the appended claims and drawings.

In one aspect of this invention, there is provided a method for an intact surface display, which comprises the steps of:

(a) preparing a surface donor selected from the group consisting of wild type spores, bacteria and yeasts , wherein the surface donor is not genetically modified; and

(b) attaching a substance of interest on the surface of the surface donor by contacting the substance of interest to the surface donor, wherein the substance of interest is proteins, sugars or nucleic acid molecules. In another aspect of this invention, there is provided a method of a bioconversion using a biocatalyst, which comprises the steps of:

(a) preparing a surface donor selected from the group consisting of wild type spores, bacteria and yeasts , wherein the surface donor is not genetically modified; (b) performing an intact surface display of a protein of interest on the surface of the surface donor by contacting the protein of interest for the bioconversion to the surface donor;

(c) recovering the surface donor displaying on its surface the protein of

interest; and (d) performing the bioconversion reaction using the surface donor displaying on its surface the protein of interest as the biocatalyst. In still another aspect of this invention, there is provided a method for producing an antibody binding specifically to an antigen in vertebrates, which comprises the steps of:

(a) preparing a surface donor selected from the group consisting of wild type spores, bacteria and yeasts, wherein the surface donor is not genetically modified; (b) performing an intact surface display of an antigen on the surface of the surface donor by contacting the antigen to the surface donor;

(c) recovering the surface donor displaying on its surface the antigen; and

(d) producing the antibody binding specifically to the antigen by injecting to vertebrates a composition containing an immunologically effective amount of the spore displaying on its surface the antigen.

In further aspect of this invention, there is provided a method for preparing a whole cell adsorber, which comprises the steps of:

(a) preparing a surface donor selected from the group consisting of wild type spores, bacteria and yeasts, wherein the surface donor is not genetically modified;

(b) performing an intact surface display of a protein having affinity for a substance of interest on the surface of the surface donor by contacting the protein to the surface donor;

(c) recovering the surface donor displaying on its surface the protein; and (d) immobilizing onto a carrier the surface donor displaying on its surface the protein.

In still further aspect of this invention, there is provided a method for preparing a protein microarray, which comprises the steps of:

(a) preparing a surface donor selected from the group consisting of wild type spores, bacteria and yeasts, wherein the surface donor is not genetically modified;

(b) performing an intact surface display of an antigen or an antigen having affinity for a protein of interest on the surface of the surface donor by contacting the antibody or the antigen to the surface donor;

(c) recovering the surface donor displaying on its surface the antibody or the antigen; and

(d) immobilizing onto a solid-phase surface the spore displaying on its surface the antibody and the antigen.

The present inventors have made intensive researches to develop novel methods for surface display of proteins, sugars or nucleic acid molecules, particularly proteins in more convenient and effective manner. As a result, we have discovered that contacting substances of interest to suitable surface donors permits to display substances of interest on the surface of surface donors in much higher efficiency. Even though the process of the present invention for surface display is relatively convenient and simple, it gives rise to unexpected results to one of skill in the art.

The present invention relates to a method for a surface display of the substance of interest, in particular to a method for the intact surface display of the substance of interest. The term used herein "method for the intact surface display" refers to displaying the substance of interest on the surface of the surface donor not transformed genetically (for examples, spores, bacteria or virus) and used as a coined term to concisely address a novel approach of the present invention. In addition, the intact surface display of the present invention comprises no transformation processes. Preferably, the intact surface display comprises no chemical and genetic modifications of the surface donor for the surface display. For instance, the Korean Pat. No. 522,398 filed by the present inventors discloses a

method for a surface display using coat proteins, essentially including transformant production steps with a vector to express fusion proteins of spore coat proteins and proteins of interest for the surface display. According to another method proposed by the present inventors (see Korean Pat. Appln. No. 2001-0002156), it includes inevitably transformation of gene carrier-containing host cells with vectors carrying genes encoding proteins of interest. In contrast, the surface donor used in the present invention is not transformed for the surface display, which is one of the most distinguished features over known methods to one of skill in the art.

According to the present invention, the substance of interest, especially the protein of interest is displayed on the surface of the surface donor. The term used herein "surface donor" refers to a subject binding to the substance of interest, particularly, a supporting material to provide the surface with the substance of interest for stable binding. The surface donor includes spores, virus, bacteria and yeasts. Preferably, the surface donor includes spores and bacteria (Gram positive bacterium and Gram negative bacterium), more preferably, spores. The spore has a following advantages (Driks, 1999): 1) a higher heat stability, 2) a significant stability to radioactivity, 3) a stability to toxins, 4) a higher stability to acid and base, 5) a significant stability to lysozyme, 6) a resistance to dryness, 7) a higher stability to organic solvents, 8) no metabolic activity, and 9) shorter time for obtaining spore, e.g. within several hours.

For using spores as the surface donor in this invention, preferably, spores are originated from spore-forming Gram negative bacterium including Myxococcusr, a spore-forming Gram positive bacterium including Clostridium, Paenibacillus and Bacillus; a spore-forming Actionmycete; a spore-forming yeast including Saccharomyces cerevisiae, Candida and Hansenula and a spore-forming fungus, but not limited to. More preferably, spores are originated from a spore-forming Gram positive bacterium, still more preferably, Bacillus, most preferably Bacillus subtilis.

In the present method, the surface donor may be prepared by the

conventional methods known to one of skill in the art, for instance, renografin gradients methods (C. R. Harwood, et al., "Molecular Biological Methods for Bacillus." John Wiley & Sons, New York, p.416(1990)). Viruses, bacteria and yeasts may be obtained by a variety of conventional culture methods known to one skilled in the art. Detailed descriptions on culture and fermentation of microorganisms are disclosed in Kubitschek, H. E., Introduction to Research with Continuous Cultures. Engle wood Cliffs, NJ.: Prentice-Hall, Inc., 1970; Mandelstam, J., et al., Biochemistry of Bacterial Growth, 3 ^rd ed. Oxford: Blackwell, 1982; Meynell, G. G., et al., Theory and Practice in Experimental Bacteriology, 2 ^nd ed. Cambridge: Cambridge University Press, 1970; Gerhardt, P., ed., Manual of Methods for General Bacteriology, Washington: Am. Soc. Microbiol, 1981, teachings of which are herein incorporated by references in their entities.

Although a natural-occurring surface donor is useful in the present invention, the surface of the surface donor may be chemically modified to permit the surface display more effectively. Preferably, the surface of the surface donor is chemically modified with a cross-linking agent for proteins. For instance, the surface of the surface donor is chemically functionalized to contain aldehyde group (-CHO) or epoxide group, such that it is allowed to have very high chemical reactivity for immobilization by covalent bonds with the substance of interest (for example, proteins). Where the surface of the surface donor is chemically modifed, any type of cells such as spores, viruses, bacteria and yeasts may be used as the surface donor. Preferably, spores are used because they maintain their integrity under conditions for extreme chemical reactions.

The substance of interest is displayed on the surface of the surface donor by contacting the substance of interest to the surface donor prepared as described above. The substance of interest displaying on the surface includes, but not limited to, proteins, polypeptides, peptides, carbohydrates (monosaccharide and polysaccharide) and nucleic acid molecules (DNA and RNA). Preferably, the

substance for the surface display is proteins. For example, the protein includes enzyme, enzyme inhibitor, hormone, hormone analogue, hormone receptor, signal transduction protein, antibody, monoclonal antibody, antigen, attachment protein, structural protein, regulatory protein, toxin protein, cytokine, transcription regulatory protein, blood clotting protein, plant defense-inducing protein and fragments thereof. The proteins include multimer as well as monomer. The surface display of multimeric proteins has been rarely reported, for instance, the surface display of alkaline phosphatase in E. coli, has resulted the display toward inner part of cell outer membrane (Stathopoulus et al., 1996). β-galactosidase used as reporter enzyme in Examples of the present invention must form tetramer to exhibit its activity and has not been published to be successful in surface display, β-galactosidase generally cannot pass across cell membrane and comprises an amino acid sequence detrimental to cell membrane, as a result, the fusion protein between surface display motif and β-galactosidase has been recognized not to be displayed on cell surface. Therefore, the surface display of β-galactosidase described in Examples proves to be surprising.

The term used herein "protein" refers to molecule consisting of peptide bond, for example including oligopeptide and polypeptide.

According to the present method, the substance of interest or the protein of interest not be modified physically and chemically is effectively displayed on the surface donor. Preferably, the substance of interest or the protein of interest could be fused with spore-specific peptides or modified chemically to increase the efficiency of the surface display. The spore-specific peptides fused with the substance of interest or the protein of interest include NHFLPKV, SpsC NHFLP, SpsA NHFYP, CotA THFLP, SLLPGLP and ATYPLPIR, but not limited to. The substance of interest or the protein of interest may be chemically modified by incorporating functional groups with higher reactivity such as aldehyde group or epoxide group.

According to a preferred embodiment, the step of contacting the substance of

interest or the protein of interest to the surface donor is performed by contacting physically the substance of interest or the protein of interest to the surface donor. More preferably, the step of contacting the substance of interest or the protein of interest to the surface donor is performed by agitating the mixture of the surface donor and the protein of interest.

The substance of interest or the protein of interest is bounded non-covalently to the surface of the surface donor. In particular, the non-covalent bindings are formed by hydrophobic interaction, ionic interaction, hydrogen bond and Van der Waals force or combinations thereof. Although the present methods is fundamentally directed to surface display via noncovalent bond between the surface donor and proteins of interest, additional covalent bond may be used, if necessary for more stabilized linkage. The stabilizing the bond between the surface of the surface donor and the protein of interest can be performed by means of forming covalent bonds to between the surface of the surface donor and the protein of interest by use of physical, chemical or biochemical methods following displaying the protein of interest on the surface of the surface donor via noncovalent bond. Among the methods to form covalent bond, a treatment of glutaraldehyde (DeSantis G. and Jones J. B. Curr. Opin. Biotechnol. 10:324-330(1999)) is preferable as chemical method, a treatment of ultraviolet (Graham L., and Gallop P.M. Anal. Biochem. 217:298-305(1994)) is preferable as physical method and a treatment of enzyme ensuring formation of covalent bond (Gao Y, and Mehta K., J. Biochem. 129:179-183(2001)) as biochemical method.

By means of these simple processes described above, the protein of interest could be displayed on the surface donor stably and effectively. As described in Example, the efficiency of the protein surface display in the present method is tens to hundreds times higher than those of the known display methods.

According to a preferred embodiment, the present method further comprises the step of contacting to the surface donor a peptide or protein with a non-specific

affinity to proteins before the step of contacting of the substance of interest or the protein of interest to the surface donor. This pre-treatment process permits more proteins to display on the surface to the surface donor.

The protein of interest displayed on spore surface according to the present methods can be demonstrated with a wide variety of methods as follows: 1) A primary antibody is bound to the protein of interest displayed on the surface donor and then reacted with a secondary antibody labeled with fluorescent chemical to stain the surface donor, followed by observation with fluorescence microscope or analysis with flow cytometry. 2) The protein of interest displayed on the surface donor is treated with protease, followed by measurement of the activity of the protein or detecting lower signal with fluorescence microscope or flow cytometry. 3) In case that the protein of interest uses a substrate with higher molecular weight, the direct measurement of the activity of the protein can provide the level of display since the substrate cannot pass across outer coat of the surface donor. In the meantime, the bioconversion process using surface-displayed enzymes requires a physiochemical stability of the surface donor in extreme conditions because the process is usually executed in high temperature and/or organic solvent. In particular, a chemical synthesis valuable in current industry is mainly carried out in organic solvent and the synthesis of chiral compound or the resolution of racemic mixture is also performed in highly severe physiochemical conditions. Therefore, the surface-displayed enzyme as well as the organisms displaying enzyme is compelled to have stability under such extreme conditions. In this connection, it is demonstrated that the methods for bioconversion using system for spore surface display is largely advantageous. The chemical processes using surface-displayed enzymes have been proposed

(Georgiou et al., 1993). However, the proposed processes have generally required immobilization of cell surface with cross-linking agent since the host displaying enzyme is very unstable during process (Freeman et al., 1996). The present

bioconversion process is free from the disadvantage mentioned above. Because the surface-displayed enzyme as well as the host displaying enzyme is largely stable, the present method avoids the immobilization.

Enzymes used in the bioconversion process of the preset invention include any type of enzyme such as oxidoreductase, transferase, hydrolyase, lyase, isomerase and ligasse. For example, any protein {e.g., β-galactosiase, lipase, protease, cellulase, glycosyltransferase, oxidoreductase and aldolase) displayed on the surface of the surface donor is useful in the present invention. In addition, the present method is useful in a single step or multi-step reaction and in aqueous or non-aqueous solution. The present bioconversion method employs the surface donor as a free or immobilized form.

Similar to DNA microarray, a protein microarray provides means for analyzing expression or expression level of target protein in certain cell. In order to fabricate protein array, the suitable proteins to be arrayed must be obtained and then immobilized on solid surface. During analysis using protein array, washing step is necessarily performed to remove unbound proteins and various treatments such as high temperature, higher salt concentration and pH adjustment are executed; therefore, it is pivotal to guarantee proteinaceous substance with higher stability under such detrimental environment. In addition, the conventional process for preparing protein array needs tedious and repetitive works such as cloning genes of several thousands to tens of thousands of proteins and immobilizing of the proteins expressed. Therefore, there remains a need to improve simplicity and rapidity of the works.

According to the method for preparing protein microarray of this invention, it is ensured that the works described-above can be performed with much greater readiness. According to the present invention, the surface donor with proteins of interest on its surface is immobilized on solid substrates. In the method for preparing protein array, the conventional steps may be used (see WO 0061806, WO

0054046, US 5807754, EP 0818467, WO 9742507, US 5114674 and WO 9635953). The protein microarray manufactured by the present invention has a variety of applicable fields including diagnosis, analysis of gene expression, analysis of interaction between proteins, analysis of interaction between protein and ligand, study on metabolism, screening novel or improved enzymes, combinatorial biochemical synthesis and biosensor.

The solid substrate suitable in the present method includes, but not limited to, glasses (e.g., functionalized glasses), Si, Ge, GaAs, GaP, SiO, SiN ₄ , modified silicone nitrocellulose, polyvinyl idene fluoride, polystylene, polytetrafluoroethylene, polycarbonate, nylon, fiber and combinations thereof. The surface donor optionally may be attached to the array substrate through linker molecules. It is preferred that the regions of the array surface not being spotted are blocked. The amount of the surface donor applied to each spot (or address) depends on the type of array. Interaction between the protein displayed on the surface donor attached to solid substrate and the sample applied can be detected based on their inherent characteristics (e.g., immunogenicity) or can be rendered detectable by being labeled with an independently detectable tag (e.g., fluorescent, luminescent or radioactive molecules, and epitopes). The data generated with protein array of this invention can be analyzed using known computerized systems such as "reader" and "scanner".

According to the method producing an antibody of this invention, a composition containing an immunologically effective amount of the surface donor, preferably, further comprises adjuvant such as incomplete and complete Freund's adjuvants. In the present method, the mode of administration is, preferably, injection and more preferably, intravenous, intraperitoneal, subcutaneous and intramuscular injections. Boosting within suitable period after the first administration is preferable to yield a sufficient amount of antibody.

Meanwhile, antibodies or polypeptides used in adsorption chromatography should be produced, purified and immobilized on a carrier. Generally, it is very difficult to prepare the bioadsorbers. The disadvantage may be overcome using whole cell displaying protein as described in Georgiou et al., 1997. Therefore, the system for the surface donor display of this invention provides a whole cell adsorber to solve the problems of the known adsorbers.

The features and advantages of the present invention are summarized as follows: (a) the present invention uses a unmodified surface donor with no genetic manipulations or introduction of foreign genes for the surface display.

(b) the present invention needs no transformation for the surface display.

(c) the substance of interest, especially the protein is displayed on the surface with much higher efficiency in the present method. (d) according to the present invention, the protein activity displayed on the surface retains with little or no loss.

(e) the present method for the surface display is carried out in a very simple manner.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 schematically illustrates the intact surface display of the present invention.

Fig. 2 represents the result of the surface display of β-galactosidase fused with spore-specific fusion peptides on the Bacillus subtilis spore-surface by the present invention.

Fig. 3 shows the surface display result of β-galactosidase on the spore-surface of Bacillus coagulans by the present invention.

Fig. 4 represents the graph to show the standard curve for /7-nitrophenol in 0.1 M potassium phosphate buffer with various pH values.

Fig. 5 represents the measuring results of the amount and activity of CaIB immobilized on the surface of the Bacillus subtilis spore depending on concentrations of CaIB.

Fig. 6 shows results of the immobilization of CaIB on Bacillus subtilis spores with varying adsorption time.

Figs. 7a-7b represent the measured amount and activity (measured using p- NPB) of CaIB immobilized on various Bacillus species spore-surfaces. Fig. 8 represents the measured amount and activity (measured using _/ P-NPP) of CaIB immobilized on various Bacillus species spore-surfaces.

Fig. 9 shows the amount of CaIB displayed on the surface of the Bacillus subtillis spore analyzed by the flow cytometry.

Fig. 10 represents results of (a) Coomassie staining and (b) Western blot for analyzing binding types of CaIB immobilization. Spores or Spore with immobilized

CaIB were treated with extraction solvents for desorption and then subjected to 10%

SDS-PAGE: M, pre-stained marker; lane 1, spore treated in IM NaCI; lane 2, CaIB coated spore treated in IM NaCI; lane 3, spore treated in IM NaCI in 0.1M KPi (pH

6.0); lane 4, CaIB coated spore treated in IM NaCI in 0.1 M KPi (pH 6.0); lane 5, spore treated in 10% formic acid in 45% CH ₃ CN; lane 6, CaIB coated spore treated in 10% formic acid in 45% CH ₃ CN.

Fig. 11 shows measurement results of the enzyme activity of CaIB at each pH.

Fig. 12 represents experimental result for verifying whether spores and CaIB enzymes were bound by charge interactions using desorption experiments with varying pi values.

Figs. 13a-13b show analysis results for (a) the activity for /3-NPB and (b) immobilization of the CaIB enzyme on the pre-treated various Bacillus spore-surfaces.

Hg. 14 represents analysis results for the activity for /J-NPP and immobilization of the CaIB enzyme on the pre-treated various Bacillus spore-surfaces. Fig. 15 represents results of esterification using CaIB immobilized on Bacillus subtilis spore-surface: lane 1, Oleic acid (Substrate, 200 mM); lane 2, B. subtilis spore (blank, 1.6 mg of spores); lane 3, Novozyme 435 (38 mg of beads); lane 4, Pre-treated B. subtilis spore, CaIB coated (0.25 mg of CaIB); lane 5, Wild-type B. subtilis spore, CaIB coated (0.45 mg of CaIB); lane 6, Free-form CaIB (1.25 mg of CaIB); lane 7, Ethyl oleate (product, 100 mM); lane 8, Ethyl oleate (200 mM); lane 9, Ethyl oleate (500 mM). Fig. 16 represents results of esterification using CaIB immobilized on various

Bacillus spore-surface: lane 1, B. subtilis spore (blank, 5 mg of spores); lane 2, Novozyme 435 (25 mg of beads); lane 3, Wild-type B. subtilis spore, CaIB coated (0.2 mg of CaIB); lane 4, Wild-type B. thuringiensis spore, CaIB coated (0.2 mg of CaIB); lane 5, Wild-type B. megaterium spore, CaIB coated (0.2 mg of CaIB); lane 6, Wild-type B. mensentericus spore, CaIB coated (0.2 mg of CaIB); lane 7, Wild-type B. licheniformis spore, CaIB coated (0.2 mg of CaIB); lane 8, Free-form CaIB (0.2 mg of CaIB); lane 9, Ethyl oleate (product, 200 mM).

The present invention will now be described in further detail by examples. It would be obvious to those skilled in the art that these examples are intended to be more concretely illustrative and the scope of the present invention as set forth in the appended claims is not limited to or by the examples.

EXAMPLES

EXAMPLE I: Bacillus subtilis non-GMO intact spore-surface display of β- galactosidase

1-1. Isolation of spores

Bacillus substilis DB104 strain (Kawamura F. and Doi R. H., J. Bacteriol., 160:442-444(1984)) was cultured for 24 hr at a shaking incubator (37 ⁰ C, 250 rpm) in GYS medium (2 g/L (NH ₄ ) ₂ SO ₄ , 2 g/L yeast extract, 0.5 g/L K ₂ HPO ₄ , 1 g/L glucose, 0.41 g/L MgSO ₄ ^■ H ₂ O, 0.08 g/L CaCI ₂ ^• 2H ₂ O, and 0.07 g/L MnSO ₄ ^• 5H ₂ O), and the only pure spores were isolated using renografin gradients method (C. R. Harwood, et al., "Molecular Biological Methods for Bacillus." John Wiley & Sons, New York, p.416 (1990)).

1-2. Attachment of β-qalactosidase to spore-surface The absorbance values of the spore suspension isolated in the above Example were measured at 580 nm and assumed the number of spores (0.3 OD _{580 nm} = 2 x 10 ⁷ spore/ml). Spores were suspended in 10 mM sodium phosphate buffer (pH 7.2) at the concentration of 2 x 10 ⁹ spores/ml. E co/Z-derived purified β-galactosidase (Sigma-Aldrich, USA) was dissolved in 10 mM sodium phosphate buffer (pH 7.2) at the concentration of 400 μg/ml and mixed with equal volume of spore suspension, and then they were cultured in a shaking incubator at 37 ⁰ C. After 30-min reaction, proteins not attached to spores were rinsed with 3 volumes of 10 mM sodium phosphate buffer (pH 7.2) and then suspended at the concentration of 2 x 10 ⁹ spores/ml. The activity of Bacillus spore-surface displayed β-galactosidase was measured by Miller method (Miller, " Experiments in Molecular Genetics", Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, p. 352-355(1972)). As a result, it could be understood that about 5.4 x 10 ⁵ molecules of β-galactosidase were attached to a spore, considering the specific activity and molecular weight of β-galactosidase (for tetrameric β-galactosidase).

The results of the intact surface display as the present invention were compared with those of known spore display technologies, "Method for Expression of Proteins on Spore Surface" using CotG motif (see Korean Pat. No. 522,398) and

"Method for Surface Display of Proteins on Genetic Carriers" (see Korean Pat. Appln. No. 2001-0002156).

As shown in Fig. 1 and Table 1, it could be measured that the number of β- galactosidase displayed per spore was tens to hundreds times larger than those of the known display methods as described above. TABLE 1

1-3. β-qalactosidase display after removing spore-surface binding proteins

When spores were isolated, their surfaces might have various proteins and peptides non-specifically bound. Therefore, β-galactosidase was displayed under the same conditions as Example 1-2 after removing peptides and proteins bound non- covalently and non-specifically on spore surface.

Bacillus spores were suspended with distilled water at the concentration of 4 x 10 ⁸ spore/ml and 0.1 M CAPS buffer (pH 11) was added, followed by sonication on ice (30% amplification, for 3 min). Spores were then rinsed three times with 10 volumes of 10 mM sodium phosphate buffer to remove free peptides and proteins.

Afterwards, β-galactosidase was displayed under the same conditions as Example 1-2 and its number attached to a spore was measured to be about 5.2 x 10 ⁴ . Such spore-display potential was about ten times lower than those of spore- display using surfaces with non-covalently and non-specifically bound peptides and proteins, demonstrating peptides and proteins present on isolated spores would be responsible in part for spore display of proteins of interest.

Therefore, the optimization of the isolation method for spores would increase the number of proteins displayed on spore-surfaces. Furthermore, it could be appreciated that pre-attachment of non-specific protein-affinity peptides and proteins on spores could increase the number of proteins of interest displayed on

spore-surfaces. Alternatively, there could be provided another strategy in which antibodies or other proteins binding specifically to a target protein are first expressed on spore surface and then the target protein is bound to with them in outside of spores, elevating the spore binding ability and the specificity.

1-4. Display of β-qalactosidase with spore-specific binding peptides

As demonstrated in Example 1-3, the efficiency of the surface display could be elevated by non-genetic modifications to alter spore surface. We contemplated that modifications of target proteins permit the efficiency of the surface display to increase. For instance, target proteins could be displayed more effectively on Bacillus spore-surface, if peptides having spore-specific attachment ability as shown in Table 2 were fused with target proteins (Turnbough Jr., C.L., Journal of Microbiological Methods 53(2): 263-271(2003)). In this Example, β-galactosidase fused with NHFLPKV at its N-terminal was expressed and partially purified, β-galactosidase activity was measured after binding to Bacillus subtilis DB104-derived spores as prepared in Example 1-1. Fig. 2 represents the comparison result of the β- galactosidase activity displayed on the spore-surface with that of CotG-β-gal surface display method (see Korean Pat. No. 522,398). The surface display technology of the present invention was revealed to be 6 times more effective than the CotG-β-gal method. Accordingly, it could be appreciated that the efficiency of the surface display could be elevated by modification of target proteins. In addition, chemical modifications of target proteins with little or no activity loss could increase an efficiency of the spore-surface display of the present invention in various manners. TABLE 2 Spore-specific binding peptides

EXAMPLE II: Bacillus coagulans spore-surf ace display of β-galactosidase

Bacillus coagulans is a reclassified spore-forming lactobacillus which was one of lactobacillus known as Lactobacillus sporogenes. Currently, its spores per se are used most widely as tonic medicines because they have excellent tonic effect.

However, where genetically modified microbes are used to improve their functions, it is necessary to provide a lot of safety data for product approval. In this regard, utilizing both the non-GMO surface display method of the present invention and merits of Bacillus coagulans for function improvement makes it possible to employ the microbe in more various fields. The present inventors identified that β- galactosidase to catalyze galactose degradation could be displayed on the surface of

Bacillus coagulans without any genetic modification as follows:

II- 1. Isolation of spores Spores for the surface display of β-galactosidase were cultured and isolated purely in the same manner as Example I.

II-2. Intact spore-surface display of β-qalactosidase

Isolated spores were suspended in 1 ml of PBS buffer to a density to be and incubated with 0, 100 or 500 units of E co/Z-derived purified β- galactosidase (Sigma-Aldrich, USA) at room temperature for surface display. As shown in Fig. 3, 90% and 56% of enzyme activity were found on spore surface where 100 and 500 units of β-galactosidase were added, respectively.

EXAMPLE III: Bacillus subtilis spore-surface display of lipase B (CaIB) IH-I. Strains and enzymes

To immobilize lipase B (CaIB) on Bacillus spores as carriers, B. subtilis (KCTC 3135), B. thuringiensis (KCTC 1034), B. megaterium (KCTC 3007), B. mensentericus (ATCC 945), B. licheniformis (KCTC 1918) or Paenibacillus polymyxa (KCTC 3627) were cultured and their spores were used after purification. Enzyme for immobilization was Candida antarct/ca-derWed CaIB (Genofocus, Inc., Korea).

3-2. Isolation of spores and characterization

DSM media were used to culture each strain. 1 L of DSM media was prepared as follows: 8 g/L bacto-nutrient broth, 1 g/L KCI and 0.12 g/L MgSO ₄ ^• 7H ₂ O were mixed well and then 0.5 mM NaOH were added up to pH 7.6, followed by autoclave. 1 mM Ca(NO ₃ ) ₄ , 0.01 mM MnCI ₂ and 0.001 mM FeSO ₄ were added to the resultant. Each strain was inoculated into 200 ml of DSM media and cultured for 72 hr at a shaking incubator (30 ⁰ C, 200 rpm) to form spores. The only pure spores were isolated by renografin (Sigma, USA) gradient method. Dry weight of each Bacillus spore used as a carrier was measured using the HB43-S moisture analyzer (Mettler- Toledo AG, Laboratory & Weighing Technologies, Swiss). Spores isolated were analyzed to measure OD values and then diluted in third distilled water to measure their dry weight. As shown in Table 3, the dry weight of Bacillus subtillis spore (OD ₅₀₀ =I) was the smallest (0.19 mg/ml), whereas that of Bacillus megaterium spore was the highest (0.93 mg/ml). TABLE 3 Dry weight of spores

III-3. Measurement of the activity and concentration of CaIB

The concentration of lipase B (CaIB) used in the immobilizing reaction was measured by a Bradford method (Bradford, MM. "A rapid and sensitive for the quantitation of microgram quantitites of protein utilizing the principle of protein-dye binding" . Analytical Biochemistry!!: 248-254. (1976)). To obtain a standard curve, 1 mg/ml of bovine serum albumin (BSA) were used. 0, 4, 8, 12, 16 and 20 μl of BSA stock (1 mg/ml) were added to 1.5 ml tube, respectively, and third distilled water was added up to 800 μl. 200 μl of protein quantification reagent (Bio-Rad Laboratories, Inc., USA) were mixed with the resulting solution. After 5 min, the OD values at 595 nm were measured using a UV-spectrophotometer. The equation was obtained using a standard curve by Sigma-Plot program (y=0.05439x + 0.04964).

Protein quantification of unknown samples was performed in the same manner as describe above and the amount of each protein was calculated according to the equation.

CaIB enzyme activity was measured according to two methods: either p- nitrophenyl butyrate or />nitrophenyl palmitate as a chromogenic substrate was used and />nitrophenol in the free form was measured with a spectrophotometer; and oleic acid and ethanol as substrates were used and ethyl oleate generated was analyzed by a thin-layer chromatography (TLC). 980 μl of 0.1 M potassium phosphate buffer (pH 7.4) and 5 μl of 100 nM /?-NPB (or 10 μl of 10 mM /HMPP) were mixed. 20 μl of enzyme solution diluted (1000-fold dilution for the free form, 10-fold dilution for the immobilized form) were added and reacted for 10-20 min (10 min for /7-NPB hydrolysis, 20 min for /?-NPP hydrolysis) at 37 ⁰ C and the reaction was stopped by adding 1 ml of -2O ⁰ C stored acetonitrile (Burdick & Jackson Co., USA). 15 μl of 1 M NaOH were added into each sample to adjust pH and centrifuged. 1 ml of the supernatant was added in a cuvette and the OD value was measured at 405 nm. To give a standard curve, 100 mM p-nitrophenol stock were used. 0.1, 0.2, 0.5,

1, 2, 5, 10, 20 and 50 μM of the /7-nitrophenol stock were added, respectively, and 0.1 M potassium phosphate buffer (with pH 4.2, 6.0, 7.4 and 9.0) was added up to the final volume of 1 ml, followed by addition of 1 ml acetonitrile. For pH 4.2, 100 μl of 1 M K ₂ HPO ₄ (pH 9.0) and 15 μl of 1 M NaOH were sequentially added to adjust pH, followed by centrifugation for measuring the OD values at 405 nm. The standard curve for the CaIB activity at each pH in 0.1 M potassium phosphate buffer was given using the Sigma-Plot program (Fig. 4).

III-4. Immobilization of CaIB with varying enzyme concentrations To immobilize CaIB on Bacillus subtilis spores, the method for adsorbing enzyme onto carriers was used. To determine the optima! enzyme concentration for displaying CaIB on spores, the concentration of Bacillus subtilis spores as carriers, was fixed to 20 mg and CaIB (0.1, 0.2, 0.5, 1, 2 and 5 mg, respectively) was added. 0.1 M potassium phosphate buffer (pH 7.4) was added to the total volume of 3 ml. Each sample was reacted in a shaking incubator for 1 hr at room temperature and spores were recovered by centrifugation. Carriers immobilized were rinsed repeatedly in 50 ml of 0.1 M potassium phosphate buffer (pH 7.4). The activities of CaIB immobilized on carriers, used at the initial immobilization, contained in supernatant recovered by centrifugation and contained in the washing solution were measured with both spectrophotometer and TLC as described above. The protein quantification was performed by a Bradford method and the CaIB protein immobilized on spores was quantified as follows (Fig. 5 and Table 4):

(The amount of the CaIB protein immobilized on spores) - (The amount of CaIB used at the initial immobilization) - (The amount of CaIB contained in the supernatant ecovered by centrifugation) - (The amount of CaIB contained in the washing solution)

TABLE 4

Amount and activity of the CaIB enzyme immobilized on Bacillus subtilis spores depending on concentrations of CaIB

Condition: 0.1 M potassium phosphate buffer (pH 7.4); total volume was 3 ml, shaking incubation at a room temperature for 1 hr at 150 rpm. ^b It was measured by p-NPB hydrolysis reaction. Recovered activity = [(Total activity of CaIB bound to carriers after coating for ,cH\IPB)/(Total activity of free CaIB used in coating for p-

NPB)] x 100. ^c Catalytic efficiency was determined by the specific activity ratio of free-form and immobilized

CaIB, and the specific activity of free CaIB for /7-NPB was 93 U/mg protein. Catalytic activity =

[(Activity of immobilized CaIB mg protein for _/ σ-WPB)/(Activity of free CaIB mg protein for p-

NPB)] x 100.

III-5. CaIB immobilization on Bacillus subtilis spores depending on adsorption time

Immobilization reaction was optimized by determining period of time for adsorption of the CaIB enzyme onto Bacillus subtilis spores. 200 mg of spores and 20 mg of CaIB were added in 50 ml tube and 0.1 M potassium phosphate buffer (pH 7.4) was added to the total volume of 30 ml. The solution was incubated in a shaking incubator for 1 hr at room temperature and samples in 3 ml were harvested at five-min intervals. After 15 min incubation, samples were taken at the interval of 15 min and centrifuged for harvesting. After washing repeatedly with 50 ml of the above buffer, the activities of CaIB molecules immobilized on carriers, contained in the supernatant and the washing solution were measured using /7-NPB as substrates. The amount of each protein was analyzed by a Bradford method as described above. The optimal time for adsorbing CaIB onto Bacillus subtilis spores was determined as 1 hr (Rg. 6).

III-6. Immobilization of CaIB depending on various Bacillus spores

To determine an optimal carrier for immobilizing CaIB, various Bacillus spores were isolated by renografin gradients method and the amount and activity of CaIB displayed to each spore were measured. 100 mg of spores and 10 mg of CaIB were added in 15 ml tube and 0.1 M potassium phosphate buffer (pH 7.4) was added to the total volume of 9.6 ml. The solution was incubated in a shaking incubator for 1 hr at room temperature and each spore immobilized CaIB was recovered by centrifugation. The activity was measured in the same manner as described above using /7-NPB and /?-NPP as substrates and the amount of immobilized protein was analyzed (Fig. 7, Fig. 8, Table 5(a) and Table 5(b)).

III-7. Fluorensce activated cell sorter (FACS ^" ) analysis

After immunofluorescence staining using Ca IB-antibody, CaIB immobilized on the spore-surface was measured using FACS (BD science, USA) to identify whether CaIB was immobilized on Bacillus subtilis spores as carriers and how many CaIB was immobilized. 0.2 mg of spores not immobilized CaIB (blank) and immobilized CaIB was added to 1.5 ml tube and were suspended by adding 200 μl of PBS buffer. 2 μl of CalB-antibody (Rabbit) were mixed with each sample and incubated for 30 min on ice. The supernatant contained remaining antibody was removed by centrifugation and the pellet was rinsed three times repeatedly with 1 ml of PBS buffer. 2 μl of fluorescein isothiocyanate(FITC)-linked secondary antibody were added to the resultant and mixed. After 30 min reaction, each carrier stained by immunofluorsence was recovered by washing as described above. Fluorosence- labeled carriers were analyzed. As a result, it was confirmed that 29.1% of CaIB were on the surface of spores (Fig. 9).

III-8. Desorption experiment to investigate where binding force CaIB is adsorbed to spore

4 ml each of (1) 1 M NaCI and extracting solvent with 10% formic acid, (2) extracting solvent with 1 M NaCI in 0.1 M potassium phosphate buffer (pH 6.0), (3) 0.1 M potassium phosphate buffer (pH 6.0) were added to 40 mg of spores immobilized CaIB with 45% acetonitril and the mixed solution was incubated at room temperature for 2 hr. The solution was centrifuged at 5,000 rpm for 10 min at room temperature to confirm how many CaIB enzymes immobilized on carriers were isolated. After centrifugation, 10% SDS-PAGE was carried out with the supernatant. And then, one gel was stained with Coomassie blue and the other was used for Western blot analysis. To confirm how CaIB is attached to spores, 0.1 M potassium phosphate buffer with extraction solvents and 1 M NaCI were added to Bacillus subtilis spores immobilized CaIB and were concentrated. Western blot analysis and SDS-PAGE were performed with this concentration. As a result, CaIB band was detected at the position of 33kD in the sample dealt with extraction solvent containing 10% formic acid with 45% acetonitrile. This result indicates that CaIB was bound to spores by hydrophobic interaction principally (Hg. 10). More understandings on how CaIB is displayed to spores as the surface donor make it practical to immobilize CaIB in much effectively manner. These experiments evidently show that hydrophobic interactions play a pivotal role in the surface display of CaIB. These results urge us to reason that pre-treatments for permitting spores to have increased hydrophobicity ensure immobilization of increased amounts of CaIB on spores.

III-9. Experiment to investigate whether CaIB is attached to spores by charge

It was known that pi value of CaIB was pH 6.0 and the spore had a negative charge generally. To desorb CaIB immobilized on spores, various pH buffers (pi) were added followed by recovering the supernatant and carriers. After the activities of CaIB were measured and it was identified how many the CaIB proteins were free from spores.

4 ml of 0.1 M potassium phosphate buffer (pH 4.3, 6.0, 7.4 and 9.0, respectively) were added in 1 g of spores and 0.015 mg of sample immobilized CaIB. The solution was incubated for 2 hr at room temperature and the supernatant was recovered by centrifugation. 50 ml of 0.1 M potassium phosphate buffer (pH 7.4) were added to pellets and spores were recovered after washing. The enzyme activity was measured using /7-NPB as substrates in order to confirm how the CaIB proteins immobilized to carriers were free from carriers.

The enzyme activity of CaIB was changed by the buffers, not by pH, i.e. pi values. CaIB activities were decreased in phosphate buffer than in Tris buffer (Rg. 11). To identify how CaIB immobilized to carriers was free from carriers depending on the pi of CaIB, though it was not a main interaction force, the charge interaction was influenced to the interaction of CaIB immobilized on Bacillus subtilis spores as carriers (Fig. 12).

III-IO. Pre-treatment process of carriers

In previous experiment, it was identified that the attachment of enzyme to spores was accomplished by hydrophobic interaction mainly. To immobilize more

CaIB proteins on spores, the surface of spore was pre-treated with the mixture of

10% formic acid and 45% acetonitrile. 500 mg of Bacillus spores prepared by renografin gradients method and the mixture of 10% formic acid and 45% acetonitrile were added in 15 ml tube. And then, the solution was incubated in a shaking incubator for 2 hr at room temperature and carriers were recovered.

Recovered carriers were rinsed with 50 ml of 0.1 M potassium phosphate buffer (pH

7.4) repeatedly and were used to the immobilization.

III-ll. Immobilization of CaIB to the pre-treated spores

CaIB was immobilized on the pre-treated spores in the same manner as described above. To determine an optimal carrier for immobilizing CaIB, various

Bacillus spores were isolated by renografϊn gradients method and the amount and activity of CaIB displayed on each spore were measured. 100 mg of various pre- treated Bacillus spores and 10 mg of CaIB proteins were added in 15 ml tube and 0.1 M potassium phosphate buffer(pH 7.4) was added to the total volume of 9.6 ml. The solution was incubated in a shaking incubator for 1 hr at room temperature followed by recovering by centrifugation in the same manner as described above. The activity was measured by the hydrolysis of p-HPB and /3-NPP as substrates and the amount of immobilized proteins was analyzed. As a result, it was shown that the immobilization efficiency and the hydrolysis reaction activities for pre-treated CaIB were higher than those for non-pretreated CaIB (Fig. 13(a), Fig. 13(b), Fig. 14, Table 5(a) and Table 5(b)). TABLE 5(a)

TABLE 5(b)

Condition: 0.1 M potassium phosphate buffer (pH 7.4); total volume was 9.6 ml, shaking incubation at a room temperature for 1 hr at 150 rpm. ^b It was measured by /HMPB and >σ-NPP hydrolysis reaction.

111-12. Esterification esterification using CaIB immobilized

Ethyl oleate was synthesized by reacting CaIB immobilized on Bacillus spores or its free-form with the mixture of oleic acid and ethanol under the constant agitation speed and the reaction temperature followed by confirmation using TLC. Ester synthesis was reacted to the total volume of 3 ml under 1,500 rpm at 37 ⁰ C in CCAIlOO reactor (EYELA, Japan). 0.5 ml of 0.1 M potassium phosphate buffer (pH 7.4), 60 mM oleic acid, 60 mM ethanol and 2.4 ml of hexane (Burdick & Jackson Co., USA) were mixed and pre-treated at 37 ⁰ C for 5 min. 0.45 mg of Bacillus subtilis spores immobilized CaIB, 0.25 mg of pre-treated Bacillus subtilis spores immobilized CaIB, 38 mg of Novozyme 435 carriers immobilized CaIB and 1.25 mg of free-form CaIB were incubated. 24 hr later, 2 μl of each sample were taken to analysis by TLC. Esterification was performed by using CaIB enzymes immobilized on various Bacillus spores in the same manner as described above. 25 mg of Novozyme 435 carriers, various Bacillus spores immobilized on CaIB correspond to the concentration of 0.2 mg CaIB and 0.2 mg of free-form CaIB were incubated with the resultant. After reaction for 24 hr, ethyl oleate was measured in the same manner as described above.

As a result, ethyl oleate was synthesized by esterification like existing Novozyme 435 carriers by CaIB enzymes immobilized on the surface of Bacillus

subtilis spores. In addition, CaIB enzymes immobilized on various Bacillus spores showed same results (Rg. 15 and Fig. 16).

As explained in detail above, according to the present invention, proteins could be displayed on the surface very effectively, and the activity of surface- displayed proteins could be maintained by themselves. Furthermore, the present invention could be performed by very simple process.

Having described a preferred embodiment of the present invention, it is to be understood that variants and modifications thereof falling within the spirit of the invention may become apparent to those skilled in this art, and the scope of this invention is to be determined by appended claims and their equivalents.

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