WO2002095076A2 - Modified polypeptides having protease-resistance and/or protease-sensitivity - Google Patents

Abstract

The present teachings relate to modifying the carboxyl terminus (C-terminus) of a target polypeptide in order to thereby adjust the stability of the polypeptide with respect to protease. The present teachings also relate to modified polypeptides having protease-resistance sequence(s) attached to the C-terminal side of the target polypeptide. Protease-resistant sequences preferably include amino acid sequences that exhibit resistance (insensitivity) to proteases.

Description

MODIFIED POLYPEPTIDES HAVING PROTEASE-RESISTANCE AND/OR PROTEASE-SENSΓΓΓVITY

[0000]

This application claims priority to Japanese patent application numbers 2001-154321 and 2001-394821, the contents of which are incorporated herein by reference. [0001]

BACKGROUND OF THE INVENTION Technical Field

The present invention relates to techniques for changing (i.e., increasing and/or decreasing) the stability of polypeptides against proteases. In one aspect, the present invention relates to techniques for modifying the C-terrninal side of an exogenous polypeptide or a polypeptide that inherently has a low stability against proteases in order to increase the resistance of the polypeptide against proteases. [0002] Description of the Relevant Art

Various techniques are known for increasing the stability of polypeptides against proteases in order to use the polypeptides as anti-microbial agents. For example, when a polypeptide that is resistant to pathogenic microbes is utilized in plant tissue, the polypeptide also should be modified to provide resistance to at least the proteases originating from the plant host. [0003]

European Patent Publication No. 0 472 987 Al describes a method for addressing this issue. According to this known art, a polypeptide is first contacted with an intercellular fluid derived from plant cells and the resulting decomposition products are analyzed to determine the cleavage site. Next, a varie of peptides are synthesized in which the amino acid sequences to the front and rear of the cleavage site are modified or substituted. The synthesized peptides are then screened in order to identify stable peptides that also have anti-microbial characteristics. [0004]

According this approach, even if only one cleavage site identified, there are approximately 400 possible amino acid sequences that must be synthesized, Thus, a tremendous amount of time and labor is required in order to synthesize and then evaluate all of the possible amino acid sequences. Therefore, a long-felt need still remains for methods for making polypeptides that have been stabilized against proteases, as well as methods for reliably and easily preparing such stabilized polypeptides. [0005] SUMMARY OF THE INVENTION

The present inventors have studied polypeptide stability within protease-containing fluids. As a result, the inventors have identified polypeptide fragments that exist in a relatively stable manner in such protease-containing flαids. Such polypeptides preferably possess one or more protease resistant sequences at the C-terminus of the polypeptide. Therefore, one object of the present teachings is to provide amino acid sequences that increase the resistance of the polypeptide to protease activity. [0006]

In another aspect of the present teachings, methods are taught for increasing the protease-resistance of a target polypeptide. Such methods may include binding a protease resistant Sequence having at least two amino acids to the C-terminal side of the target polypeptide. The protease resistant sequence may preferably include a positively charged amino acid bound to a negatively charged amino acid by a peptide bond. Representative protease resistant sequences include Lys-Asp (K-D)₅ Arg-Asp (R-D), Lys-Glu ( -E), Arg-Glu (R-E), Asp-Lys (D-K), Asp-Arg (D-R), GIu-Lys (E-K) and Glu-Arg (E-R). A particularly preferred protease resistant sequence comprises Lys-Asp ( -D), Arg-Asp (R-D), Lys-Glu (K-E), Arg-Glu (R-E) and Asp-Lys (D-K). Fuπher preferred protease sequence comprises the sequence Lys-Asp (K-D). [0007]

In another aspect of the present teachings, methods are taught for increasing the proccasc-scnsitivϊty of a target polypeptide. Such methods may include binding a protease- sensitive sequence having at least two amino acids to the C-terminal side of the target polypeptide. Representative protease-sensitive sequences include Leu-Val, Asn-Leu, Arg-Asn, Leu-Arg and Phe-Leu. [0008]

In another aspect of the present teachings, polypeptides are taught chat include a protease resistant sequence having at least two amino acids bound to the C-terminal side of a target polypeptide. The protease resistant sequence preferably includes a positively charged amino acid bound to a negatively charged amino acid by a peptide bond. As noted above, representative protease resistant sequence include Lys-Asp (K-D), Arg-Asp (R-D), Lys-Glu (K-E), Arg-Glu (R- E), Asp-Lys (D-K), Asp-Arg (D-R), Glu-Lys (E-K) and Glu-Arg (E-R), A particularly preferred protease resistant sequence comprises Lys-Asp (K-D), Arg-Asp (R-D), Lys-Glu (K-E), Arg-Glu (R-E) and Asp-Lys (D-K). Further preferred protease sequence comprises the sequence Lys-Asp (K-D). [0009]

^• In another aspect of the present teachings, the protease resistant sequence may include a hydrophobic amino acid on the N-tem inal side of the protease resistant sequence, Further,. the protease resistant sequence may include one or more spaced repeats of Lys-Asp (K-D), Arg-Asp (R-D), Lys-Glu (K-E), Arg-Glu (R-E), Asp-Lys (D-K), Asp-Arg (D-R), Glu-Lys (E-K) and/or Glu-Arg (E-R). [0010]

In another aspect of the present teachings, the target polypeptide may contain a α-helix structure. In another aspect of the present teachings, the target polypeptide may be a cell-killing peptide, a portion thereof or a derivative thereof. [0011]

In another aspect of the present teachings, the target polypeptide may include at least one polypeptide sequence selected from the group consisting of CAP 18, magainins, melittins, alamethicins, poly ixins, cecropins, tachyplesins, dermaseptins, bombinins, and thionϊns, portions thereof and derivatives thereof. More preferably, the target polypeptide may include at least one polypeptide sequence selected from the group consisting of CAP18₁₀₆._I42, CAP18₁₀₆.₁₃₇, and CAP_10S.₁₂₉ originating from a rabbit, and CAP18_IM._μo, and CAP 18 _m._l3it originating from¹ a human. In this case, the protease resistant sequence may preferably include the sequence Lys-Asp (K-D). In the alternative, the target polypeptide may include at least one polypeptide sequence selected from the group consisting of the entire length of CAP18, a portion of CAP S, and derivatives of CAP 18, originating from a rabbit. A particularly preferred target polypeptide includes a polypeptide sequence from rabbit CAP18_]0&._]29. [0012]

In another aspect of the present teachings, bioactive polypeptides are taught that include a protease sensitive sequence bound to the C-teraiinal side of a polypeptide. Preferably, the protease-sensitive sequence comprises.at least two amino acids. Representative protease sensitive sequences include Leu-Val, Asn-Leu, Arg-Asn, Leu-Arg, and Phe-Leu. [0013]

The present teachings also provide the amino acid sequences indicated by SEQ ID NO: 5, 6, 8, 9, 10, 11 and 12 and amino acid sequences that are substantially, functionally equivalent thereto. [0014]

In another aspect of the present teachings, DNA constructs are taught that comprise at least one DNA sequence encoding any of the above-noted polypeptides, Further, vectors are also taught that comprise such DNA constructs. In addition, the present teachings provide transfσrrnants that carry such DNA constructs in a manner that enables the target polypeptide to be expressed in a host cell. In one preferred embodiment, the iransformant is a transformed plant cell and thus, the host cell is a plant cell. Furthermore, plant bodies comprising such transformed plant cells are also taught, as well as plant reproductive mediums comprising such transformed plant cells. [0015]

In another aspect of the present teachings, methods are taught for producing plant bodies. For example, such methods include regenerating a transformed plant cell carrying one or more of the above-noted DNA constructs. Such methods also may include cultivating one or more of the above-noted plant bodies. In cither case, preferred plant bodies include sweet potatoes. [0016]

In another aspect of the present teachings, methods are taught for making polypeptides. Such methods may include culturing one or more of the above noted transformants. [0017]

In another aspect of the present teachings, methods are taught for selecting or identifying protease resistant sequences. Such methods may include contacting a target polypeptide with a protease. Then, changes over time in the C-terminal amino acid sequences of the polypeptide fragments generated by decomposing the target polypeptide may be monitored. Thereafter, polypeptide fragments) may be selected for which decomposition of the C-terminal side is relatively slow. [0018]

Thus, in one aspect of the polypeptide stabilization techniques of the present teachings, one or more protease resistant sequence(s) is (are) preferably provided on the C-terminal side of a polypeptide in order to increase the stability of the polypeptide and preferably to impart resistance against proteases to the polypeptide. Such polypeptides preferably have, e.g., improved resistance against intercellular fluids and intracellular fluids, particularly against such fluids derived from plants. Therefore, by providing a polypeptide stabilized according to the present teachings in a useful cell or useful organism body including a plant cell or plant body, the organism can be effectively modified to exhibit the function inherently possessed by the polypeptide. [0019]

In another aspect of the polypeptide stabilization technology of the present teachings, a cell or organism can be effectively modified to exhibit the function inherently possessed by a polypeptide by expressing a DNA construct. The DNA construct preferably includes a DNA sequence that encodes a stabilized polypeptide for the particular cell or organism. The DNA construct preferably may be made, e.g., using recombinant DNA technology. [0020]

In another aspect of the polypeptide stabilization technology of the present teachings, disease resistance may be imparted to plants or other organisms. For example, the plant cells may be modified so as to carry a DNA construct in such a manner that a particular polypeptide can be expressed. The particular polypeptide preferably has been stabilized against the proteases of the particular plant. By stabilizing the particular polypeptide with respect to proteases, increased resistance to pathogenic organisms may be imparted to the plant. The DNA construct preferably includes a DNA sequence that encodes a protease-resistant polypeptide. According to these techniques, plants may be efficiently and safely produced that do not require the use of pesticides against pathogenic organism during growth. [0021]

Additional objects, features and advantages of the present invention will be readily understood after reading the following detailed description together with the accompanying drawings and the claims. [0022] BRIEF DESCRIPTION OF THE DRAWINGS FIG. I shows a chromatograph obtained by performing reverse phase chromatography on a reaction fluid containing CAP32 and sweet potato intercellular fluid.

FIG. 2 shows the decomposition over time of CAP32 (0 to 4.5 hours) in the sweet potato intercellular fluid.

FIG, 3 shows the decomposition over time of CAP3 (0 to 16 hours) in the sweet potato intercellular fluid.

FIG. 4 is a graph showing the stability over time of CAP32 and three CAP32 derivatives in a sweet potato intercellular fluid.

FIG. 5 is a table showing the amino acid sequences and the origins of all tested polypeptides in Example 14.

FIG. 6 is a table showing the anti-microbial properties and stability against protease degradation of indicated polypeptides in Fig. 5,

FIG. 7 is a graph showing the stability over time of rCAP24, rCAP24KD and Thionin in a sweet potato intracellular fluid, [0023] DETAILED DESCRIPTION OF THE INVENTION

The present teachings relate to techniques for modifying the carboxyl terminus (C- terminus) of a polypeptide in order to thereby control the stability of the polypeptide. In addition, the present teachings also rejate to polypeptides having a protease-resistant C-terminal side. For example, an amino acid sequence may be added to the C-terminal side of a target polypeptide in order to increase resistance (insensϊtivity) to proteases. According to the present teachings, "protease-resistant amino acid sequences" are intended to include sequences having at least two amino acids that exhibit resistance against decomposition (cleavage) from the C-terminal side caused by carboxy exoproteases, [0024]

Protease resistant amino acid sequences (hereinafter "protease-resistant sequences") may include, e,g., amino acid sequences having the following properties. For example, when attached to the C-terminus of a target polypeptide, the protcasc-rcsistancc sequence preferably increases the half-life of the target polypeptide as compared to target polypeptides that do not possess such protease-resistant sequences when each are exposed to a protease-containing fluid under identical conditions. Therefore, the protease-resistant sequences may be, e.g., a C-terminus sequence that increases the stability of the C-terminus of the target polypeptide to a protease-containing fluid. Further, the protease-resistant sequences include sequences that will become exposed at the C- terminus sequence of a fragment of the target polypeptide, thereby increasing the stability of the fragment with respect to proteases. The present protease-resistant sequences may, e.g., block protease from cleaving or degrading the target polypeptide. Thus, the stability of the target polypeptide may be substantially increased, even when the modified polypeptide is contacted with a protease-containing system. [0025]

In addition, polypeptides are taught that contain one or more protease-sensitive sequence(s) on the C-terminal side of the polypeptides. A protease-sensitive sequence is preferably an amino acid sequence that exhibits low resistance (sensitivity) to protease activity. [0026]

If one or more protease-resistant sequences are added (linked or bound) to the C-terminal side of a target polypeptide, degradation (cleavage) of the peptide chain by proteases can be significantly reduced. Consequently, even a exogenous polypeptide or a polypeptide that is easily decomposed.by protease can stably exist in a host cell or tissue and perform its functions in the host cell, because the exogenous polypeptide will be resistant to proteases originating from the host or a pathogen. Further, because the C-terminal side of a polypeptide typically contains a binding region for various physiologically active compounds and biomcmbranes, the physiological activity of the target polypeptide can be effectively protected (preserved) by appropriately protecting the C-terminal side of the target polypeptide from protease cleavage, [0027]

Polypeptides having a protease-resistant sequence on their C-terminal side are particularly useful, e.g., in a recombinant DNA expression system, in which an exogenous polypeptide will be produced (synthesized) inside a host cell. For example, such polypeptides may be particularly useful in an expression system for expressing polypeptides having anti-microbial activity and other functions inside the host cells or in a hose tissue having intercellular tissue between the host cells. In such cases, the functions of the polypeptide in the host cells or host tissue can be stably exhibited, because the exogenous polypeptide has an increased resistance to host proteases, [0028] Polypeptides with the proteasc-resϊsiant sequcnce(s) are also useful for short polypeptides and polypeptides that are expressed in small quantities in a recombinant expression system. The polypeptides can stably exist in sufficient quantities in order to effectively perform their function. [0029]

Representative polypeptides and representative methods for preparing polypeptides according to the present teachings will now be explained in further detail. For example, modified polypeptides may be prepared by attaching a protease-resistant sequence and/or a protease- sensitive sequence to the C-terminal side of a target polypeptide. Thus, modified polypeptides may include protease-resistant sequence(s) and or protease-sensitive sequence(s) on its C-terminal side. Also, the N-tcrminal side of the modified polypeptide may include an amino acid sequence for at least one polypeptide that increases the stability against proteases. In this specification, the term "modified polypeptide" is intended to encompass polypeptides that include at least two amino acids for increasing or decreasing the stability of a target polypeptide against proteases. Further, the terms "functional polypeptide," "bioactive polypeptide" and "target polypeptide" are intended to encompass polypeptides having a desirable bioactivity. These terms are intended to have substantially the same meaning and may be utilized interchangeably [0030]

Representative bioactive polypeptides include a variety of physiologically active polypeptide such as antibodies, hormones, cytokines, growth factors, inhibition factors, enzymes, and anti-microbials. Such polypeptides can be utilized for medical treatment, or as a diagnosis material, a medicinal material, an industrial material, or an agricultural material, which includes a disease resistant agent, Bioactive polypeptides may interact with the inner surface of a membrane (including a cell wall) of a target celL For example, either the membrane-operating polypeptide acting alone or the membrane-operating polypeptide acting together with a membrane component will impart some type of function to the cell, [0031]

For example, several types of bioactive polypeptide may be advantageously modified according to the present teachings. For example, cell-killing polypeptides kill target cells by exerting some type of effect on the cell such that the cell is disabled or the mitosis (cell division) functions of the cell are inhibited. Such cell-killing polypeptides include anti-microbial polypeptides, such as membrane-operating anti-microbial peptides that exhibit the ability to kill at the surface layer of target cells. Thus, such cell-killing polypeptides (anti-microbial polypeptides) may be advantageously modified according to the present teachings.,

[0032]

Representative examples of such membrane-operating cell-killing peptides, which may be advantageously modified according to the present teachings, include: CAP18 originating from humans or rats (James et al., Anti-microbial Activity of Human CAP18 Peptides, Immunotechnology 1 (1995), 65-72); Andropin, BLP-1, Bombinin, Bombolitin, Cecropins, Ceratotoxin A, Clavanin, CRAMP, Dcrmascptin 1 , Enbocinm, FALL-39, Lycotoxin I, Magainin I, Mejittin, Misgrin, PGLa, Pleurocidin, Se inalplasmin, Styelin, Abaecin, Apidaecin lA, Bactenecin 5, Diptericin, Drosocin, Enkelytin, Formaecinsin 1, Histatin I, Indolicidin, Lebocin 1, Mectchnikowin, PR-39, Prophenin, Pyrrhocoricin, Tenecin, Bovine dodccapeptide, Brevinin-1, Brevinin-IE, Esculcntin, Pipinin, Ranalexin, Thanatin, 11- k D polypeptide, etc. (Andru and Rivas, Animal anti-microbial peptides, Bipolymers (Peptide Science) VoL47, 415-433 (1998), Thionins, Defensins, Lipid Transfer Proteins (Garacia-Olemedo et al., Plant Defense Peptides, Bipolymers (Peptide Science) Vol.47, 479-491 (1998)), anti-microbial peptides originating from Amphibia of the genus Bombina, anti-microbial peptides originating from Xenopus Laevis, anti-microbial peptides originating from Amphibia of the genus Rana, anti-microbial peptides originating from Amphibia of the genus Phyllonedusa, anti-microbial peptides originating from Australian frogs (Anti-microbial peptides from Amphibian Skin: What Do They Tell Us?, Simmaco ct al., Bipolymers (Peptide Science) Vol.47, 435-450 (1998)), Cecropins, Magainins, Dermaseptins, Ala ethicϊns, Pardaxins (Oren ct al., Mode of Action of Linear Amphipafhic α-Helical Antimicrobial Peptides, Biopolymers (Peptide Science) Vol.47, 51-463 (1998)); and Insect Defensins, Drosomycin, Tachyplesins, Thahatins, Androctonins, Lysozymes, Androctonin, Proteglins, Penaeidins, Tachycitins, Mytilins, and Mytimycin (Dimarcq et al, Cystein-Rich, Anti-microbial Peptides in Invertebrates Biopolymers (Peptide Science) Vol.47, 465-477 (1998)). [0033]

Particularly preferred arc: CAP1S originating from humans or rodents (e.g., rat and rabbit), Magainins, Melittins, Alamcthicins, Polymixins, Cecropins, Tachyplesins, Dermaseptins, Bombinins, and Thionins, as well as the functional (i.e., cell-killing) portions of any of these polypeptides or derivatives of any of these polypeptides. [0034] Also preferred are bioactive polypeptides that have the ability to bind to a component substance found on the surface layer of a targeted cell. For example, such bioactive polypeptides may possess an ability to bind to a component substance of the cell wall. Representative examples of such component substances include: lipids, proteins, saccharides (including polysaccharides), glycoproteins, lipopolysaccharides, and glycolipids. Regardless of whether the component substance is a polysaccharide, a glycoprotein, a glycolipid, or a lipopolysaccharide, the binding target may preferably be a Sugar chain contained within the component substance. [0035]

Representative sugar chains may preferably be selected from a group that includes one or more of the following: acidic polysaccharides including teichoic acids and teichuronic acids, peptidoglycans, chitins, celluloses, hemicelluloses, lignins, pectins, and cholesterols. For example, if the target cells of the bioactive polypeptides are fungi (including filiform fungi), insects and/or crustaceans, chitjn is a preferred target component. Thus, chitin-binding polypeptides may be advantageously utilized as the bioactive or target polypeptide in such situations, because chitin does not typically exist in the surface layer of many types of cells. However, chitin is present with specificity and or in large quantities in cell walls of filiform fungi and other fungi, as well as the outer skins of insects and crustaceans.

If the target of the bioactive polypeptides is a gram-positive bacteria, peptidoglycan- binding polypeptides may be advantageously utilized, because peptidoglycans are present in large quantities in the cell wall of the bacteria. [0036]

Representative examples of such membrane-binding polypeptides include: various types of lectins, agglutinins, enzymes, anti-microbial peptides, etc. For example, polypeptides possessing the ability to bind chitin include: lectins, hevein, chitinase, Ac-AMP, and potato WIN. For example, such membrane-binding polypeptides include: wheat germ agglutinin isolectins, barley lectin, rice lectin, Uritica Dioica agglutinin, potato Iectin, tryptic peptide from potato lectin, hevein, wound-induced proteins from potato chitinase (WIN 1 , WIN2), bean basic chitinase, tobacco basic chitinase; lection, poplar wound-induced chitinase, Arabidopusis thaliana basic chitinase, rice basic chitinase, bean acidic pathogenesis-related 4 chitinase, maize seed chitinase, and Amaranthus caudatus anti-microbial peptide (Ac-AMP) (Lee et al., Structure and Functions of chitin-binding proteins, Annu.Rev.Plant.Physiol.Plant.Mol.Biol.1993.44:591-615). Moreover, peptidoglycan recognition protein (PGRP) (Proc, Natl. Acad. Sci. USA, Vol. 95, issue 17, pages 10078-10082, August 18, 1998) is another example of a polypeptide that binds to peptidoglycan. Membrane-penetrating proteins and GPI anchors that can bind to the above-described component substances also may be advantageously modified and utilized with the present teachings. [0037]

Other preferred bioactive polypeptides include modified polypeptides possessing an α- helix structure, because the C-teirminus of an α-helix structure generally has a low resistance to proteases. Thus, the present teachings may be advantageously utilized to increase the stability of such bioactive polypeptides against proteases. The majority of the above-noted physiologically active polypeptides possess a α-helix structure, [0038]

Furthermore, bioactive (target) polypeptides according to the present teachings are not limited to naturally occurring polypeptides. For example, artificially-synthesized polypeptides prepared, e.g., using recombinant DNA techniques or chemical synthesis, also may be advantageously modified according to the present teachings. Moreover, the entire sequence of a functional polypeptide may be utilized or only a partial sequence (sub-sequence) of the entire sequence may be utilized. If a partial sequence is utilized, the partial sequence preferably exhibits the desired functionality of the parent peptide. For example, the specific ρortion(s) of a peptide that exhibits a particular function (e.g., an anti-microbial function) can be used alone as the functional polypeptide in the present teachings. Derivatives thereof can also be used. [0039]

Representative examples of the above-noted CAP 18 include the LPS binding domain thereof, e.g., CAP18_106-M2, CAP18_10fi.₁₃₇, and CAP18_106-129 from rabbit and CAP18_I04.,₄₀, and CAP18_J04.,_3S from human. Particularly preferred CAP18 derivatives include CAPI8_{lW |29} from rabbit and CAP18_ιω._]3s from human. [0040]

Preferred bioactive polypeptides may also include polypeptides that are functionally equivalent (substantially identical) to one of the above-noted polypeptides or a functional portion thereof (e,g., the membrane-operating portion of the polypeptide). For example, such peptides preferably exhibit identical, or substantially identical, bioactivity to a reference (parent) peptide, but differ from the reference peptide in that one or more amino acids have been eliminated, replaced with a substitute, added and/or inserted. Preferred modified polypeptide that are functionally equivalent to a parent peptide can be produced using amino acid mutation technology or genetic engineering that is well known in this technical field. [0041]

Preferred polypeptides may possess two or more of bioactive polypeptides (regions). The two or more bioactive polypeptides can be the same type of polypeptide or a combination of different types of polypeptides. If a modified polypeptide includes two or more bioactive polypeptides (active sites), a cleavage site may be provided between two bioactive polypeptides so that the peptide regions can be separated from each other, e.g., by cleavage. Further, if the bioactive polypeptide is desired to operate in a linked condition, a Unking portion may be provided between polypeptides such that the two-dimensional structure or the three-dimensional structure required for the function of the target polypeptide is maintained. [0042]

Next, protease-resistant sequences and protease-sensitive sequences will be explained in further detail. According to research conducted by the inventors, the minimum sequence unit for a protease-resistant sequence includes at least two amino acids. The minimum sequence unit preferably is positioned close to the C-tcrminus of the target polypeptide in order to impart protease-resistance to the entire target polypeptide (modified polypeptide). [0043]

Generally speaking, amino acids can be separated into four classifications based upon the side chain R groups thereof. That is, amino acids may be classified as (1) hydrophobic amino acids, (2) polar amino acids that do not carry an electric charge (herein "polar amino acids¹"). (3) polar amino acids that do carry a positive charge at physiological pHs (herein "positively charged amino acids"), and (4) polar amino acids that carry a negative charge at physiological pHs (herein "negatively charged amino acids"). Based upon this classification, the minimum sequence unit according to the present teachings preferably includes a positively charged amino acid bound to a negatively charged amino acid. The two amino acids are preferably attached by a peptide bond. The arrangement of the two amino acids is not limited to one particular order. For example, the two amino acids may be arranged in the sequence of "positively charged amino acid - negatively charged amino acid" (sequence type I) or the sequence of "negatively charged amino acid - positively charged amino acid" (sequence type II). These arrangements are identified according to the order in the direction of approaching the C-terminal side from the N-terminal side. ' In the present specification, all explanations concerning amino acid sequences will follow this directionality convention, unless otherwise noted, [0044]

In one embodiment of the present teachings, type I units may include a Lys-Asp (K-D) sequence, an Arg-Asp (R-D) sequence, a Lys-Glu (K-E) sequence and an Arg-Glu (R-E) sequence. Type II units may include an Asp-Lys (D-K) sequence, an Asp-Arg (D-R) sequence, a Glu-Lys (E-K) sequence and a Glu-Arg (E-R) sequence. [0045]

Protease-resistant sequences according to the present teachings also may contain one or more arnino acids linked or bound to the N-terminal side of the minimum sequence unit. For example, a sequence of three amino acids (e.g., hydrophobic amino acid - positively charged amino acid - negatively charged amino acid) may be advantageously utilized. Such a sequence includes one hydrophobic amino acid disposed on the N-terminal side of the minimum sequence unit, Valine (Val) and isojeucine (lie) are noted as representative examples of appropriate hydrophobic amino acids according to this aspect of the present teachings. Furthermore, in such three amino acid sequences, the order of the positively charged amino acid and the negatively charged arnino can be changed into a type II sequence, as was discussed above. [0046]

Moreover, two hydrophobic amino acids may be provided on the N-tcrminal side of the minimum sequence unit. For example, a Lysinc (Lys)-Isolcucinc (Ac) sequence or a Leucine (Leu) - Valine (Val) sequence can be added to the minimum sequence unit according to the present teachings. [0047]

Protease-resistant sequences also may contain two or more of the above-noted minimum sequence units linked in tandem (e.g„ spaced repeats). In one representative example, the minimum sequence units may be directly Jinked or bound to each other and/or a suitable number of arnino acids may be disposed between the respective minimum sequence units. In the latter case, the interposed amino acids preferably include one or more hydrophobic amino acids, such as Leucine and/or Valine. More preferably, a Leu-Val sequence may be interposed between respective minimum sequence units. By interposing one or more arnino acids between respective minimum sequence units, protease resistance may be imparted to, e.g., an -heliχ structure of the bioactive (target) polypeptide and/or to the entire structure of the modified polypeptide. [0048]

As noted above, one or more protease-resistant sequences are preferably provided on the C-terminal side of the bioactive (target) polypeptide. The protease-resistant sequences are preferably provided on the C-terminal side adjacent to a bioactive region contained in the modified polypeptide. In this case, the active region can be reliably protected from cleavage or degradation by proteases. [0049]

Thus, protease-resistant sequences according to the present teachings may be directly linked Or bound to the bioactive region or a suitable number of a ino acids may be interposed between the bioactive region and the protease-resistant sequence(s). In addition, a portion of the protease-resistant sequence(s) also may be contained within the C-terminal side of the bioactive region. In this case, the protease-resistant sequence(s) preferably do not significantly reduce the bio-activity (functionality) of the bioactive (functional) region of the target polypeptide. Moreover, the protease-resistant sequence(s) preferably do not inhibit or significantly change the primary structure, the secondary structure and/or higher-level structures of the target polypeptide, which structures may be required to exhibit the function of the bioactive region. [0050]

For example, one or more of the following sequences may be advantageously utilized to impart protease-resistance to an α-helix structure in the target polypeptide: Lys-Asp (K-D), Arg- Asp (R-D), Lys-Glu (K-E), Arg-Glu (R-E), Asp-Lys (D-K), Asp-Arg (D-R), Glu-Lys (E-K) and/or Glu-Arg (E-R). The sequence Lys-Asp-Leu-Val-Lys-Asp is particularly preferred for imparting protease-resistance to α-helix structures. The protease-resistant sequences may comprise more than one sequence selected from this group or may consist (essentially or exclusively) of one Sequence selected from this group. [0051]

Peptide sequences that have a high sensitivity to protease activity typically include one of the following minimum sequence units: "hydrophobic amino acid - hydrophobic amino acid," "polar amino acid - hydrophobic a ino acid," "positively charged arnino acid - polar arnino acid," or "hydrophobic amino acid - positively charged amino acid." For example, representative minimum sequence units of protease-sensitive peptides include: Leu- Val, Asn-Leu, Arg-Asn, Leu-Arg, and Phe-Leu. These sequences have a higher sensitivity to protease activity than the minimum sequence units (protease-resistant sequences) according to the present teachings (e.g., Lys-Asp). Thus, by attaching one or more of the minimum sequence units of the present teachings to the C-terminal side of a bioactive polypeptide that is prone to protease cleavage or degradation, the stability modified polypeptide can be decreased. [0052]

In order to modify (adjust or change) the protease-resistance or protease-sensitivity of the functional polypeptide, the protease-resistant sequcncc(s) (i.e., one or more minimum sequence unit) and/or protease-sensitive sequence(s) are preferably provided on the C-tcrminal of the functional polypeptide. That is, the protease-resistant sequence(s) and/or protease-sensitive sequence(s) are preferably exposed at the C-tcrminal end of the resulting modified polypeptide. [0053]

However, modified polypeptides of the present teachings arc not limited to such an arrangement. For example, a suitable number of spacer arnino acids may be interposed between the protease-resistant sequence(s) and/or the protease-sensitive sequences and the C-terminal of the modified polypeptide. In one embodiment, the C-terminus sequence may function as a masking sequence, which masking sequence may include one or more a ino acid sequences having appropriate resistance and/or sensitivity toward proteases. By utilizing such a masking sequence, it is possible to increase or decrease the amount of time until proteases will degrade the protease-resistant sequences or control (modify or adjust) the activation timing of the bioactive polypeptide. Representative examples of such masking sequences include FLRNLV and PRTES, which amino acid sequences are represented according to the common convention for identifying arnino acids. [0054]

The masking sequence may include an amino acid sequence that is sensitive to endoproteascs on the C-terminal side of the protease-resistant sequence(s) or protease-sensitive scquence(s). In this case, the expected effect of the protease-resistant sequences and/or protease- sensitive sequences can be delayed until after an endoprotease has attacked (cleaved or degraded) the protease-sensitive site. Furthermore, an amino acid sequence ensitive to endoproteases may be provided on the N-terminal side of the protease-resistant sequences of a target polypeptide. In this case, the modified polypeptide will be stabilized by the protease-resistant sequcncc(s) before the endoprotease attacks the protease-sensitive site. Moreover, after the endoproteases have cleaved the protease-sensitive site, the active C-terminal region of the bioactive polypeptide will be exposed, Therefore, it is possible to suitably protect the C-terminal active region of the bioactive polypeptide and change or modify the timing at which the C-terminal active region exhibits its functionality. [0055]

Representative methods for selecting suitable protease-resistant sequences and protease- sensitive sequences will now be described in further detail. In one preferred method, such Sequences may be obtained by first selecting a target polypeptide, or a derivative thereof. The target or bioactive polypeptide is preferably a polypeptide for which it is desired to adjust the stability of the polypeptide against proteases. The target polypeptide may then be contacted with a protease. For example, the target polypeptide may be contacted with an aqueous solution that comprises the protease in a manner that will substantially replicate the intended environment to which the modified polypeptide will be exposed. Then, the protease-mcdiatcd degradation of the target polypeptide may be monitored over time and the resulting polypeptide fragments may be isolated. The resulting polypeptide fragments may then be analyzed in order to determine the arnino acid sequences on the C-terminal side of the fragments. [0056]

In other words, the reaction may be checked at various times after the protease has contacted the target polypeptide. Then, it is possible to identify polypeptide fragment(s) for which decomposition of the C-terminus is relatively slow. By identifying polypeptide fragments having relatively slow decomposition rates in the presence of protease, information concerning possible protease-resistant sequences can be obtained. Moreover, information regarding protease-sensitive sequences also can be obtained by identifying the polypeptide fragments having relatively fast decomposition rates. [0057]

In order to more precisely evaluate protease-resistance and protease-sensitivity, the decomposition half-lives of the respective polypeptide fragments having relatively slow and fast decomposition rates can be compared. For example, selected polypeptide fragments may be again contacted with the protease and the half-lives may be compared. Based upon this evaluation, preferred polypeptide fragments (and the C-tcrrninal sequences thereof) can be identified in order to obtain more precise information concerning protease-resistant sequences and protease-sensitive Sequences. [0058]

In another embodiment, protease-resistant sequences and protease-sensitive sequences can also be identified by truncating the C-cerminal side of one or two target polypeptides and exposing these truncated polypeptides to a protease. The decomposition half-life of the fragments may be compared and the C-terminal sides of the respective fragments may be sequenced in order to identify protease-resistance sequences and protease-sensitive sequences. [0059]

Appropriate protease-containing systems will differ depending on the type of target polypeptide being evaluated and the environment in which the target polypeptide will be expressed and/or will function. For example, if an anti-microbial peptide will be expressed and/ or will function in a plant cell or plant tissue, suitable protease-containing systems preferably include plant intracellular proteases, intercellular fluid proteases, proteases from pathogens, and/or other proteases. Such proteases may be extracted using known methods in order to prepare the protease-containing system. [0060]

Thus, the present methods for selecting protease-resistant sequences and/or protease- sensitive sequences differ from known techniques. For example, according to the present teachings, such sequences are identified based upon changes in degradation rates over time when a target polypeptide (either naturally-occurring or artificially-synthesized) is disposed in an appropriate protease containing system. On the other hand, in known techniques, the stability of target polypeptides was investigated by identifying protease recognition (cleavage) sites, generating (synthesizing) a variety of modified polypeptides, and evaluating the stability of the respective modified polypeptides. Therefore, the present methods permit more efficient identification and evaluation of protease-resistant and protease-sensitive sequences (especially for protease-resistant sequences), Further, the selected sequence(s) preferably exhϊbit(s) sufficient protease resistance or sensitivity with high reliability. [0061]

The protease-resistant sequences and protease-sensitive sequences according to the present teachings can function specific to polypeptides on its C-terminal side. Therefore, if the sequences are provided on the C-terminal sides of other N-terminal arnino acid sequences, the function can be exhibited in the same or similar manner. Thus, the sequences and methods of the present teachings permit the simple and quick modification of the stability of a target polypeptide and a wide range of stabilities can be attained according to the present teachings, [0062]

Representative methods for preparing modified polypeptides according to the present teachings will now be explained. For example, modified polypeptides may be obtained by manipulating a bioactive polypeptide such that a protease-resistant sequence and or a protease- sensitive sequence is attached to the C-terminal side of the target polypeptide. Various known methods may be utilized with the present teachings and the present teachings are not particularly limited in this respect. [0063]

In one representative example, modified polypeptides may be obtained by adding a protease-resistant sequence and/or a protease-sensitive sequence to the C-tejrminal end of a bioactive (target) polypeptide. A protease-resistant sequence and/or a protease-sensitive sequence may be included within the C-terminal side by inserting, substituting, adding, and/or eliminating a ino acids to and from the amino acid sequence on the C-terminal side of the bioactive polypeptide. Furthermore, if the C-terminal side of the bioactive polypeptide naturally includes a protease-resistant sequence and or a protease-sensitive sequence, the protease-resistant Sequences and/or the sensitive sequence can be exposed at the C-terminus by truncating the C-terminal side. In any of these cases, the protease-resistant sequences preferably do not reduce, or substantially reduce, the activity of the active sites included within the bioactive (target) polypeptide. In one representative embodiment, amino acids may be substituted, inserted, added, and or eliminated without disturbing the secondary and tertiary structures of the bioactive (target) polypeptide. [0064]

Although modified polypeptides, of course, may be prepared by chemical synthesis, preferred methods include culturing protein production cells obtained by genetic manipulation (i.e., recombinant DNA techniques). In the latter case, the target polypeptide (or a derivative thereof) may be first obtained from the culture and then the protease-resistant sequence(s) and/or protease-sensitive sequence(s) may be added by a chemical synthesis process after the unprotected portions (bioactive polypeptide) have been separated/purified from the cultured cells. Furthermore, the modified polypeptides also may be prepared by introducing a vector into a host cell. The vector may carry a DNA sequence encoding the full length of the modified polypeptide. In this case, the entire modified polypeptide can be expressed by culturing the host cells. [0065]

Representative DNA constructs according to the present teachings may include nucleotide sequences that encode the bioactive (target) polypeptide and one or more protease- resistant sequencc(s) and/or protease-sensitive sequences. Representative DNA constructs also may include one or more nucleotide sequences that encode the amino acids that link (bind) the bioactive (target) polypeptide to the one or more protease-resistant sequence(s) and/or protease- sensitive sequences, as well as other amino acids. As was described above, DNA constructs according to the present teachings are not limited to naturally-occurring DNA. For example, DNA constructs according to the present teachings may include a DNA sequence that has been modified by addition, insertion, elimination, and/or replacement of one or more of the naturally occurring nucleotides. Naturally, chemically-synthesized DNA also may be utilized with the present teachings. [0066]

Furthermore, in addition to DNA sequences encoding one or more modified polypeptides according to the present teachings, the DNA construct also may include a secretion signal sequence that is suitable for the host cell. In this case, it will be easier to localize the desired polypeptide on the cell surface for simple transfer to the outside of the cell or to secrete the desired polypeptide to the outside of the cell, [0067]

DNA constructs according to the present teachings also may include one or more DNA sequences for expressing the DNA sequence that encodes the modified polypepride(s). For example, a cis-regulatory element (e.g., a promotor sequence) may be disposed upstream of the sequence encoding the target or modified polypeptide, a termination sequence may be disposed downstream of the encoding sequence, and/or a polyadenylation signal may be utilized. No particular restrictions are placed on the cis-regulatory elements and various types of cis-regulatory elements may be utilized with the present teachings. However, the cis-regulatory element(s) preferably include a sequence that causes the encoded sequence to be expressed in the host cell. An enhancer can also be included. Preferably, a marker gene for selecting cells carrying the DNA construct also may be introduced into the DNA construct at the same time. The selection marker gene sequence may be linked or bound directly to the DNA construct or may be introduced by co-transfection. [0068]

The DNA construct may be integrated into an appropriate vector. Thus, a vector may carry the DNA construct and the vector may serve as an expressing vector for the modified polypeptide. Appropriate vectors may be selected based upon the type of host cell and gene introduction method, Generally speaking, plasmid vectors, virus vectors, YAC, B AC, PAC, MAC, etc., may be advantageously utilized with the present teachings. Suitable prokaryotic cell vectors, eukaryotic cell vectors, animal cell vectors, and plant cell vectors are well known in the field and need not be discussed in detail herein. [0069]

Once a DNA construct or a vector has been constructed, it can be introduced into an appropriate host cell using an appropriate method, e.g„ transformation, transfection, conjugation, protoplast fusion, electroporation, particle gun, calcium phosphate precipitation, agrobacterium method, or direct microinjection. After the polynucleotide or vector has been introduced, the host cell may be cultured with a selective medium, [0070]

There are no particular restrictions on the types of host cells that may used to obtain the transformed cell. All types and modes of cells may be utilized, including various types of prokaryotic cells of bacteria, eukaryotic cells, such as fungi, yeasts, animals, and plants; plant and animal hosts, eukaryotic cells, and tissue and organs of eukaryotic cells. Viruses also may be utilized. Furthermore, reproductive media (e.g., seeds, rhizomes, fruit, and grains) also may be utilized. [0071]

Transformed cells producing the modified polypeptide can be obtained by selecting cell(s) carrying the DNA construct using an appropriate selection method. The genes introduced into the transformed cells can be maintained either inside or outside the chromosome in the host cell. Gene expression may be either temporary or stable, When the transformed cells are cultured and multiplied under appropriate culturing conditions, the transformed cells will produce polypeptides within the cells, at the cell surface, or on the outside of the cells. The produced polypeptides can then be collected and purified using an appropriate method.

[0072]

Furthermore, transformed plant cells, tissue, organs, individuals, and reproductive media are useful for expressing modified polypeptides of the present teachings. For example, modified polypeptides of the present teachings can be produced and can express their inherent function effectively in plant cells, tissues, organs and reproductive media. If the modified polypeptide contains an anti-pathogenic polypeptide for an N-terminal side amino acid sequence, the modified polypeptide will exhibit resistance against pathogenic organisms and components derived from such pathogenic organisms inside the plant cells and/or tissues. In particular, it has been a serious problem that anti-microbial polypeptides easily decompose due to protease being present in intracellular fluid and intercellular fluid of plants, as well as proteases derived from pathogens. However, the present modified polypeptides, which include one or more protease-resistant sequences, possess a resistance against these proteases and can exhibit microbial properties during the initial stage of pathogen infection, thereby effectively preventing chain-reaction disorders thereafter. [0073]

For example, plant transformants may be prepared from plant cells, such as various types of plant cells having or not having regeneration capabilities within plant bodies. Cells having suitable regeneration capabilities are not limited in the present teachings, and may include: cultured cells, protoplasts, shoot primordiums, multiple-bud bodies, hair root, and callus. [0074]

Also, although no particular restrictions arc placed on the type of plant species that may be transformed using the present teachings, plants utilized with the present teachings preferably include cultivated products, and more preferably, agricultural products. For example, preferred plants include: grains, such as rice, barley, wheat, rye, maize, or sugarcane; rhizomes or root tubers, such as potato or sweet potato; bean plants, such as soybeans, kidney beans, lima beans, or peas; seed producing plants, such as peanuts, sesame, rape seed, cottonseed, sunflower seed, or safϊlower seed; fruit producing plants, such apple, melon, or grape; or other plants, such as tomato or eggplant. Thus, flower plants and other garden plants may be used with the present teachings. [0075] By regenerating the transformed plant cells, the cells can be converted into a plant body. Regeneration methods differ depending on the type of plant and a variety of publicly known methods can be used for this aspect of the present teachings. [0076]

If the transformed plant body is an agricultural food product, the plant itself will comprise a food product or nutritional food product that can be administered to vertebrates, including humans. [0077]

The present teachings also provide methods for controlling plant pathogenic organisms. Such methods may include introducing a modified polypeptide of the present teachings into an environment in which plant pathogenic organisms exist. Such environments include an environment in which a plant suffers from a disorder or there is a possibility that the plant will be attacked by a plant pathogenic organism. Therefore, the present modified polypeptides may be utilized for both prevention and extermination. For example, the modified polypeptides of the present teachings can be introduced into various environments, such as a cultivation area for rice, sweet potatoes, or other cultivated products; a cultivation area for decorative garden plants; a home vegetable garden; and mountains and forests. [0078]

The modified polypeptides of the present teachings may be introduced into an environment by administering the polypeptide according to one or more of the following methods: scattering, spraying, or applying as is to the soil and plants in a cultivation region; cattering transformed cells or causing transformed cells to multiply; transforming plants for cultivation such that the modified polypeptide can be expressed in the transformed plants, and administering by cultivating. Modified polypeptides can also be administered in a post harvest manner to harvested products. Therefore, the present teachings also provide methods for producing cultivated plants that involve introducing one or more modified polypeptides of the present teachings to the cultivation environment of the cultivated plants by any of these various methods. [0079]

Anti-microbial polypeptides may posses anti-microbial activity against a microbe, such as bacteria (e,g., Escherichia coli) and fungi (e.g„ filiform fungi). For example, the anti-rnicrobial polypeptide may possess activity against pathogenic microorganisms, such as anti-microbial or resistance against Ceratocystϊs fimbriata, which causes purple blotch in sweet potatoes. [0080]

Such polypeptides having anti-microbial and other disease-resistant properties preferably may be highly stable against proteases in order to maintain their anti-microbial activity. The minimum sequence unit of the above-noted protease-resistant sequences (e.g., Lys-Asp (K-D), Arg-Asp (R-D), Lys-Glu (K-E), Arg-Glu (R-E), Asp-Lys (D-K), Asp-Arg (D-R), Glu-Lys (E-K) and Glu-Arg (E-R)) can stabilize a variety of polypeptides and derivatives thereof without reducing the activity thereof or may provide a substantially equivalent activity. For instance, bioactive polypeptides, such as various CAP-18s, the LPS binding domain and anti-microbial domain on the C-terminal side of CAPlδs (CAP18_{10 42}, CAP18_1Q6._I37, CAP18_K)6.₁₂₉ from rabbit and CAP18_lw._la0, CAP18_1(>a.i₃₅ from human), may be advantageously modified according to the present teachings. Furthermore, by introducing a protease-resistance sequence, the activity of CAPI8 and the above-noted active regions thereof can be substantially maintained and the active regions can be stabilized (i.e., protease-resistance may be imparted). In addition, these protease- resistant sequences may also maintain (or stabilize) the α-helix structure of the active regions. Preferably, the protease-resistant sequence(s) is (are) placed as close as possible to the C-terminal side of the active regions so that the α-helix structure of the active domains can be maintained. For example, protease-resistant sequences may be directly linked or bound to the C-terminal of the active region, or a portion of the C-terminal of the active region may be modified such that the ^■ sequence is provided therein. [0081]

For example, useful modified peptides may be obtained from a polypeptide that includes human CAP18 or a derivative thereof (e.g., CAP18_ιω._U0 and CAP18_Iθ4._I35) or rabbit CAP18 or a derivative thereof (e.g., CAP18_lofi.₁₂₉). Modified polypeptides preferably include the bioactive (target) polypeptide and at least one minimum sequence unit, e.g., Lys-Asp, Asp-Lys, Arg-Asp, Lys-Glu, Arg-Glu, or Lys-Asp-Leu-Val- Lys-Asp, that is preferably attached to the C-terminal side of the bioactive polypeptide in a manner that exposes the minimum sequence unit(s). Preferred polypeptides include human CAP18_104.135 rabbit CAP18_ια6_,₂g ^anc* derivatives thereof as the bioactive (target) polypeptide. One or more protease-resistant sequences are preferably linked or bound directly to these active domains. Also, Lys- Asp-Leu- Val-Lys- Asp may preferably be substituted at position no. 132 or higher in the active domain of human CAP 18. [0082]

Suc anti-microbial polypeptides are particularly useful against Ceratocystis fimbriata, which causes purple blotch in sweet potatoes, and/or Escherichia coli, and can be used as antimicrobial agents against these bacteria, [0083]

The effectiveness of the modified polypeptide can be further increased according to the present teachings. For example, after purifying the target polypeptide from a transformed cell, the polypeptide can be modified such that an active secondary or tertiary structure can be maintained. It is also possible to change the electric charge of the polypeptide or bind the polypeptide to another peptide. [0084]

Each of the additional examples and method steps disclosed above and below may be utilized separately or in conjunction with other features and method steps to provide improved protease-resistant and or protease-sensitive polypeptides, Representative examples of the present invention, which examples utilize many of these additional features and method steps in conjunction, will now be described in detail with reference to the drawings. This detailed description is merely intended to teach a person of skill in the art further details for practicing preferred aspects of the present teachings and is not intended to limit the scope of the invention. Only the claims define the scope of the claimed invention. Therefore, combinations of features and methods disclosed In the following detail description may not be necessary to practice the invention in the broadest sense, and are instead taught merely to particularly describe some ^' representative examples of the invention, which detailed description will now be given with reference to the accompanying drawings, [0085] Example ϊ Human peptide - CAP 18 (L104 to V 135) (hereinafter "CAP32")

The CAP32 peptide derived from human has the amino acid sequence indicated at SEQ ID NO: 1 of the attached Sequence Listing. This CAP32 peptide was synthesized for the applicants by Sawaday Technology Co., Ltd. based upon the applicant's instructions. The CAP32 peptide utilized in the following examples had a purity of 90% Or higher. [0086] Example 2

Extraction of intercellular fluid from sweet potato leaves

A leaf from a sweet potato (kokei No. 14) was cut into approximately 5-mm squares and then washed with a cleansing fluid (10 M KH₂PO₄, pH 6.0). The cleaned pieces of cut leaves were soaked in an extract liquid (50 mM MgCl₂) and subjected to reduced pressure for 10 minutes using a vacuum pump. After the pressure reduction treatment, the leaves were placed in a 5-mL syringe mounted within a centrifuge tube and centrifuged for 10 minutes at 4500 g. The liquid extracted from the leaves in the syringe was collected and used as a sweet potato intercellular fluid (ICF) in the following examples. [0087] Example 3 C AP32 decomposition in sweet potato ICF

45 micrograms of the anti-microbial protein CAP32, which was prepared in Example I , were added to 300 microlitcrs of 12.5% of sweet potato ICF solution, which was prepared in Example 2, and permitted to react for 2.5 hours at 26.5^* C The resulting survival rate of intact CAP32 was 10% or less. [0088] Example 4 Separation of decomposition products by reverse phase chromatography

Similar to Embodiment 3, 45 micrograms of CAP32 were added to 300 microliters of 12.5% of sweet potato ICF solution and allowed to react for 2.5 hours at 26.5^"C. The resulting products were then separated by reverse phase chromatography under the conditions described in the following paragraph. As a result, three purified CAP32 decomposition products (PI, P2, and P3) were obtained, The chromatograph of these three decomposition products is shown in FIG,'l. [0089]

The conditions of the reverse chromatography were as follows. A Source 15RPC ST 4,6/100 column (Amasharm Pharmacia Biotech) was used for the separation. Solution A (5% acetonitrile, 0.1% TEA) was used as an initial buffer and a gradient was applied starting from 100% solution A to 100% solution B (70% acetonitrile, 0,1 TFA) in order to dissolve the decomposition products. Polypeptide detection was performed at UV214nm. [0090] Example 5

Identification of N-terminal amino acid sequences of decomposition products

Decomposition products PI, P2, P3, which were purified in Example 4, were each freeze-dried and then dissolved in acetonitrile. The dissolved decomposition products were then sequenced in a known manner using a protein sequencer (Applied Biosystems, ABI373 A) in order to determine the N-tcrminal amino acid sequences. [0091] Example 6 Mass spectroscopy of decomposition products and identification of decomposition products

Decomposition products PI, P2, P3, which were purified in Example 4, were each freeze-dried and then mass spectroscopy (machine used; MALDI-TOF/MS (Matrix Assisted Laser Desorption lonization)) of the products was performed by Sawaday Technology Co., Ltd. based upon the applicant's instructions. Together with the N-terminal amino acid results, the amino acid sequences of the decomposition products PI, P2, and P3 were determined and are shown in Table I . Thus, PI was identified as CAP (L104-D129) (hereinafter "CAP26"). P2 was identified as CAP (L104-R132) (hereinafter "CAP29"). Finally, P3 was identified as CAP (LI04-N133) (hereinafter "CAP30"). The respective a ino acid sequences of CAP26, CAP29 and CAP30 are indicated at SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4 of the attached Sequence Listing. [0092] Table 1

[0093]

Example 7

Decomposition of CAP32 into decomposition products CAP30, CAP29, and CAP26

15 micrograms of CAP32 were added to 100 microliters of 12.5% sweet potato ICF solution (Example 2) and permitted to react for 16 hours at 26.5^° C. The peak area values of CAP32, CAP30, CAP29, and CAP26 based upon reverse phase chromatography were calculated in order to determine the respective quantities of each of these components over time. The time- based changes in the accumulated amounts of CAP32, CAP30, CAP29, and CAP26 were then investigated. For example, CAP30 was detected in the initial stage of the decomposition and showed a peak accumulated amount at 1.5 hours. Further, the accumulated amount of CAP29 rose and peaked after 2 hours. Finally, the accumulated amount of CAP26 also rose. FIG. 2 shows the time-based changes that occurred between 0 and 4.5 hours. [0094]

During the reaction period (16 hours), the accumulated amount of CAP26 peaked at 8 hours, None of CAP32, CAP30, or CAP29 was detected at that time. FIG. 3 shows the time- based changes that occurred between 0 and 16 hours. These results suggest that CAP32 decomposes sequentially from its C-tcrrninus. Further, CAP26 shows high stability, thereby suggesting that the KD sequence existing on the C-terminus of CAP26 functions as a stabilizing (i.e., protease-resistant) sequence in sweet potato intercellular fluid. [0095] Example 8 Synthesis of CAP32KD and CAP32KDKD

CAP32KD (SEQ ID NO: 5) and CAP32KDKD (SEQ ID NO: 6) were synthesized by Sawaday Technology Co., Ltd. based upon applicant's instructions. Both synthesized polypeptides had a purity of 90% or higher, [0096] Example 9 Evaluation of stability (protease-resistance) of CAP32, CAP32KD, CAP32KDKD, and CAP26

15 micrograms of CAP32, CAP32KD, CAP32KDKD, and CAP26 were respectively added to separate tubes each containing 100 microliters of 12.5% of sweet potato ICF solution (Example 2) and permitted to react for 16 hours at 26.5 . For each test specimen, reaction fluids were collected after predetermined periods of time had elapsed. Reverse phase chromatography was performed under the same conditions as in Example 4 and the peak area values for CAP32, CAP32KD, CAP32KDKD and CAP26 were determined. In addition, the amount of intact peptide remaining within the respective reaction fluid after reacting for a prescribed period was quantified by utilizing the concentration of the intact peptide in the pre-treatmeπt sample as the 100% reference value. As a result, it was determined that the amount of time for 50% decomposition

(half-life) of each component was 6 minutes for CAP32, 5-8 hours for CAP32KD, and 8 hours for

CAP32KDKD. The latter two peptides respectively showed stabilities of 50X and 80X as compared to CAP32. CAP26 also had a half-life of 8 hours, which is probably due to the KD sequence at its C-terminus. These results are shown in FIG. 4.

[0097]

Example 1

Evaluation of anti-microbial properties of CAP32, CAP32KD, CAP32KDKD, and CAP26

A.¹ Filiform Fungi

30 microlitcrs each of the respective solutions of CAP32, CAP32KD, CAP32KDKD, and CAP26, which were diluted to their respective concentrations, 10 microliters of 200-mM MES buffer fluid (2-(N-morpholino) ethanesulfonic acid) having a pH of 6.0, and 60 microliters of a conidiospore suspension (1 * 1Q⁵ spores/mL in 80% potato dextrose broth) of Ceratocystis fimbriata IF0 30501 (the fungus that causes purple blotch in sweet potatoes) were placed into respective wells of a 96-welI micro-plate and cultured at 26" C. Using a micro-plate reader (Bio- Rad), the OD4I5 was measured 48 hours later and the minimum inhibition concentration ("MIC") was calculated. [0098] B. Bacteria

E. coli (DFO 3301) was inoculated into a bullion culture medium (Nissui Pharmaceuticals Co,, Ltd.) and cultured at 37 °C until the OD660 value became 0,3. Then, the cultured medium was diluted so as to be 0,01 of OD660 with said bullion culture medium. 5 microliters of this bacterial fluid, 500 microliters of the bullion culture medium, and 10 microliters of each of the above-noted peptide solutions (diluted to a prescribed concentration) were added to a test tube and then cultured for 16 hours at 37^*C. After culturing, the OD660 was measured and the minimum inhibition concentration was calculated. These results are shown in Table 2. [0099] Table 2

[0100]

As shown in Table 2, the MIC with respect to filiform fungi was 30 to 100 micrograms/mL, i.e,, roughly the same for all of the peptides. The MIC with respect to filiform fungi of CAP26, which Is a decomposition product of CAP32, was also 30 to 100 micrograms/mL, thereby indicating that the anti-microbial activity of CAP32 was maintained in decomposition product CAP26. [0101]

Further, with respect to the anti-microbial activity to bacteria, CAP32KD showed a slight decline jn anti-microbial activity, while CAP32KDKD maintained an equivalent antibacterial activity. CAP26 showed a large decline in its antibacterial activity against bacteria. [0102]

Therefore, both CAP32KD and CAP32KDKD maintain a level of anti-microbial activity against filiform fungi that is equivalent to the anti-microbial activity of CAP32. Further, both CAP32KD and CAP32KDKD have the same effectiveness against filiform fungi as CAP32, In addition, both polypeptides CAP32KD and CAP32KDKD have improved stability against proteases. Finally, it was learned that CAP32KDKD particularly maintains anti-microbial activity against bacteria equivalent to CAP32 and is even more useful than CAP32. [0103] Example 11 Synthesis of analogs rCAP24 and rCAP24 rCAP24 is an active fragment of the polypeptide CAP18, which was originally derived from rabbit, and has the amino acid sequence indicated at SEQ ID NO: 7 of the attached Sequence Listing. rCAP24 and derivatives thereof (rCAP24-KD, rCAP24-DK, rCAP24-KE, rCAP24-RD, rCAP24-RE) were synthesized by Shimazu Scientific Research Inc. based upon applicant's instructions. The amino acid sequences of these derivatives are respectively indicated at SEQ ID No: 8., 9, 10, 11 and 12 of the attached Sequence Listing. Each synthesized polypeptide had a purity of 90% Or higher.

[0104]

Example 12

Evaluation of tability (protease-resistance) of rCAP24 and derivatives thereof

15 micrograms of each of rCAP24 and its derivatives were respectively added to separate tubes each containing 100 microliters of 12.5% of sweet potato ICF solution and were permitted to react for 16 hours at 26.5" C. For each test specimen, reaction fluid was collected after predetermined periods of time had elapsed. Reverse phase chromatography was performed under the same conditions, as in Example 4 and the peak area values for rCAP24 and its derived were obtained. In addition, the amount of intact peptide remaining within the respective reaction fluids after reacting for a prescribed period was quantified by utilizing the concentration of intact peptide in the pre-treatment sample as a 100% reference value. As shown in Table 3, the results show that after 5 hours in the sweet potato ICF, the remaining amount of (the percent of intact) rCAP24 was 0%. However, the amount of intact rCAP24 analogs (derivative) was 70% or higher in each case. Also, it was learned that rCAP24DK has a particularly high stability (protease-resistance) among the tested analogs (derivatives). [0105] Table 3

[0106]

Example 13

Evaluation of anti-microbial properties of rCAP24 and its analogs

Evaluations of the anti-microbial properties to Fungi and Bacteria were performed under the same conditions as in Example 10 and the results are also shown in Table 3. Specifically, as shown in Table 3, rCAP24 and its analogs respectively had a MIC with respect to filiform fungi of 3 to 10 micrograms/mL. Further, rCAP24 and its analogs respectively had a MIC with respect to bacteria of 1 to 3 micrograms/mL. Accordingly, there were no significant differences in antimicrobial activities between rCAP24 and its analogs,

[0107] Example 14

Evaluation of anti-microbial properties and Stability (protease-resistance) in sweet potato ICP of rCAP24KD and other anti-microbial polypeptides rCAP24KD as well as other microbial polypeptides (commercially or synthetically available) were evaluated with respect to anti-microbial properties and stability against protease degradation. The amino acid sequences and the origin of all tested polypeptides are indicated in Fig. 5. With the exception of Thionin (Takara, Corystein (product name)), and MYP30 (chemically synthesized), all anti-microbial polypeptides were supplied by American Peptide Company.

[0108]

In addition, evaluations of anti-microbial properties to Fungi and Bacteria were performed under the same conditions as in Example 10. In order to prepare appropriate test solution to determine the anti-microbial properties, the respective polypeptides were diluted to prescribed concentrations with sterilized water. Evaluation of the stability in ICF was performed under the same conditions as in Example 12. The results were showed in Fig. 6.

[0109]

As shown in Fig. 6, rCAP24KD had the highest anti-microbial activity and stability among the 14 types of anti-microbial polypeptides. Consequently, rCAP24KD is clearly a preferred modified polypeptide in view of all requirements for an anti-microbial polypeptide. [0110] Example 15 Extraction of intracellular fluid from sweet potato leaves

A leaf of a sweet potato (kokei No. 14) was cut into approximately 5-mm squares and washed with a cleansing liquid (10 rnM, KH₂PO₄, pH 6.0). The cleaned pieces of cut leaves were soaked in an extract liquid (50 mM MgCI₂) and subjected to reduced pressure for 10 minutes using a vacuum pump. After the pressure reduction treatment, the leaves were placed into a 5-mL syringe mounted in a centrifugϊng tube and centrifuged for 10 minutes at 4500 g. The liquid extracted from the leaves was removed from the syringe and then the remaining leaves were used for another extraction. An extracting buffer (50mM Tris-HCl (pH 8.0), 5mM EDTA, 0.25 mM

Sucrose, lOmM DTT) was added into the remaining leaves in an amount twice the volume of the remaining leaves and the resulting mixture was ground in a mortar. Thereafter, the mixture was centrifuged for 10 minutes at 15,000 rpm. The supernatant was collected and used as an intracellular fluid.

[0111]

Example 16

Evaluation of stability (protease resistance) of rCAP24, rCAP24KD and Thionin

15 micrograms each of rCAP24, rCAP24KD and Thionin were respectively added to separate tubes each containing 100 microliters of 12.5% of sweet potato intracellular fluid and permitted to react for 24 hours at 26.5°C. For each test specimen, reaction fluid was collected after predetermined periods of time had elapsed. Reverse phase chromatography was performed under the same conditions as in Example 4 and the peak area values for rCAP24, rCAP24KD and Thionin were obtained. In addition, the amount of intact peptide remaining within the respective reaction fluid after reacting for a prescribed period was quantified by utilizing the concentration of intact peptide in the pre-treatment sample as a 100% reference value. [0112]

As shown in FIG. 7, the amount of remaining intact polypeptide after reacting for 24 hours was 80% or higher for rCAP24KD and Thionin and was 0% for rCAP24.

[0113]

Sequence Listing Free Text

SEQ ID NO: 5

Modified polypeptide based upon the CAP18 derived from human

SEQ ID NO: 6

Modified polypeptide based upon the CAP18 derived from human

SEQ ID NO: 8

Modified polypeptide based upon the CAP18 derived from rabbit

SEQ ID NO: 9 Modified polypeptide based upon the CAP 18 derived from rabbit

SEQ ID NO; 1.0

Modified polypeptide based upon the CAP 18 derived from rabbit

SEQ ID NO: 11

Modified polypeptide based upon the CAP 18 derived from rabbit

SEQ ID NO: 12

Modified polypeptide based upon the CAP 18 derived from rabbit

[0114]

All literature references identified in this specification are hereby incorporated by reference as if fully set forth herein.

Claims

1. A method for increasing protease-resistance of a target polypeptide comprising: binding at least one protease resistant sequence to the C-terminal side of the target polypeptide, the at least one protease resistant Sequence comprising at least two amino acids.

2. A method as in claim 1, wherein the protease resistant sequence includes a positively charged amino acid bound to a negatively charged amino acid, e.g., by a peptide bond.

3. A method as in claim 1 or 2, wherein the protease resistant sequence comprises at least one sequence selected from the group consisting of Lys-Asp (K-D), Arg-Asp (R-D), Lys-Glu (K-E), Arg-Glu (R-E), Asp-Lys (D-K), Asp-Arg (D-R), Glu-Lys (E-K) and Glu-Arg (E-R).

4. A method as in claim 1, wherein the protease resistant sequence comprises the sequence Lys- Asp (K-D).

5. A method for increasing protease-sensitivjty of a target polypeptide comprising: binding at least one protease-sensitive sequence to the C-terminal side of the target polypeptide, the at least one protease-sensitive sequence comprising at least two amino acids.

6. A method as in claim 5,^' wherein the protease-sensitive sequence comprises at least one sequence selected from the group consisting of Leu- Val, Asn-Leu, Arg-Asn, Leu-Arg and Phe- Leu.

7. A polypeptide comprising: at least one protease resistant sequence bound to the C-terrninal side of a target polypeptide, the at least one protease resistant sequence comprising at least two amino acids.

8. A polypeptide as in claim 7, wherein the protease resistant sequence includes a positively charged amino acid bound to a negatively charged amino acid, e.g., by a peptide bond.

9. A polypeptide as in claim 7 or 8, wherein the protease resistant sequence includes at least one sequence selected from the group consisting of Lys-Asp (K-D), Arg-Asp (R-D), Lys-Glu (K-E), Arg-Glu (R-E), Asp-Lys (D-K), Asp-Arg (D-R), Glu-Lys (E-K) and Glu-Arg (E-R).

10. A polypeptide as in claim 7 or 8, wherein the protease resistant sequence includes at least one sequence selected from the group consisting of Lys-Asp (K-D), Arg-Asp (R-D), Lys-Glu (K-E), Arg-Glu (R-E) and Asp-Lys (D-K).

11. A polypeptide as in claim 7 or 8, wherein the protease resistant sequence is Lys-Asp (K-D).

12. A polypeptide as in any of claims 7-11, wherein the protease resistant sequence further includes a hydrophobic amino acid attached to the N-terminal side of the protease resistant sequence.

13. A polypeptide as in any of claims 7-12, wherein the protease resistant sequence comprises at one or more spaced repeats consisting of one or more sequences selected from the group consisting of Lys-Asp (K-D), Arg-Asp (R-D), Lys-Glu (K-E), Arg-Glu (R-E), Asp-Lys (D-K), Asp-Arg (D- R), Glu-Lys (E-K) and Glu-Arg (E-R).

14. A polypeptide as recited in any one of claims 7-13, wherein the target polypeptide contains a α-helix structure.

15. A polypeptide as recited in any one of claims 7-14, wherein the target polypeptide comprises at least one of a cell-killing peptide, a portion thereof or a derivative thereof.

16. A polypeptide as recited in any one of claims 7-15, wherein the target polypeptide comprises at least one polypeptide sequence selected from the group consisting of CAP 18, magainins, melittins, alamethicins, polymixins, cecropins, tachyplesins, dermaseptins, bombinins, and thionins, portions thereof and derivatives thereof.

17. A polypeptide in any one of claims 7-14, wherein the target polypeptide includes at least one polypeptide sequence selected from the group consisting of CAP18_lπfi.142, CAP18₁₀₍₁.₁₃₇ and CAP18₁₀₅_₁₂₃ originating from rabbit, and CAP18_]CJ4._]40 and CAP18_1M._I3S originating from human, Wherein the protease resistant sequence optionally includes the sequence Lys-Asp (K-D).

18. A polypeptide in any one of claims 7-15, wherein the target polypeptide comprises at least one polypeptide sequence selected from the group consisting of the entire length of CAP18, a portion of CAP 18, and derivatives of CAP 18, each originating from rabbit,

19. A polypeptide in any one of claims 7-15, wherein the target polypeptide includes a polypeptide sequence from rabbit CAP18₁₀₆.₁₂₉,

20. A polypeptide comprising: at least one protease sensitive sequence bound to the C-terminal side of a target polypeptide, the protease-sensitive sequence comprising at least two amino acids.

21. A polypeptide as recited in claim 20, wherein the protease sensitive sequence includes at least one sequence selected from the group consisting of Leu- Val, Asn-Leu, Arg-Asn, Leu-Arg, and Phe-Leu.

22. A polypeptide consisting of the arnino acid sequence indicated by SEQ ID NO: 5, or an amino acid sequence that is substantially, functionally equivalent thereto.

23. A polypeptide consisting of the amino acid sequence indicated by SEQ _.ID NO; 6, or an amino acid sequence that is substantially, functionally equivalent thereto.

24. A polypeptide consisting of the amino acid sequence indicated by SEQ ID NO: 8, or an amino acid sequence that is substantially, functionally equivalent thereto.

25. A polypeptide consisting of the amino acid sequence indicated by SEQ ID NO: 9 or an amino acid sequence that is substantially, functionally equivalent thereto.

26. A polypeptide consisting of the amino acid sequence indicated by SEQ ID NO: 10 or an amino acid sequence that is substantially, functionally equivalent thereto.

27. A polypeptide consisting of the amino acid sequence indicated by SEQ ID NO: 11 or an amino acid sequence that is substantially, functionally equivalent thereto.

28. A polypeptide consisting of the amino acid sequence indicated by SEQ ID NO: 12, or an amino acid sequence that is substantially, functionally equivalent thereto.

29. A DNA construct comprising a DNA sequence encoding the polypeptide recited in any one of claims 7-28.

30. A vector comprising the DNA construct recited in claim 29-

31. A transformant carrying the DNA construct recited in claim 29 in a manner that enables said polypeptide to be expressed in a host cell.

32. A transformed plant cell carrying the DNA construct recited in claim 29 in a manner that enables said polypeptide to be expressed in the transformed plant cell.

33. A plant body comprising the transformed plant cell recited in claim 32,

34. A plant reproductive medium comprising the plant cell recited in claim 32.

35. A method of producing plant body comprising: regenerating a transformed plant cell carrying the DNA construct recited in claim 29.

36. A method of producing plant body comprising: cultivating the plant body recited in claim 33.

37. A method as in claim 35 or 36, wherein said plant body is a sweet potato.

38. A method for making a polypeptide comprising: culiuring the transformant recited in claim 31 αr 32.

39. A method for selecting a protease resistant sequence comprising: contacting a target polypeptide with a protease, monitoring changes over time in C-terminal amino acid sequences of polypeptide fragments generated protease-cleavage of the target polypeptide and selecting polypeptide fragmcnt(s) for which decomposition of the C-cerminal side is relatively slow.