# Patent

## Title
Method for expression of proteins on spore surface

## Abstract

The present invention relates to a method for display of proteinson spore surface and a method for improving protein with rapidity using the same, which comprises the steps of (i) preparing a vector for spore surface display comprising a gene construct containing a gene encoding spore coat protein and a gene encoding a protein of interest, wherein, when expressed, the gene construct expresses a fusion protein between the spore coat protein and the protein of interest, (ii) transforming a host cell with the vector for spore surface display; (iii) displaying the protein of interest on a surface of a spore of the host cell; and (iv) recovering the spore displaying on its surface the protein of interest.

## Background
This application is the U.S. national phase of international application PCT/KR01/02124 filed Dec. 7, 2001 which designated the U.S.
FIELD OF THE INVENTION
The present invention relates to a method for display of proteins on spore surface, in particular to a method for surface display using spore coat proteins as surface display motif and a high throughput method for improving protein.
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). 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., 106-109 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 1010 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 108 cells a hour. 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 105, 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 intracellularly 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 et al., 1997; and Lee et al., 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. coli (Martineau et al., 1991). As described, the desired peptide is displayed on the protruding region of MalE 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 Absorber
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 bioabsorber.
As absorber, a whole cell displaying absorption protein has been developed. The whole cell absorber 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).
Therefore, the surface display motif or novel recombinant protein, which meets the requirements described above, should be selected or prepared to develop novel surface display system overcoming disadvantages of the known systems. In addition, the selection of a suitable host cell to display is very pivotal.
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).
In the developed phage display system, the concentration of the desired clone from phage library has been found to be difficult and the antibody selected from phage library displaying has usually exhibited very low expression rate. 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., 1987; and Agterberg et al., 1990) but also drop of the stability of cell outer membrane and its viability. In addition, in surface display system of yeast, because the vector used has usually shown a low rate of transformation, which is unfavorable to surface display of library.
The surface display systems developed have been cooperatively used each other. For example, to screen antibody variant with enhanced binding affinity, a primary screening is performed using phage surface display system and additionally, the secondary screening is carried out using cell surface display system (Georgiou, 2000). However, the phage display technology is encountered to difficulty in concentration of the desired clones from phage library. The reason is that the antibody displayed on phage surface does not show the elution pattern depending exactly on its binding affinity, which is ascribed to avidity of antibody displayed on phage surface. Therefore, there remains a need of novel methods ensuring screening the desired antibody from antibody library.
E. 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). To be from the shortcomings, ice-nucleation protein with no effect on viability has been used as display motif, and has been applied to bioconversion process, surface display of enzyme library and screening enzyme variants (Jung et al., 1998a, 1998b; and Kim et al., 1998, 1999, 2000).
The size of library displayed on surface depends on the transformation efficiency of host cell with vector; thus E. coli as host has an advantage in view of the size of library to be displayed. Gram positive bacteria as host are relatively rigid and permit stable display of the desired protein; however, transformation efficiency is exhibited low, which results in smaller size of library than E. coli. 
The host organisms having been developed are likely to be sensitive to a variety of physiochemical treatments, which makes it impossible to select proteins displayed on surface by virtue of direct physiochemical treatment. For example, in screening a variant of antibody with enhanced binding affinity, abrupt change of pH or adjustment of the concentration of base is generally performed to elute the variant, which are found to decrease the viability of phage or bacteria in medium.
In addition, the host organisms used conventionally have a complicated and weak structure of cell surface, which drops adaptability to extreme environment such as high temperature and high pressure. To employ E. coli displaying on its surface enzyme in bioconversion reaction, the cells must have represent stability in bioconversion system. In this context, the surface of E. coli displaying on, its surface enzyme is generally subject to immobilization, which does not lead to satisfying results (Freeman et al., 1998).
As described above, the known surface display technologies, based on applying fields, have used bacteriophage, Gram negative or positive bacterium, yeast, cilium or mammalian cell as host organism and surface proteins of each organism as surface display motif. However, in the surface display methods having been developed, the host organism does not have resistance to chemicals and physiochemical change such as pH change, and displaying protein on its surface in excess leads to disadvantages in cell surface, finally reducing the viability of host cell largely (Georgiou et al., 1996).
DETAILED DESCRIPTION OF THIS INVENTION
Under such situation, the present inventors have made intensive studies to be from the shortcoming of conventional display methods, and as a result, we have developed novel display system using a spore as host and a coat protein as motif of surface display. Surprisingly, the developed display system has been found to have excellent stability to a variety of physiochemical stresses in surrounding environment and have much broader applicability.
Accordingly, it is an object of this invention to provide a method for displaying a protein of interest on spore surface using a system for spore surface display.
It is another object of this invention to provide a method for improving a protein of interest using a system for spore surface display.
It is still another object of this invention to provide a method for bioconversion using a system for spore surface display.
It is further object of this invention to provide a method for preparing protein microarray using a system for spore surface display.
It is still further object of this invention to provide a method producing an antibody to antigen in vertebrates using a system for spore surface display.
It is another object of this invention to provide a method for preparing a whole cell absorber using a system for spore surface display.
It is still another object of this invention to provide a microbial transformant for spore surface display of a protein of interest.
It is further object of this invention to provide a spore for spore surface display of a protein of interest.
It is still further object of this invention to provide a vector for spore surface display.
The principle of the present invention lies in the employment of microbial spore as host for surface display and spore coat protein as surface display motif. The present inventors have been compelled to select a system for spore surface display since 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) a fusion protein between a surface display motif and a protein of interest is displayed on spore surface immediately after expression without secretion in host cell, 9) no metabolic activity, and 10) shorter time for obtaining spore, e.g. within several hours.
In particular, the spore coat proteins used in this invention circumvent a necessity for passage across cell membrane, so that they do not need secretion signal and target signal which are prerequisites of surface display motif, thereby ensuring a surface display of protein such as β-galactosidase, in orderly fashion, which is difficult to pass across cell membrane.
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. However, as disclosed, a purification method for demonstrating surface display of protein is not recognized to isolate spores specifically. Furthermore, the activity of enzyme expressed has been very low and the display of enzyme on spore surface has never been demonstrated by means of reliable methods such as biochemical, physical and immunological methods. In addition to this, the inner coat protein, cotD is enclosed by outer coat protein of 70-200 nm thickness, which makes it difficult to be exposed to spore surface. In case of fusion protein expression using outer coat protein, cotC, the activity of enzyme is increased by four-fold in comparison with that of cotD; however, the activity, 0.02 U, is considered negligible, in particular, in consideration of industrial scale. Therefore, the matter disclosed in the document above cannot be considered to use and recognize a system for spore surface display. In other words, the patent document cannot be recognized to describe a system for spore surface display. U.S. Pat. Nos. 5,837,500 and 5,800,821 also indicate cotC and cotD as a preferable surface display motif, and therefore the patent documents cannot be recognized to describe a system for spore surface display because of the reasons mentioned above.
Furthermore, according to the purification method of spore proposed in U.S. Pat. No. 5,766,914, half of the purified resultant has been observed under microscope to have the complex forms between cells harboring spores and cell-lysis matters bound to spores (see FIG. 1; cells with blackish color and long side are those not forming spore and spores is observed to be white and circular), which has been demonstrated by the present inventors. The facts hereinabove reveals possibility to bring about the false results by measuring of the activity of reporter enzyme or analyzing of reporter enzyme with flow cytometry in vegetative cells rather than on spore surface. In contrast, the renografin gradient centrifugation as demonstrated in Examples below allows for the perfect purification of spores (see FIG. 2), thereby measuring the activity of enzyme displayed on spore surface solely.
Observations on lower enzyme activity in several documents including the patents above are likely to be resulted from the following reasons. First, it is suggested that the expression level of coat protein itself is low. The maximum expression levels of CotC and CotD are 40 and 147 Miller Units, respectively, which is considered to be largely low, in particular, in consideration of CotE of 6021 Miller Units (Zheng L and Losick R., J. Mol. Biol. 212:645-660(1990)). Furthermore, it is notable that the amount of enzyme displayed on spore surface has not been reported. Secondly, it is possible that the protein displayed on spore surface is cleaved by protease in host cell. Such suggestion is made based on the fact that at spore-forming stage of Bacillus subtilis a variety of proteases are expressed and reconstitution for spore formation is occurred. The suggestion can be demonstrated in Examples below in which a variant lack protease exhibits a much higher enzyme activity displayed on spore surface (see FIG. 7).
Using gene of GFP (green fluorescence protein) as reporter linked to cotE and spoIVA, the studies on gene expression and localization of the expressed protein in spore has been attempted (Webb et al., 1995; Lewis et al., 1996). The publications disclose that the fusion protein expressed is found in spore by means of observation under fluorescence microscope using fluorescence of GFP; however, they never describe if the fusion protein is displayed and linked on spore surface.
As another example of spore surface display using coat protein, U.S. Pat. No. 5,800,821 discloses a spore as delivery system of antigen. However, the publication does not disclose that the antigen expressed is displayed on spore surface and the spore containing antigen administered can induce immunization reaction in host.
The present inventors have recognized the shortcomings of the conventional arts described above and developed an efficient and optimized system for spore surface display, which have been confirmed by enzymological, immunological and physiochemical methods using various spore coat proteins.
In one aspect of this invention, there is provided a method for displaying a protein of interest on spore surface, which comprises the steps of: (i) preparing a vector for spore surface display comprising a gene construct containing a gene encoding spore coat protein and a gene encoding a protein of interest, wherein, when expressed, the gene construct expresses a fusion protein between the spore coat protein and the protein of interest; (ii) transforming a host cell with the vector for spore surface display; (iii) displaying the protein of interest on a surface of a spore of the host cell; and (iv) recovering the spore displaying on its surface the protein of interest.
In another aspect of this invention, there is provided a method for improving a protein of interest, which comprises the steps of: (i) constructing a gene library of the protein of interest; (ii) preparing a vector by linking the gene library to a gene encoding spore coat protein; (iii) transforming a spore-forming host cell with the vector; (iv) forming a spore in the transformed host cell and displaying the protein of interest on a surface of the spore; (v) recovering the spore displaying on its surface the protein of interest; and (vi) screening the spore displaying a variant of the protein of interest having a desired property.
In still another aspect of this invention, there is provided a method for improving a protein of interest using a resistance property of spore, which comprises the steps of: (i) constructing a gene library of the protein of interest; (ii) preparing a vector by linking the gene library to a gene encoding spore coat protein; (iii) transforming a spore-forming host cell with the vector; (iv) forming a spore in the transformed host cell and displaying the protein of interest on a surface of the spore; (v) treating the spore displaying on its surface the protein of interest with one or more selected from the group consisting of organic solvent, heat, acid, base, oxidant, dryness, surfactant and protease; (vi) recovering the spore displaying on its surface the protein of interest; and (vii) screening the spore displaying a variant of the protein of interest having a resistance to the treatment.
In further aspect of this invention, there is provided a method for bioconversion, which comprises the steps of: (i) preparing a vector for spore surface display comprising a gene construct containing a gene encoding spore coat protein and a gene encoding a protein of interest conducting a bioconversion reaction, wherein, when expressed, the gene construct expresses a fusion protein between the spore coat protein and the protein of interest; (ii) transforming a host cell with the vector for spore surface display; (iii) displaying the protein of interest on a surface of a spore of the host cell; (iv) recovering the spore displaying on its surface the protein of interest; and (v) performing the bioconversion reaction using the spore displaying on its surface the protein of interest.
In still further aspect of this invention, there is provided a method for preparing protein microarray, which comprises the steps of: (i) preparing a vector for spore surface display comprising a gene construct containing a gene encoding spore coat protein and a gene encoding antibody or antigen having binding affinity to a protein to be analyzed, wherein, when expressed, the gene construct expresses a fusion protein between the spore coat protein and the antibody or antigen; (ii) transforming a host cell with the vector for spore surface display; (iii) displaying the antibody or antigen on a surface of a spore of the host cell; (iv) recovering the spore displaying on its surface the antibody or antigen; and (v) immobilizing onto a solid surface the spore displaying on its surface the antibody or antigen.
In another aspect of this invention, there is provided a method producing an antibody to antigen in vertebrates, which comprises the steps of: (i) preparing a vector for spore surface display comprising a gene construct containing a gene encoding spore coat protein and a gene encoding the antigen, wherein, when expressed, the gene construct expresses a fusion protein between the spore coat protein and the antigen; (ii) transforming a host cell with the vector for spore surface display; (iii) displaying the antigen on a surface of a spore of the host cell; (iv) recovering the spore displaying on its surface the antigen; and (v) administering to vertebrates a composition containing an immunologically effective amount of the spore displaying on its surface the antigen.
In still another aspect of this invention, there is provided a method for preparing a whole cell absorber, which comprises the steps of: (i) preparing a vector for spore surface display comprising a gene construct containing a gene encoding spore coat protein and a gene encoding a protein having a binding affinity to a certain substance, wherein, when expressed, the gene construct expresses a fusion protein between the spore coat protein and the protein; (ii) transforming a host cell with the vector for spore surface display; (iii) displaying the protein on a surface of a spore of the host cell; (iv) recovering the spore displaying on its surface the protein; and (v) immobilizing onto a carrier the spore displaying on its surface the protein.
According to preferred embodiments of this invention, the gene encoding spore coat protein is derived from a spore-forming Gram negative bacterium including Myxococcus; a spore-forming Gram positive bacterium including Bacillus; a spore-forming Actionmycete; a spore-forming yeast including Saccharomyces cerevisiae, Candida and Hansenulla or a spore-forming fungus, but not limited to. More preferably, the gene encoding spore coat protein is derived from a spore-forming Gram positive bacterium, most preferably, Bacillus including Bacillus subtilis and Bacillus polymyxa, etc.
The gene of spore coat protein useful in this invention includes cotA, cotB, cotC, cotD (W. Donovan et al., J. Mol. Biol., 196:1-10(1987)), cotE (L. Zheng et al., Genes & Develop., 2:1047-1054(1988)), cotF (S. Cutting et al., J. Bacteriol., 173:2915-2919(1991)), cotG, cotH, cotJA, cotJC, cotK, cotL, cotM, cotS, cotT (A. Aronson et al., Mol. Microbiol., 3:437-444(1989)), cotV, cotW, cotX, cotY, cotZ (J. Zhang et al., J. Bacteriol., 175:3757-3766(1993)), spoIVA, spoVID and sodA, but not limited to.
In addition, the gene encoding spore coat protein useful in this invention is a modified form or a recombinant form of one selected from the group consisting of cotA, cotB, cotC, cotD, cotE, cotF, cotG, cotH, cotJA, cotJC, cotK, cotL, cotM, cotS, cotT, cotV, cotW, cotX, cotY, cotZ, spoIVA, spoVID and sodA, in which the modified form or the recombinant form has a more compatibility for spore surface display relative to wild type genes. The modified form of the gene is obtained by DNA shuffling method (Stemmer, Nature, 370: 389-391(1994)), StEP method (Zhao, H., et al., Nat. Biotechnol., 16: 258-261 (1998)), RPR method (Shao, Z., et al., Nucleic acids Res., 26: 681-683 (1998)), molecular breeding method (Ness, J. E., et al., Nat. Biotechnol., 17: 893-896 (1999)), ITCHY method (Lutz S. and Benkovic S., Current Opinion in Biotechnology, 11: 319-324 (2000)), error prone PCR (Cadwell, R. C. and Joyce, G. F., PCR Methods Appl., 2: 28-33 (1992)), point mutagenesis (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989), nucleotide mutagenesis (Smith M. Annu. Rev. Genet. 19: 423-462 (1985)), combinatorial cassette mutagenesis (Wells et al., Gene 34: 315-323 (1985)) and other suitable random mutagenesis.
Further to this, the gene encoding spore coat protein is selected from the group consisting of cotA, cotB, cotC, cotD, cotE, cotF, cotG, cotH, cotJA, cotJC, cotK, cotL, cotM, cotS, cotT, cotV, cotW, cotX, cotY, cotZ, spoIVA, spoVID and sodA, in which the gene has a substituted promoter for its promoter to enhance spore surface display relative to wild type genes. The promoter for enhancing surface display, for example, includes the promoters of cotE or cotG genes, which show higher expression level.
In preferred embodiments of this invention, the gene encoding spore coat protein is selected from the group consisting of cotA, cotE, cotF, cotG, cotH, cotJA, cotJC, cotK, cotL, cotM, cotS, cotT, cotV, cotW, cotX, cotY, cotZ, spoIVA, spoVID and sodA, more preferably, cotE or cotG and most preferably, cotG.
According to the present methods, as linking a gene of coat protein and a gene of the protein of interest, the overall sequence, fragments, two or more repeated sequences of the gene of coat protein are useful. In two or more repeated sequences, the repeated sequences may be the same or different each other. The overall sequence, two or more repeated sequences of the gene of the protein of interest are also useful in the fusion sequence. In two or more repeated sequences, the repeated sequences may be the same or different each other. Other combinations also may be useful in the fusion sequence.
It is understood by one skilled in the art that the gene construct may exist as plasmid in host cell independently or as integrated form into chromosome of host cell. Additionally, in the gene construct, it is recognized by one skilled in the art that the gene of coat protein may be followed or preceded by the gene of the protein of interests Integrated form into the counterpart gene may be useful.
It is recognized by one skilled in the art that the expression of the fusion protein between coat protein and protein of interest can be induced by virtue of promoters of coat protein gene and protein of interest or other suitable promoters inducible in host cell
The present methods is applicable to any protein, for example, including 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 applicable 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.
The host cell suitable in this invention, includes spore-forming Gram negative bacterium including Myxococcus, a spore-forming Gram positive bacterium including Bacillus, a spore-forming Actionmycete, a spore-forming yeast and a spore-forming fungus, but not limited to. Preferably, the host cell is a spore-forming Gram positive bacterium, more preferably, Bacillus. In particular, Bacillus subtilis is advantageous in the senses that genetic knowledge and experimental methods on its spore forming as well as culturing method are well known.
According to the present methods, the spore may be reproductive or non-reproductive. In the method for improving a protein, the recovered coats are subject to reproduction but the methods using a spore as delivery means of protein of interest obviate the necessity for reproduction of spore. It is considerable that the organisms genetically engineered is likely to be regulated under laws and rules; hence non-reproductive spore is preferable. For example, Bacillus subtilis lack of cwlD gene is preferably used due to being non-reproductive.
According preferred embodiments of this invention, the recovery of spore is performed in such a manner that the display of the protein of interest on the spore surface is maximized by controlling culture time, after which culturing is terminated and the spore is then recovered. Suitable culture time is varied depending upon the type of cell used, for example, in case of using Bacillus subtilis as host, the culture time of 16-25 hours is preferred.
In the present methods, the recovery of spore may be carried out according to the conventional methods known to one skilled in the art, more preferably, renografin gradients methods (C. R. Harwood, et al., “Molecular Biological Methods for Bacillus.” John Wiley & Sons, New York, p. 416(1990)).
As demonstrated in Examples, the stability of spore displaying the foreign protein of interest an its surface is very high in the present invention, indicating maintenance of the integrity of spore surface structure formed by cooperation of coat proteins while the foreign protein is displayed.
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 spore surface and then reacted with a secondary antibody labeled with fluorescent chemical to stain the spore, followed by observation with fluorescence microscope or analysis with flow cytometry. 2) The protein of interest displayed on spore surface 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 spore.
In the method for improving protein, the construction of gene library for the protein of interest is performed by a mutagenesis of the gene encoding the protein of interest of wild type, in which the mutagenesis includes DNA shuffling method (Stemmer, Nature, 370: 389-391(1994)), StEP method (Zhao, H., et al., Nat. Biotechnol., 16: 258-261 (1998)), RPR method (Shao, Z., et al., Nucleic acids Res., 26: 681-683 (198)), molecular breeding method (Ness, J. E., et al., Nat. Biotechnol., 17: 893-896 (1999)), ITCHY method (Lutz S. and Benkovic S., Current Opinion in Biotechnology, 11: 319-324 (2000)), error prone PCR (Cadwell, R. C. and Joyce, G. F., PCR Methods Appl., 2: 28-33 (1992)), point mutagenesis (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989), nucleotide mutagenesis (Smith M. Annu. Rev. Genet. 19: 423-462 (1985)), combinatorial cassette mutagenesis (Wells et al., Gene 34: 315-323 (1985)) and other suitable random mutagenesis.
In the method for improving protein, the screening is performed in a rapid manner by means of measuring an activity of the protein or flow cytometry (Georgiou, 2000). In case of using an activity of the protein, the screening is carried out by measuring growth of host expressing the protein or colorimetrical reaction catalyzed by the protein. In the method for improving protein using a resistance property of spore, the screening is carried out in a rapid manner by virtue of measuring an activity of the protein or using the structural stability of the protein.
The methods for improving protein provide in a high-throughput manner, from wild type, 1) enzymes catalyzing non-biological reaction (e.g., Diels-Alder condensation), 2) enzymes with non-natural steroselectivity or regioselectivity, 3) enzymes with activity in organic solvent or organic solvent-aqueous solution two-phase system, and 4) enzymes with activity in extreme conditions such as high temperature or pressure.
In addition, to select a variant of antibody with enhanced binding affinity, it is general that pH is abruptly changed or the concentration of base is adjusted to elute the variant. In a method using phage or bacteria as carrier, such elution conditions are likely to decrease the viability of phage or bacteria in medium. However, the methods for improving protein using system of spore surface display overcome the drawback.
In the meantime, the bioconversion process using surface-displayed enzymes requires a physiochemical stability of surface displaying host 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 in 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. In Examples described hereinafter, the bioconversion reaction with β-galactosidase is exemplified and thus it is understood by one skilled in the are that the present method can be also applied to any type of enzyme such as lipase, protease, cellulase, glycosyltransferase, oxidoreductase and aldolase. In addition, the present method is useful in single step or multi-step reaction and in aqueous or non-aqueous solution. The present bioconversion method employs spore as free or immobilized form and can be performed with other microbes or enzymes.
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 in 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 cane be performed with much greater readiness. In the present method, a gene construct containing a gene encoding spore coat protein and a gene encoding the desired protein is introduced into host cell and the spore displaying on its surface the desired protein is isolated, followed by immobilization of the isolated spore onto a solid surface. In the me-hod for preparing protein array, the conventional steps may be used (see Wo 0061806, WO 0054046, U.S. Pat. No. 5,807,754, EP 0818467, WO 9742507, U.S. Pat. No. 5,114,674 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, SiN4, modified silicone nitrocellulose, polyvinylidene fluoride, polystylene, polytetrafluoroethylene, polycarbonate, nylon, fiber and combinations thereof. The spore 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 spores applied to each spot (or address) depends on the type of array. Interaction between the protein displayed on spore 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 spore, 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, in the process for preparing absorption chromatography, antibody or polypeptide is produced, purified and immobilized on a carrier. Generally, it is very difficult to prepare the bioabsorbers. The disadvantage may be overcome using whole cell displaying protein as described in Georgiou et al., 1997. Therefore, the system for spore surface display of this invention provides a whole cell absorber to solve the problems of the known absorbers.
In further aspect of this invention, there is provided a microbial transformant for spore surface display of a protein of interest, characterized in that the transformant is produced by transformation with a vector for spore surface display containing (i) a gene encoding a protein of interest and (ii) a gene encoding spore coat protein is selected from the group consisting of cotA, cotB, cotC, cotD, cotE, cotF, cotG, cotH, cotJA, cotJC, cotK, cotL, cotM, cotS, cotT, cotV, cotW, cotX, cotY, cotZ, spoIVA, spoVID and sods, in which when expressed, a fusion protein between the spore coat protein and the protein of interest is expressed.
According to preferred embodiment, the transformant is derived from a variant mutated to enhance spore surface display. For example, the mutation to enhance spore surface display eliminates a production of extracellular secretory protease in the transformant, so that the protein of interest displayed on spore surface is stably maintained. In addition, the mutation to enhance spore surface display eliminates a production of intracellular protease in the transformant. It is also preferred that a gene or genes involved in spore forming is subject to mutation in order to the rate of spore forming (Perego, M., et al., Mol. Microbiol. 19: 1151-1157 (1996)).
In still further aspect of this invention, there is provide a spore for spore surface display of a protein of interest, characterized in that the spore displays the protein of interest on its surface.
According to the present invention, the spore may be reproductive or non-reproductive one which is selected based on its application field. Preferably, the non-reproductive spore can be obtained by virtue of one or more methods selected from the group consisting of genetic method (Popham D. L., et al., J. Bacteriol., 181: 6205-6209 (1999)), chemical method (Setlow T. R., et al., J. Appl. Microbiol., 89: 330-338 (2000)) and physical method (Munakata N, et al., Photochem. Photobiol., 54: 761-768 (1991)). The genetic method to make the spore non-reproductive is accomplished by, for example, deleting a gene of host cell involved in reproduction of spore.
In the present invention, it is preferred that the spore is derived from a variant mutated to increase its agglutination property because in bioconversion performed in industrial scale, the separation between the resulting product and spores is rendered easier. The increase of the agglutination property in the spore is accomplished by one or more methods selected from the group consisting of genetic method, chemical method and physical method. As example of the physical method, the heat treatment can be proposed (Wiencek K. M., et. al., Appl. Environ. Microbiol., 56: 2600-2605 (1990)).
In another aspect of this invention, there is provided a vector for spore surface display, characterized in that the vector comprises a replication origin, an antibiotic-resistance gene, a restriction site, a gene encoding a spore coat protein, a gene encoding a protein of interest and a promoter operatively linked to the gene encoding a spore coat protein, in which when expressed, a fusion protein between the spore coat protein and the protein of interest is expressed.
According to preferred embodiment, the gene encoding a spore coat protein is selected from the group consisting of cotA, cotB, cotC, cotD, cotE, cotF, cotG, cotH, cotJA, cotJC, cotK, cotL, cotM, cotS, cotT, cotV, cotW, cotX, cotY, cobZ, spoIYA, spoVID and sodA, more preferably, cotE or cotG, and most preferably, cotG.
In the vector of this invention, the replication origin can include various origins known to one skilled in the art, for example, when the vector is introduced into a spore-forming yeast, 2μ, ARS, ARS1 or ARS2 can be used as replication origin. In case of using Bacillus as host, ori 322, ColE1 origin, Rep1060, etc. can be used. The antibiotic-resistance gene used as selective marker, when prokaryote such as Bacillus is used as host, is a resistance gene to antibiotics acting to prokaryotes, for example, including kanamycin, ampicillin, carbenicillin, chloramphenicol, streptomycin, geneticin, neomycin and tetracycline. The promoter used in the present vector includes a promoter of the gene of spore coat protein and a known promoter operable in host cell.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a microscopic photograph showing spores of Bacillus subtilis purified according to method described in U.S. Pat. No. 5,766,914;
FIG. 2 is a microscopic photograph showing spores of Bacillus subtilis purified according to renografin gradients method;
FIG. 3 is a genetic map of the recombinant vector pCotE-lacZ of the present invention;
FIG. 4 is a genetic map of the recombinant vector pCotG-lacz of the present invention;
FIG. 5 represents screening results demonstrating the preferred surface display motif in the present invention;
FIG. 6 is a graph showing the affect of protease to β-galactosidase displayed on spore surface;
FIG. 7 is a graph showing the activity of β-galactosidase displayed on spore surface in accordance with culture time;
FIG. 8 is a graph representing the heat stability of Bacillus subtilis DB104 strain displaying on its surface the protein;
FIG. 9 is a genetic map of recombinant vector pCSK-cotG-CMCase of this invention;
FIG. 10 is a graph showing analysis of spore surface-displayed carboxymethylcellulase using flow cytometry;
FIG. 11 is a graph showing analysis of spore surface-displayed levansucrase using flow cytometry;
FIG. 12 is a graph showing the activity of spore surface-displayed levansucrase;
FIG. 13 is a graph representing analysis of spore surface-displayed monoclonal antibody using flow cytometry;
FIG. 14 is a graph demonstrating selectivity to spore displaying single chain Fv;
FIG. 15 is a graph representing analysis with flow cytometry of monoclonal antibody library to have binding affinity to Pre-S region of hepatitis B virus;
FIG. 16 is a graph showing analysis of spore surface-displayed GFP using flow cytometry; and
FIGS. 17a to 17d are graphs representing isolation with flow cytometry of spores displaying improved GFP.



The following specific examples are intended to be illustrative of the invention and should not be construed as limiting the scope of the invention as defined by appended claims.
EXAMPLES
Example I
Isolation of the Gene Encoding Coat Proteins
I-1: Construction of the Vector for Spore Surface Display
To isolate the most appropriate coat protein for spore surface display among coat proteins consisting of spore, the recombinant vector having the gene encoding a fusion protein between coat protein and β-galactosidase was constructed as follow:
To begin with, the DNA was extracted from the Bacillus subtilis 168 strain provided from Dr. F. Kunst (Kunst F., et al., Nature, 390: 249-256(1997)) by Kalman's method (Kalman S., et al., Appl. Environ. Microbiol. 59, 1131-1137(1993)), and the purified DNA was served as template for PCR to spoIVA primers (SEQ ID NOs: 1 and 2), cotB primers (SEQ ID NOs: 3 and 4), cotC primers (SEQ ID NOs: 5 and 6), cotD primers (SEQ ID NOs: 7 and 8), cotE primers (SEQ ID NOs: 9 and 10), cotG primers (SEQ ID NOs: 11 and 12), cotH primers (SEQ ID NOs: 13 and 14), cotM primers (SEQ ID NOs: 15 and 16), cotV primers (SEQ ID NOs: 17 and 18), cotX primers (SEQ ID NOs: 19 and 20) and cotY primers (SEQ ID NOs: 21 and 22). Taq polymerase purchased from Boehringer Mannheim was used for total 35 cycles of PCR under condition of denaturation for 30 sec at 94° C., annealing for 30 sec at 55° C. and extension for 1 min at 72° C.
After then, each amplified PCR products were digested with BamHI and SalI and cloned between BamHI and SalI sites of plasmid pDG1728 which is a gratuitous gift by Dr. P. Stragier (Geurout-Fleury, A. M., et al., Gene, 180: 57-61(1996)), thus the constructed vectors express the fusion protein of coat protein and β-galactosidase. FIG. 3a shows the genetic map of pCotE-lacZ expressing fusion protein of CotE protein and β-galactosidase and FIG. 3b shows the genetic map of pCotG-lacZ expressing Fusion protein of CotG protein and β-galactosidase.
SEQ ID NO:23 shows the sequence of cotE-lacZ fused genes and SEQ ID NO:24 shows the amino acid sequence of CotE-LacZ fusion protein. In SEQ ID NO:23, promoter for cotE is 1-329, CotE structural gene is 330-872, restriction site is 873-878 and LacZ structural gene is 879-3902.
SEQ ID NO:25 shows the sequence of cotG-lacZ fused genes and SEQ ID NO:26 shows the amino acid sequence of CotG-LacZ fusion protein. In SEQ ID NO:25, promoter of cotG is 1-460, CotE structural gene is 461-1045, restriction site is 1046-1051 and LacZ structural gene is 1052-4075.
I-2: Pure Isolation of Spores
Constructed recombinant expression vectors were transformed into Bacillus subtilis DB104 (Kawamura F. and Doi R. H., J. Bacteriol. 160: 442-444(1984)) using natural transformation (C. R. Harwood, et al., Molecular Biological Methods for Bacillus, John Wiley & Sons, New York, p. 416(1990)).
Other methods such as conjugation or trnasduction can be applied for introduction of the recombinant vectors into Bacillus strain.
Subsequently, each Bacillus strain comprising the fused gene between coat protein and β-galactosidase was cultured for 24 hr at a shaking incubator (37° C., 250 rpm) in GYS medium ((NH4)2SO4 2 g/l, Yeast extract 2 g/l, K2HPO4 0.5 g/l, glucose 1 g/l, MgSO45H2O 0.07 g/l), 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)).
I-3: Display of Proteins on Spore Surface
The spores isolated in the above-described Example and the cell pellet of Bacillus subtilis DB104 were subjected to evaluation of the activity of β-galactosidase using Miller's method (Miller, “Experiments in Molecular Genetics”, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory, p. 352-355(1972)) and the results are shown in FIG. 5. In FIG. 5, the gray bar indicates cell pellet, the black bar indicates the activity of β-galactosidase in purely isolated spores and ‘1’ relates to result of control Bacillus subtilis DB104; ‘2’ to result of SpoIVA-LacZ; ‘3’ to result of CotB-LacZ; ‘4’ to result of CotC-LacZ; ‘5’ to result of CotD-LacZ; ‘6’ to result of CotE-LacZ; ‘7’ to result of CotG-LacZ; ‘8’ to result of CotH-LacZ; ‘9’ to result of CotM-LacZ; ‘10’ to result of CotV-LacZ; ‘11’ to result of CotX-LacZ; and ‘12’ to result of CotY-LacZ fusion protein, respectively.
As shown in FIG. 5, it is known that Deits TL (U.S. Pat. No. 5,766,914) fails to induce the sufficient surface display of cotC and cotD since the expression levels of cotC and cotD are as low as the control. However, the expression level of cotE and cotG are comparatively high and especially, expression level of cotG is remarkably high comparing to other coat proteins. In addition, in the isolated spores, the surface display using cotG shows the highest enzyme activity, which demonstrates that CotG-LacZ fusion proteins are the highest level of display on spore surface.
Considering the expression level and the amount of fusion proteins displayed on spore surface, it is known that the cotG is the most preferable surface display motif. It is known to one skilled in the art that these results exclude other coat proteins other than cotG from applying to spore surface display.
I-4: Effect of Proteases on the Surface-Displayed Enzymes
To confirm whether the surface-displayed β-galactosidase is degraded or not, the purely isolated spore displaying CotG-LacZ was resuspended into 100 μl of PBS solution, and each 10 mg/ml of protease K, protease type XIV or trypsin was treated. Thereafter, the activity of β-galactosidase was measured as described above and the results are shown in FIG. 6. As shown in FIG. 6, the activity of spore surface-displayed β-galactosidase is decreased with some variations in each result. These results give the evidence for the localization of β-galactosidase on spore surface.
DB104 strain lacking neutral and alkaline protease and WB700 strain (Ye, R., et al., Biotechnology and Bioengineering, 62:87-96(1999)) lacking 7 proteases among proteases secreted from Bacillus subtilis were transformed with the pCotG-lacZ expression vector using natural transformation method as described in example I-1, and the activity of β-galactosidase in cell pellet and spores was measured as described in example I-3 (FIG. 7). As shown in FIG. 7, while the enzyme activity is abruptly decreased in DB104 strain as time goes, WB700 strain shows slight decrease in enzyme activity. These results indicate that the displayed β-galactosidases on spore surface are degraded in DB104 strain by the proteases secreted extracellularly; however, the displayed β-galactosidases in WB700 are stably maintained because of lack of the proteases secreted extracellularly. Therefore, the results also support the localization of β-galactosidase on spore surface.
Example II
Spore Production Depending on Culture Time
As shown in FIG. 7, it is required to stop incubation on a specific time point and isolate spores. In DB104, the enzyme activity of spores after 38 hr of incubation is significantly low comparing to that after 24 hr of incubation. Thus, it is demonstrated that the adjustment of incubation time makes it possible to yield spores displaying enzyme on its surface with the greatest enzyme activity.
Example III
Characterization of Spores Dispalying β-Galactosidase
Heat resistance was measured as follow in spores displaying β-galactosidase: 100 μl of spores isolated by renografin gradients in Example I-2 were heated for 15 min and then spread on LB plates to evaluate viability of spores (FIG. 8). As shown in FIG. 8, spores displaying CotG-LacZ show similar heat resistance to spores without surface protein. In a result, the display of the foreign protein fused to coat protein on spore surface does not affect on its inherent characteristics such as heat resistance. Moreover, these results provide the promising usage of spore displaying on its surface enzyme in chemical reactions at high temperature. In addition, from these results, it is suggested that the spores transformed according to the present invention remain their inherent resistances to lysozyme, a bacterial cell wall-degrading enzyme and solvent.
Example IV
Displaying Various Enzymes on Spore Surface
IV-1: Construction of Recombinant Vectors
To use spores displaying various enzymes, it is prerequisite to confirm whether various enzymes in addition to β-galactosidase can be surface-displayed. Firstly, plasmid pHPS9 (Haima, et al., Gene, 86:63-69(1990)) was digested by EcoRI and HindIII and manipulated into blunt ends using Klenow enzyme. Then, DNA fragment containing multiple cloning sites, which was obtained from plasmid p123T (EMBL Z46733) with BssHII, was ligated to the blunt-ended pHPS9 plasmid to use as virgin vector named pCSK1 in the following experiments. The pCSK-cotG plasmid was prepared by restricting PCSK1 plasmid with BamHI and PstI and ligating PCR-amplified cotG gene. In the course of PCR for cotG gene amplification, a linker between cotG gene and target gene was incorporated using cotG-linker 5 primer (SEQ ID NO:27) and 3 primer (SEQ ID NO:12) with template of DNA in Bacillus subtilis. 
In other experiments, genes encoding carboxymethyl cellulase, levansucrase and lipase was prepared as follows: Carboxymethyl cellulase cloned in pBSI plasmid (S. H. Park et al., Agric. Biol. Chem., 55: 441-448(1991)) was directly employed. The pBS1 plasmid contains the gene encoding carboxymethylcelluase cloned from Bacillus subtilis BSE616 strain. In the present Example, PCR was performed with the pBS1 as template using primer represented by SEQ ID NOs:28 and 29. In the case of PCR for levansucrase, pSSTS110 plasmid (Jung, H.-C., et al., Nat. Biotech., 16; 576-580(1998)) was used as template and primers represented by SEQ ID NOs:30 and 31 were used. In PCR for lipase, pTOTAL (Ahn, J.-H., et al., J. Bacteriol., 181: 1847-1852(1999)) was added as template and primers of SEQ ID NOs: 32 and 33 were used. All PCRs were performed in the same condition as described in Example I-1.
Recombinant vectors containing gene coding for fusion between CotG and the carboxymethylcelluase, levansucrase or lipase were prepared by cloning into pCSK-cotG using PstI and BamHI restriction enzymes both in vector and in the PCR-amplified inserts. As an example of the above construction, FIG. 9 shows pCSK-cotG-CMCase which is the recombinant vector encoding fusion protein between CotG and carboxymethylcellulase. Transformed Bacillus subtilis DB104 with pCSK-cotG-CMCase was named Bacillus subtilis GFSD18 and deposited at the Korean Collection for Type Cultures (KCTC, KR) with accession No. KCTC 0887BP (Nov. 16, 2000).
SEQ ID NO:34 shows nucleotide sequence of fused cotG-CMCase genes and SEQ ID NO:35 shows amino acid sequence of CotG-CMCase encoded by SEQ ID NO:34. In SEQ ID NO:34, promoter for cotG is 1-460, structural gene for CotG is 461-1045, linker is 1046-1084, and structural gene for CMCase is 1085-2491.
IV-2: Expression of Recombinant Vectors and Verification
The above-prepared recombinant vectors were employed for transformation of Bacillus subtilis DB104 with the same procedures as described in Example I-2. Subsequently, each transformed Bacillus strains was cultured for 24 hr at a shaking incubator (37° C., 250 rpm) in GYS midium, the only pure spores were isolated using renografin gradients method, and enzyme activity of carboxymethylcellulase (Kim, et al., Appl. Environ. Microbial., 66:788-793(2000)), levansucrase (Jung, et al., Nat. Biotech., 16:576-580(1998)) or lipase was evaluated. The activity of lipase was evaluated as follow: The spores suspended in 10% PBS was mixed with 10% olive oil, reacted for 48 hr, treated with 0.2 ml cupric acid on supernatant solution and the observance of OD was performed at 715 nm.
In the case of carboxymethylcellulase, the activity of enzyme displayed on spore was 175 mU comparing to 0 mU in control. In other verifying method, carboxymethylcellulase-specific andtibody (Kim, et al., Appl. Environ. Microbiol., 66:788-793(2000)) was probed for flow cytometry (FACSORT (Cell Sorter Flow Cytometer, Becton Dickinson, USA) and the carboxymethylcellulases were detected on the surface of spores transformed by pCSK-cotG-CMCase (FIG. 10).
The activity of levansucrase was also high in spores transformed by recombinant vector (FIG. 12) and the levansucrases were detected on the surface of transformed spores as verified with flow cytometery using levan sucrase-specific antibody (Jung, et al., Nat. Biotech., 16:576-580(1998)) in the same procedures as above-described % in carboxymethylcellulase (FIG. 11).
The activity of lipase was measured as A715=0.14 in spores transformed with recombinant vector.
On the basis of these results, it is demonstrated that various enzymes as well as β-galactosidase can be displayed on the surface of spore according to the present invention.
Based on the results in these examples and example I, it is known to one skilled in the art that the gene construct containing gene encoding fusion protein between coat protein and protein of interest may exist as plasmid in host cell independently or as integrated form into chromosome of host cell and both forms may lead to successful spore surface display. It is also recognizable that the gene of coat protein may be followed or preceded by the gene of the protein of interest. In addition, it is recognized that in the gene construct, the overall sequence, fragments, two or more repeated sequences of the gene of coat protein are useful. In two or more repeated sequences, the repeated sequences may be the same or different each other. The overall sequence, two or more repeated sequences of the gene of the protein of interest are also useful in the fusion sequence. In two or more repeated sequences, the repeated sequences may be the same or different each other.
It is recognized by one skilled in the art that the expression of the fusion protein between coat protein and protein of interest can be induced by virtue of promoters of coat protein gene and other suitable promoters operable in host cell. Any vector carrying the present gene construct may be used in this invention, which is recognized by one skilled in the art referring to these results.
It is known that both monomric and multimeric enzyme can be applied for the present invention since the β-galactosidase used in example I is tetramer (U. Karlsson et al., J. Ultrastruct. Res., 10:457-469(1964)) and the enzymes described in this Example are monomers.
Example V
Display of Antibody on Spore Surface and Screening for Directed Evolution
On the purpose of application of other proteins in addition to enzymes, the experiment to display antibody on spore surface was performed as follows:
V-1: Construction of Recombinant Vector for Surface Display of Single Chain Fv
Gene encoding single chain Fv, against Pre-S2 domain (SEQ ID NO:36) of hepatitis B virus (HBV) was linked to cotG gene encoding surface protein of Bacillus subtilis spore. Single chain Fv gene was amplified by PCR with pAScFv101 (WO 9737025) as template and with primers described in SEQ ID NOs:37 and 38. Taq polymerase purchased from Bioneer (Korea) was used for total 30 cycles of PCR under condition of denaturation for 30 sec at 94° C., annealing for 30 sec at 55° C. and extension for 1 min at 72° C. And then, each PCR product was restricted by ApaI and NheI, cloned into pCSK-CotG between the same restriction sites (pCSK-CotG-scFv) and transformed into JM109 using transformation method by Inoue, et al. (Inoue, H., et al., Gene, 96:23-28(1990)). The amplified vectors for displaying on spore surface were isolated by alkaline extraction method (Sambrook et al., Molecular Cloning: A laboratory Manual, Cold Spring Harbor, N.Y., 1989) and transformed into Bacillus subtilis DB104 by natural transformation as described in Example I-1.
V-2: Verification of Single Chain Fv Display on Spore Surface Using Flow Cytometry
Affinity of the displayed single chain Fv against the Pre-S2 of HBV was evaluated by FACSORT (Cell Sorter Flow Cytometer, Becton Dickinson, U.S.A.) as the following procedures.
Firstly, Pre-S2 peptide was labeled with fluorescein (PanVera, USA) using fluorescein succinidimyl ester coupling method.
The transformed strains were inoculated into LB broth containing 5 μg/ml chloroamphenicol, pre-cultured for 8-10 hr at 37° C., 1% of seed culture was inoculated into GYS broth for sporulation, cultured for 24 hr at 37° C. and the cultured media was harvested. The pure spores were isolated using renografin gradients method, 100 μl pure spores were blocked with PBS containing 3% skim milk to inhibit non-specific binding and reacted with 10 μl of fluorescein labeled Pre-S2 peptide. Thereafter, the spores bound to fluorescein labeled Pre-S2 peptide were detected in the same procedures as described in example IV (FIG. 13). As shown in FIG. 13, it is demonstrated that the monoclonal antibody against Pre-S2 peptide is successfully displayed without reduction of the affinity to its antigen.
According to the above results, it is recognized that the present methods may be applicable to any protein, for example, enzyme, hormone, hormone analogue, enzyme inhibitor, signal transduction protein or its fragment, antibody or its fragment, antigen protein, attachment protein, structural protein, regulatory protein, toxin protein, plant defense-inducing protein.
V-3: Selection of Spores Displaying Single Chain Fv using Flow Cytometry
Whether the displayed single chain Fv has affinity to Pre-S2 of HBV was verified with FACSORT (Cell Sorter Flow Cytometer, Becton Dickinson, U.S.A.)as follows:
The transformed strains were inoculated into LB broth containing 5 μg/ml chloroamphenicol, pre-cultured for 8-10 hr at 37° C., 1% of seed culture was inoculated into GYS broth for sporulation, cultured for 24 hr at 37° C. and the cultured media was harvested. And then, 50 ml of harvested culture medium was centrifuged at 10,000 g for 10 min supernatant was discarded, bacteria were resuspended in 500 μl of 20% renografin (Sigma, USA). 100 μl of resuspended cell was carefully flowed onto 500 μl of 50% renogrant in microtube to form layer, the microtube was centrifuged at 10.000 g for 30 min and pure spores were isolated from pellet.
To discard remained renografin, spores were rinsed 3 times with DW and resuspended in PBS buffer. And then, spores displaying single chain Fv were mixed with wild type spores at a ratio of 1:103 and 1:105 and the spores with affinity to Pre-S2 of HBV were harvested using fluorescein-labeled Pre-S2 peptide and FACSORT (Cell Sorter Flow Cytometer, Becton Dickinson, U.S.A.).
The selectivity was evaluated by colony-forming assay on LB agar plates and LB agar plates containing 5 μl/ml of chloroamphenicol comparing to wild type. Spores displaying surface single chain Fv are resistant to chloroamphenicol owing to chloroamphenicol resistant gene contained in the recombinant vectors.
FIG. 14 shows the selectivity of spores displaying single chain Fv in each ratio (selectivity=ratio of spores displaying single chain Fv after flow cytometry/ratio of spores displaying single chain Fv before flow cytometry). In the case that the ratio of spores displaying single chain Fv before flow cytometry is 10−5, the selectivity was over 4,000, which indicates that spores with enhanced affinity can be selected by flow cytometry among spores displaying various antibody libraries.
V-4: Directed Evolution of Single Chain Fv Displayed on Spore Surface
To display single chain Fv library on spore surface, the gene encoding single chain Fv against Pre-S2 of HBV was amplified by error prone PCR. PCR was carried out using pAScFv101 plasmid described in the example V-1 as template and SEQ ID NOs:37 and 38 as primer. PCR mixture was prepared by mixing 0.3 μM of each primers, 5 ng of DNA template, PCR solution (10 mM Tris(pH 8.3), 50 mM KCl, 7 mM MgCl2, 0.01% (w/v) gelatin), 0.2 mM dGTP, 0.2 mM dATP, 1 mM dTTP, 1 mM dCTP, 5 U Taq polymerase from Bioneer (Korea) and DW up to 100 μl. Total 13 cycles of PCR was performed under condition of denaturation for 30 sec at 94° C., annealing for 30 sec at 50° C. and extension for 1 min at 72° C.
Subsequently, restricted PCR products with ApaI and NheI were cloned into pCSK-CotG, vector for displaying on spore surface, between the same restriction sites and library was prepared by transforming the cloned vectors into JM109 E. coli with the method of Inoue et al.
The vectors for displaying on spore surface were isolated by alkaline extraction method and transformed into Bacillus subtilis DB104 by natural transformation. And then, single chain Fv library against Pre-S2 of HBV was displayed on spore surface as described in example V-2 (FIG. 15).
As shown in FIG. 15, spores with increased fluorescence (i.e., increased affinity) were isolated. This result demonstrates the applicability of the present invention to prepare and select protein variants with improved characteristics.
Example VI
Bioconversion Using Spores Displaying Protein of Interest
Forte of transglycosylation by enzyme is the capability of formation of site-specific glycosidic linkage without protection/de-protection step. There have been studied for formation of glycosidic linkage by 1) induction of reverse hydrolysis in non-aqueous system using glycosidase which is conventionally available glycosidic hydrolyzing enzyme and 2) transglycosylation in which glycosidic linkage is substituted with receptor alcohol instead of hydrolysis of glycosidic linkage by water (G. Ljunger et al., Enzyme Microb. Technol., 16:1808-1814(1994); T. Usui et al., Carbohytdr. Res., 244:315-323(1993); and R. Lopez et al., J. Org. Chem., 59:737-745(1994)). The above conventional methods usually use organic solvent to increase synthetic yield and inhibit hydrolysis. However, because the organic solvent inactivates enzyme, it is difficult to accomplish the high yield. Thus, it is necessary to inhibit the inactivation of glycosidase in organic solvent for higher glycosylation yield.
The purpose of the Example is to exemplify the higher glycosylation yield with improved enzyme stability even in organic solvent by virtue of displaying glycosidase on the surface of hydrophobic Bacillus spores.
VI-1: Stability of β-Galactosidase Displayed on Surface of Spores in Organic Solvent
Each of β-galactosidase in free form (Sigma, USA) and the β-galactosidase displayed on surface of Bacillus spore was dispersed into 500 μl of Tris-HCl buffer (pH 7.5), added the same volume of the various solvents described in Table 1, mixed for 37° C. for 1 hr and the remained enzymatic activity was measured by Miller method described in Example I-3 (Table 1).










TABLE 1









Residual activity (%)











Free form
Surface-Displayed



β-galactosidase
β-galactosidase















Control
100
100



Hexane
84.3
100



Ether
48.2
77.2



Toluene
4.2
51.9



Ethylacetate
0.1
9.6



Acetonitril
0.0
0.8



Ethanol
0.0
0.0










As shown in Table 1, the displayed β-galactosidase shows higher stability than that of free form β-galactosidase in various organic solvents.
VI-2: Transglycosylation Reaction in Water-Organic Solvent Two-phase System Using β-galactosidase Displayed on Spore Surface
To perform transglycosylation in two-phase system, β-galactosidase, which is one of conventional glycosidase, is used as a model for glycosylation reaction (Scheme 1).




At first, 1 ml of 1 M lactose in 10 mM phosphate buffer (pH 5.1) was mixed with 10 ml of 10 mM 5-phenyl-1-pentanol in hexane for reaction solution. And then, β-galactosidase displayed on spore surface (240 U; 1 U=the amount of enzyme capable of hydrolysis of 1 μmol ONPG (o-nitrophenyl-β-D-galactopyranoside) for 1 min at 37° C.) and free form β-galactosidase (240 U) was added into the above reaction solution, respectively, and reacted for 48 hr at 30° C. while stirring.
In results, the yield of 5-phenylpenthyl-β-D-galtopyranoside was 21% by β-galactosidase displayed on spore surface; however, in free form β-galactosidase, the hydrolysis of lactose only occurred with no transglycosylation. Such result is ascribed to the increased stability, in organic solvent, of β-galactosidase displayed on spore surface. Actually, after 72 hr reaction, about 5% of enzyme activity was detected in the displayed β-galactosidase while measured the complete inactivation in free form β-galactosidase. Another advantage of the displayed β-galactosidase owes to hydrophobicity of Bacillusspores. In other words, the distribution of displayed β-galactosidase at interface between water and organic solvent phase inhibits the hydrolysis comparing to free form β-galactosidase.
Based on the results of this Example, it is understood that variants and modifications thereof falling within the spirit of the invention may become apparent to those skilled in the art, for example, any enzymes in addition to β-galactosidase such as lipase and protease can be employed for bioconversion of the present invention. In addition, the present bioconversion is useful in single step or multi-step reaction and in aqueous or non-aqueous solution. The present bioconversion method can employ spore as free or immobilized form and can be performed with other microbes or enzymes.
Example VII
Display of Antigen on Spore Surface
By displaying antigen on spore surface, antigen capable of inducing immune response in vivo can be applied as live vaccine. Bacillus subtilis has been considered as safe strain to human body since it has been employed in food fermentation for a long time (Sonenshein A. L., et al., Bacillus subtilis and other gram-positive bacteria. American society for Microbiology, Washington, p871(1993)).
Gene for CotE-antigen fusion protein is constructed by cloning the gene for surface antigen of HBV into pCotG-lacZ vector constructed in Example I-1. Thereafter, the constructed recombinant vector is transformed into Bacillus substilis and the transformants are cultured in GYS medium. And then, the antigen-displaying spores are purely isolated from culture medium by renografin gradients method.
Example VIII
Protein Improvement Using Spore Displaying Protein of Interest
For example of application of the present invention to high-throughput screening of target protein and to protein improvement, GFP (Green Fluorescence Protein) was used as follows:
VIII-1: Construction of Vector for GFP Display on Spore Surface
gfp gene was cloned into pCSK-CotG vector constructed in Example IV-1 and the following sub-cloning procedures were performed for display on spore surface. Each primer was prepared for the purpose of fusing cotG gene to EGFP and GFPuv genes. The fluorescence intensity of EGFP (Excit./Emis. Maxima (nm): 488/509; Clontech, USA) has 35-fold stronger than that of wild type GFP and thus results in detection even in FITC filter and GFPuv (Excit./Emis. Maxima (nm): 395/509; Clontech, USA) is detectable with UV. For further manipulation, NheI and HindIII restriction sites were inserted into primers for egfp gene (SEQ ID NOs:39 and 40) and PstI and EcoRI restriction sites were inserted in primers for gfpvu gene (SEQ ID NOs:41 and 42).
Each of egfp (800 bp) and gfpuv (720 bp) genes was amplified by PCR (MJ Research PTC-100TM programmable Thermal Controller; 95° C. 30 sec, 55° C. 30 sec, 72° C. 2 min, 25 cycles) using Pfu Turbo polymerase (Stratagene, USA) and pEGFP-C1 (Clontedch, USA) or pGFPuv (Clontech, USA) as template.
Thereafter, pCSK-CotG-EGFP or pCSK-CotG-GFPuv vectors were constructed by cloning the restricted PCR products into NheI/HindIII (egfp gene) or PstI/EcoRI (gfpuv gene) restriction sites of pCSK-CotG vector.
VIII-2: Display and Confirmation of GFP on Spore Surface
The constructed vectors were transformed into Bacillus subtilis DB104 by natural transformation. Transformants were selected on LB agar plate containing 5 μg/ml chloroamphenicol. Through the selection, Bacillus subtilis DB104-SDG-EGFP strain for display of EGFP and Bacillus subtilis DB104-SDG-GFPuv strain for display of GFPvu on spore surface were obtained. As control strains, Bacillus subtilis DB104-SDC strain transformed with only pCSK vector and Bacillus subtilis DB104-SDG strain transformed for expressing only CotG protein were prepared.
For analysis of GFP display on spore surface, the above Bacillus subtilis DB104-SDC, -SDG, -SDG-EGFP and -SDG-GFPuv were inoculated into LB broth containing 5 μg/ml chloroamphenicol and spores were then purified as described in Example V-4.
Subsequently, the display of GFP on spore surface was analyzed by measuring GFP fluorescence with flow cytometry in similar manner to Example IV (FIG. 16). In FIG. 16, curves (1)-(4) indicate the results of spores of DB104-SDC DB104-SDG, DB104-SDG-GFPuv and DB104-SDG-EFGP, respectively.
As shown in FIG. 16, the fluorescent intensity of spores derived from DB1047-SDG-EGFP (recombinant strain for EGFP-spore surface display) and DB104-SDG-GFPuv (recombinant strain for GFPuv-spore surface display) is significantly higher than that of DB104-SDC and DB104-SDG as control. In above results, the successful display of EGFP or GFPuv is validated by noticeable change of peaks indicating fluorescence in spore on its surface displaying EGFP or GFPuv comparing to controls.
VIII-3: Improvement of GFP
For the purpose of GFP improvement, error prone PCR was performed with template of pGFPuv vector (Clontech, USA) containing gfpuv gene using primers of SEQ ID NOs: 42 and 43. PCR mixture was prepared by mixing 0.3 μM of each primers, 5 ng of DNA template, PCR solution 10 mM Tris (pH 8.3), 50 mM KCl, 7 mM MgCl2, 0.01% (w/v) gelatin), 0.2 mM dGTP, 0.2 mM dATP, 1 mM dTTP, 1 mM dCTP, 0.15 mM MnCl2, 5 U Taq polymerase from Bioneer (Korea) and DW up to 100 μl. Total 13 cycles of PCR was performed under condition of denaturation for 30 sec at 94° C., annealing for 30 sec at 50° C. and extension for 1 min at 72° C.
Subsequently, the gfpuv genes were discarded from pCSK-CotG-GFPuv vectors by restriction with PstI/EcoRI, the above PCR-amplified inserts were cloned into the vectors with the same restriction sites and Bacillus substilis DB104 was transformed with the cloned vectors by natural transformation to construct gfpuv library displayed on spore surface. Then, the prepared library was inoculated into GYS medium for sporulation and pure spores were isolated as described in Example V-4. Transformant spores displaying improved GFP variant were screened by measuring GFP fluorescence with flow cytometry (FIGS. 17a to 17d). FIGS. 17a to 17d indicates the analysis of flow cytometry from Bacillus subtilis DB104-SDC, DB104-SDG-GFPuv, DB104-SDG-EGFP and DB104-SDG-GFP with gfp library subject to error prone PCR, respectively.
To isolate spores with higher fluorescent intensity than spores derived from DB104-SDG-EGFP and DB104-SDG-GFP control strains, the isolation of spores with higher fluorescence (region R1) among spores displaying GFP library was repeated several times.
It is understood that using the above method, the improved GFP protein exhibiting higher fluorescence intensity or fluorescence with different wavelength can be screened in a high-throughput manner.
Example VIII
Protein Array Using Spores Displaying on its Surface Protein of Interest
106-109 spores displaying monoclonal antibodies against surface antigen of HBV are attached onto glass substrate for protein array (BMS, Germany) with aldehyde functional group on its surface using automated array apparatus. The attachment is made in a form of covalent linkage, which is Schiff base between amino group of protein on spore surface and aldehyde group on surface of slide glass. Although the displayed proteins attached on solid surface may be inactivated, they may have an orientation.
The protein array kit manufactured according to 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.
Example IX
Production of Antibody Using Spores Displayig Antigen
The spores on its surface displaying surface antigen of HBV isolated in Example VII are suspended in PBS and the same volume of complete Freund's adjuvant is added. Thereafter, the mixture is well agitated to make emulsion formulation and the emulsion is injected i.v. into BALB/c mice with age of 6-8 week. After 4 weeks of the injection, the secondary administration is performed. Then, the additional boosting injection is performed about 2-3 times for induction of antibody.
As described above, the display method on spore surface of the present invention provides improvements in: a resistance against physiochemical change in environment of display host, a diversity of displayable proteins, a viability of display host and rapidity of screening.
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.
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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## Claims

1. A method for displaying carboxymethylcellulase on spore surface, which comprises the steps of:
(i) preparing a recombinant vector pCSK-cotG-CMCase for spore surface protein display comprising a vector pDG1728 and a gene encoding for fusion between CotG and carboxymethylcellulase
(ii) transforming a host cell with the recombinant vector for spore surface protein display;
(iii) displaying the carboxymethylcellulase on a surface of a spore of the host cell; and
(iv) recovering the spore displaying on its surface the carboxymethylcellulase.

2. The method according to claim 1 wherein the gene encoding a spore coat protein is derived from Bacillus.

3. The method according to claim 1 wherein the gene encoding a spore coat protein is cotG.

4. The method according to claim 1, wherein the host cell is a spore-forming Gram positive bacterium.

5. The method according to claim 4, wherein the host cell is Bacillus.

6. The method according to claim 1 wherein the recovering is performed in such a manner that the display of the carboxymethylcellulase on the spore surface is maximized by regulating culture time, after which culturing is terminated and the spore is then recovered.