U.S. patent application number 11/937611 was filed with the patent office on 2008-05-08 for magnetic substance-biosubstance complex structure, peptide fragment capable of linking to magnetic substance and gene therefor, and process for producing the complex structure.
This patent application is currently assigned to CANON KABUSHIKI KAISHA. Invention is credited to Tsutomu Honma, Takeshi Imamura, Shinya Kozaki, Tsuyoshi Nomoto, Akiko Tsuchitani, Tetsuya Yano.
Application Number | 20080108123 11/937611 |
Document ID | / |
Family ID | 33410377 |
Filed Date | 2008-05-08 |
United States Patent
Application |
20080108123 |
Kind Code |
A1 |
Imamura; Takeshi ; et
al. |
May 8, 2008 |
MAGNETIC SUBSTANCE-BIOSUBSTANCE COMPLEX STRUCTURE, PEPTIDE FRAGMENT
CAPABLE OF LINKING TO MAGNETIC SUBSTANCE AND GENE THEREFOR, AND
PROCESS FOR PRODUCING THE COMPLEX STRUCTURE
Abstract
A magnetic substance-biosubstance complex structure comprises a
magnetic substance-containing carrier and a biosubstance
immobilized on the carrier, the biosubstance being immobilized
through a spacer comprising an amino acid sequence on a surface of
the carrier.
Inventors: |
Imamura; Takeshi;
(Chigasaki-shi, JP) ; Yano; Tetsuya; (Atsugi-shi,
JP) ; Nomoto; Tsuyoshi; (Tokyo, JP) ; Kozaki;
Shinya; (Tokyo, JP) ; Honma; Tsutomu;
(Atsugi-shi, JP) ; Tsuchitani; Akiko; (Yamato-shi,
JP) |
Correspondence
Address: |
FITZPATRICK CELLA HARPER & SCINTO
30 ROCKEFELLER PLAZA
NEW YORK
NY
10112
US
|
Assignee: |
CANON KABUSHIKI KAISHA
3-30-2, Shimomaruko, Ohta-ku,
Tokyo
JP
|
Family ID: |
33410377 |
Appl. No.: |
11/937611 |
Filed: |
November 9, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10546404 |
Aug 19, 2005 |
|
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PCT/JP04/06350 |
Apr 30, 2004 |
|
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11937611 |
Nov 9, 2007 |
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Current U.S.
Class: |
435/183 ;
530/400; 530/402; 536/121; 536/23.1; 554/74 |
Current CPC
Class: |
C12N 9/1025 20130101;
Y10S 530/811 20130101; C12N 11/14 20130101; C12N 11/06 20130101;
C07K 2319/00 20130101; G01N 33/54326 20130101; C12N 15/1013
20130101; G01N 33/54353 20130101 |
Class at
Publication: |
435/183 ;
536/121; 554/074; 530/400; 536/023.1; 530/402 |
International
Class: |
C12N 9/00 20060101
C12N009/00; C07H 23/00 20060101 C07H023/00; C11C 3/00 20060101
C11C003/00; C07K 1/00 20060101 C07K001/00; C07H 21/04 20060101
C07H021/04; C07K 14/00 20060101 C07K014/00 |
Foreign Application Data
Date |
Code |
Application Number |
May 2, 2003 |
JP |
2003/127504 |
Claims
1. A magnetic substance-biosubstance complex structure comprising:
a magnetic substance-containing carrier; and a biosubstance
immobilized on the carrier, the biosubstance being immobilized
through a spacer comprising an amino acid sequence that binds to
the magnetic substance on a surface of the carrier and, wherein the
magnetic substance comprises an MO.Fe.sub.2O.sub.3 structure (M:
bivalent metal) or an Fe.sub.2O.sub.3 structure, the biosubstance
is one or more biosubstances selected from the group consisting of
proteins, carbohydrates, lipids, and complexes thereof and the
amino acid sequence is selected from the group consisting of SEQ ID
NO:16, SEQ ID NO:17 SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ
ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25,
SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID
NO:30: and complexes thereof.
2. (canceled)
3. (canceled)
4. (canceled)
5. (canceled)
6. The structure according to claim 1, wherein the protein is a
polyhydroxyalkanoate-synthesizing enzyme.
7. (canceled)
8. A peptide fragment that binds to a magnetic substance,
comprising an amino acid sequence selected from the group
consisting of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID
NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ
ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28,
SEQ ID NO:29, and SEQ ID NO:30.
9. A DNA sequence encoding a peptide fragment that binds to a
magnetic substance, the peptide fragment comprising an amino acid
sequence selected from the group consisting of SEQ ID NO:16, SEQ ID
NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ
ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26,
SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30.
10. A fusion type polyhydroxyalkanoate-polymerizing enzyme protein,
which is a fusion type protein formed from a peptide capable of
linking to a magnetic substance and a
polyhydroxyalkanoate-polymerizing enzyme protein; the peptide
comprising a amino acid sequence selected from the group consisting
of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID
NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ
ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28,
SEQ ID NO:29, and SEQ ID NO:30; and the
polyhydroxyalkanoate-polymerizing enzyme protein being SEQ ID NO:1
of SEQ ID NO:3.
11. A process for producing a structure having a biosubstance
immobilized on a magnetic substance-containing carrier through a
spacer comprising an amino acid sequence, the process comprising:
(1) a step of preparing a biosubstance-spacer complex formed by
linking the spacer and the biosubstance; and (2) a step of bringing
the biosubstance-spacer complex with the carrier; wherein the
biosubstance is immobilized by linking a portion of the spacer of
the biosubstance-spacer complex to the surface of the carrier.
12. The process for producing the structure according to claim 11,
wherein the spacer comprises a peptide structure comprising two or
more amino acid units.
13. The process according to claim 12, wherein the biosubstance
contains a protein, the step of producing a biosubstance-spacer
complex includes an operation of expressing a fusion type protein
constituted of the peptide structure and the protein joined
together according to joined gene having a base sequence formed by
joining a base sequence coding amino acid sequence of the protein
and a base sequence of coding the amino acid sequence of the
peptide structure; and the biosubstance-spacer complex is prepared
by the fusion type protein.
14. The process for producing the structure according to claim 13,
wherein the protein included in the biosubstance is a
polyhydroxyalkanoate-synthesizing enzyme.
15. The process for producing the structure according to claim 11,
wherein the magnetic substance comprises an MO.Fe2O3 structure (M:
bivalent metal) or an Fe2O3 structure, and the amino acid sequence
in the spacer is one or more of amino acid sequence and/or
complexes selected from the group consisting of amino acid
sequences represented by SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17,
SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID
NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ
ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30; and complex
thereof.
Description
TECHNICAL FIELD
[0001] The present invention relates to a magnetic
substance-biological substance complex type structure, a peptide
fragment and a gene having an amino acid sequence capable of
linking to the magnetic substance for preparation of the structure,
and a process for producing the magnetic substance-biological
substance complex type structure. (Hereinafter the "biological
substance" is referred to as a "biosubstance".) In particular the
present invention relates to a structure containing a biosubstance
immobilized on a magnetic substance-containing carrier which is
useful in biochemical fields and medical field as carriers of
diagnostic agents, carriers for separation of bacteria or
biological cells, carriers for separation and purification of
nucleic acids or proteins, carriers for drug delivery, carriers of
enzyme reaction, carriers for cell culture, and carriers for drug
screening; and to a process for producing the structure.
BACKGROUND ART
[0002] A magnetic substance-containing structure, which is readily
collectable by magnetic force, is promising for use mainly in
biochemical fields as carriers of diagnostic agents, carriers for
separation of bacteria or biological cells, carriers for separation
and purification of nucleic acids or proteins, carriers for drug
delivery, carriers for enzyme reaction, carriers for cell culture,
and carriers for like uses. Further, the structure is promising as
a carrier in drug-screening fields for selecting efficiently a
target substance having an intended physiological activity or
pharmacological activity from possible drug substances containing a
biopolymer such as nucleic acids, peptides, proteins, and
carbohydrates.
[0003] In such applications, in utilizing the magnetic
substance-containing structure as a carrier, a means is necessary
for immobilizing and holding the physiologically active substance
on the surface of the magnetic substance-containing structure.
Methods for the immobilization are disclosed in literature as
below.
[0004] Japanese Patent Application Laid-Open No. H05-209884
discloses use of magnetic particles extracted from a magnetic
bacteria as a carrier for immobilizing an antibody fragment on the
surface of the magnetic particles. The magnetic particles derived
from a magnetic bacterium are coated with a lipid film on the
surface. On this lipid film, an antibody fragment is immobilized by
utilizing N-succinimidyl 3-(2-pyridylthio)propionate.
[0005] Japanese Patent Application Laid-Open No. H05-080052
discloses immobilization of anti-rabbit IgG on ferrite particles
prepared by coating of polymer particles mainly composed of
polystyrene with Fe.sub.3O.sub.4 as the carrier modifying the
particle surface by a
--(CH.sub.2).sub.3NHCO(CH.sub.2).sub.3CONH(CH.sub.2).sub.6NH.sub.2
group by use of a coupling agent.
[0006] Japanese Patent Application Laid-Open No. H07-063761
discloses a process for producing a fine particulate magnetic
substance for immobilizing a physiologically active substance in
which fine magnetic particles of average diameter of 0.3-1.0 .mu.m
are fixed onto resin particles of average diameter of 1.0-10 .mu.m
as the nuclei by a high-speed gas stream impact method; the fixed
magnetic fine particles are surface-treated with a silane coupling
agent; and a physiologically active substance is bonded thereon
directly or through another functional group introduced
thereto.
[0007] U.S. Pat. No. 5,776,360 discloses a method in which
magnetite fine particles are surface treated for aminosilanation,
and HCG (human chorionic gonadotropin) antibody is immobilized
thereon with glutaraldehyde by utilizing the introduced amino
group.
[0008] The aforementioned prior art techniques utilize a chemical
covalent bond between the biosubstance and the magnetic substance
for immobilizing a biosubstance on a magnetic substance contained
in a carrier. Such a technique is liable to cause conversion or
denaturation of the biosubstance depending on the covalent bond
formation conditions (such as temperature, pH, and reagent). For
example, the site exhibiting an inherent function of the biological
material (e.g., molecular recognition site, and catalyst site) can
be modified by introduction of a reagent to affect adversely the
inherent function of the biosubstance. Otherwise, the covalent bond
can be formed near the active site of the inherent function of the
biosubstance to impair the inherent function exhibition.
[0009] As the results of the above adverse effects, the obtained
biosubstance-holding carrier may not achieve the intended inherent
function as the carrier of a medical diagnostic drug, the carrier
for separation of bacteria or cells, the carrier for separation and
purification of nucleic acids or proteins, the carrier for drug
delivery, the carrier for enzyme reaction, the carrier for cell
culture, or the carrier for drug screening.
[0010] The present invention provides a novel method for
immobilizing a biosubstance like a protein on a carrier containing
a magnetic substance, wherein the biosubstance is immobilized on
the magnetic substance surface with the function kept active, and
enabling immobilization of the intended biosubstance selectively
onto the magnetic substance surface. The present invention provides
also a magnetic substance-biosubstance complex structure, and a
process for producing thereof.
DISCLOSURE OF THE INVENTION
[0011] After comprehensive investigation to solve the above
problems, the inventors of the present invention found that a
peptide having a specified amino acid sequence can be linked to a
magnetic substance surface stably with high reproducibility, and
further found that a biosubstance-spacer complex which is formed
from the biosubstance and a spacer having a peptide fragment
containing an amino acid sequence linkable to the aforementioned
magnetic substance can be immobilized on the magnetic substance by
the linking ability of the spacer portion. Furthermore, the
inventors of the present invention confirmed that the
biosubstance-spacer complex itself having the spacer preliminarily
linked thereto can be prepared in a state so as to perform
effectively the inherent function, and consequently, the
biosubstance-spacer complex, when immobilized on the surface of the
magnetic substance, can be held with its function kept active.
[0012] The inventors of the present invention found also that the
peptide fragment having the amino acid sequence linkable to a
desired magnetic substance can readily be obtained from a random
peptide library in dependence upon the linking ability to the
magnetic substance, and with the amino acid sequence of the peptide
fragment, the spacer containing the peptide fragment having an
amino acid sequence capable of linking to the magnetic substance
can be designed readily. In addition to the above findings, the
inventors of the present invention found that a biosubstance-spacer
complex retaining the inherent function can be prepared from
various biosubstances, and the structures which are obtained by
immobilizing a biosubstance through a spacer containing a peptide
fragment having an amino acid capable of linking directly to the
magnetic substance are useful for various applications and
purposes. The present invention has been accomplished based on the
above findings.
[0013] The magnetic substance-biosubstance complex structure of the
present invention comprises a magnetic substance-containing carrier
and a biosubstance immobilized on the carrier, the biosubstance
being immobilized through a spacer comprising an amino acid
sequence on a surface of the carrier. The spacer comprises
preferably a peptide structure comprising two or more amino acid
units.
[0014] The peptide structure comprising two or more of amino acid
units is preferably a peptide fragment comprising an amino acid
sequence capable of linking to the magnetic substance. The amino
acid sequence capable of linking to the magnetic substance can be
selected from a random peptide library in dependence upon
capability of linking to the magnetic substance.
[0015] In the magnetic substance-biosubstance complex structure of
the present invention, the biosubstance is preferably one or more
biosubstances selected from the group consisting of nucleic acids,
proteins, carbohydrates, lipids, and complexes thereof. For
example, the protein is suitably a
polyhydroxyalkanoate-synthesizing enzyme.
[0016] In the magnetic substance-biosubstance complex structure of
the present invention, the magnetic substance comprises preferably
an MO.Fe.sub.2O.sub.3 structure (M: bivalent metal) or an
Fe.sub.2O.sub.3 structure, and for the magnetic substance the amino
acid sequence in the spacer is one or more of amino acid sequences
and/or complexes selected from the group consisting of amino acid
sequences represented by SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17,
SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID
NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ
ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30; and complex
thereof.
[0017] Correspondingly, the present invention provides also peptide
fragments having an amino acid sequence capable of linking to the
magnetic substance and being useful as the peptide structure
contained in the spacer. The peptide fragment having an amino acid
sequence capable of linking to the magnetic substance is
exemplified by peptide fragments containing an amino acid sequence
selected from the group consisting of SEQ ID NO:15 through SEQ ID
NO: 30.
[0018] Further, the genetic DNA of the present invention for coding
a peptide fragment having an amino acid sequence capable of linking
to the magnetic substance is exemplified by genetic DNAs comprising
a DNA coding the peptide fragments containing an amino acid
sequence capable of linking to the magnetic substance selected from
the group consisting of SEQ ID NO:15 through SEQ ID NO: 30.
[0019] The biosubstance-spacer complex of the present invention
which is prepared by linking of a spacer containing a peptide
fragment having an amino acid sequence capable of linking to the
magnetic substance is exemplified by fusion type
polyhydroxyalkanoate polymerization enzyme proteins which are
fusion type proteins prepared by fusion of a peptide comprising an
amino acid sequence selected from the group of SEQ ID NO:15 through
SEQ ID NO:30 with a polyhydroxyalkanoate polymerization enzyme
protein represented by SEQ ID NO:1 or SEQ ID NO:3.
[0020] The process of the present invention for producing the a
structure having a biosubstance immobilized on a magnetic
substance-containing carrier through a spacer comprising an amino
acid sequence comprises
(1) a step of preparing a biosubstance-spacer complex formed by
linking the spacer and the biosubstance, and
(2) a step of bringing the biosubstance-spacer complex with the
carrier;
wherein the biosubstance is immobilized by linking a portion of the
spacer of the biosubstance-spacer complex to the surface of the
carrier. In the process, the spacer comprises preferably a peptide
structure comprising two or more amino acid units.
[0021] In utilizing the spacer having a peptide structure
constituted of two or more amino acid units, the biosubstance
contains a protein, the step of producing a biosubstance-spacer
complex includes an operation of expressing a fusion type protein
constituted of the peptide structure and the protein joined
together according to joined gene having a base sequence formed by
joining a base sequence coding amino acid sequence of the protein
and a base sequence of coding the amino acid sequence of the
peptide structure; and the biosubstance-spacer complex can be
prepared by the fusion type protein. For instance, the above
constitution can be employed suitably, when the protein included in
the biosubstance is a polyhydroxyalkanoate-synthesizing enzyme.
[0022] The process for producing the magnetic
substance-biosubstance complex structure of the present invention
is useful when the magnetic substance comprises an
MO.Fe.sub.2O.sub.3 structure (M: bivalent metal) or an
Fe.sub.2O.sub.3 structure. With such a magnetic substance, the
amino acid sequence in the spacer is one or more of amino acid
sequence and/or complexes selected from the group consisting of
amino acid sequences represented by SEQ ID NO:15 through SEQ ID
NO:30.
[0023] The term "a magnetic substance-biosubstance complex
structure" as the object of the present invention means a structure
which is constituted of a carrier containing a magnetic substance,
and a biosubstance in a form of a complex immobilized in a state of
an immobilized layer on the surface of the carrier.
BEST MODE FOR CARRYING OUT THE INVENTION
[0024] Being different from conventional chemical method of fixing
chemically a biosubstance onto a magnetic substance surface through
a covalent bond, the magnetic substance-biosubstance complex
structure of the present invention is prepared, for example,
through the steps below: a peptide fragment capable of linking to a
desired magnetic substance is obtained simply by screening of
random peptide library for the capability of linking to the
magnetic substance; a spacer is designed which contains the peptide
fragment having an amino acid sequence capable of linking to the
magnetic substance corresponding to the amino acid sequence; a
biosubstance-spacer complex is prepared by linking the spacer with
the biological material; and the resulting biosubstance-spacer
complex is immobilized on the surface of the magnetic substance
surface.
[0025] Therefore, in this method, the biosubstance-spacer complex
can be confirmed preliminarily to have the inherent function of the
biosubstance. Therefore, in subsequent operation of immobilization
of the biosubstance-spacer complex on the magnetic substance
surface, the biosubstance immobilized on the magnetic substance
surface is kept in a state to exhibit the inherent function when
biosubstance is immobilized on the magnetic substance surface,
since no chemical reaction with a reagent or the like affecting the
function of the biosubstance is employed. Further, the amino acid
sequence having linking capability can be selected by screening
corresponding to the employed magnetic substance, and the linking
state of the spacer to be linked preliminarily to the biosubstance
and the amino acid sequence capable of linking to the magnetic
substance contained in the spacer can be designed. Therefore, the
magnetic substance-biosubstance complex structure can employ in
wide ranges of the used magnetic substance and the objective
biosubstance.
[0026] The present invention is described below in more detail.
<Magnetic Substance>
[0027] The "magnetic substance" for constituting the
magnet-containing carrier in the present invention may be suitably
selected without limitation, provided that the peptide having an
amino acid sequence of the spacer is capable of linking with the
carrier by its affinity. The kind and structure of the magnetic
substance may be suitably selected in correspondence with the
conditions of immobilization of the biosubstance through the
linking spacer and with the application conditions of the produced
magnetic substance-biosubstance complex structure. The magnetic
substance-containing carrier of the present invention includes
structures in shapes of granules, fibers, needles, flat plates, and
films containing a magnetic substance as the constituent
therein.
[0028] The magnetic substance for constituting the carrier of the
present invention includes metals and metal compounds having
magnetism: specifically ferrites such as iron tritetraoxide
(Fe.sub.3O.sub.4), .gamma.-sesquioxide (.gamma.-Fe.sub.2O.sub.3),
MnZn-ferrite, NiZn-ferrite, YFe-garnet, GaFe-garnet, Ba-ferrite,
and Sr-ferrite; metals such as iron, manganese, cobalt, nickel, and
chromium; alloys of iron, manganese, cobalt, nickel, and the like,
but not limited thereto. For instance, for deposition and
immobilization of a biosubstance complex, or for administration of
the structure formed by depositing and immobilizing a biological
complex to a living body, various ferrite compositions are useful
which are prepared by substituting at least a part of the metal
element of magnetite by other metal element in place of magnetite
(Fe.sub.3O.sub.4) well adaptable to living bodies. The shape of the
magnetic substance varies depending on the formation conditions,
taking a shape such as a polyhedron, octahedron, hexahedron,
sphere, bar, and scale. A less anisotropic structure of the
magnetic substance is preferred as the carrier for the stable
performance of the function. The primary particle size of the
magnetic substance constituting the magnet-containing carrier of
the present invention may be selected depending on the usage
thereof, and may be, for instance, in the range from 0.001-10
.mu.m.
[0029] The magnetic substance constituting the magnet-containing
carrier of the present invention may be super-paramagnetic. For
instance, ferrite particles having a small particle size of not
more than 20 nm become superparamagnetic owing to thermal
disturbance effect and lose residual magnetization and coercivity.
Even if the particles are superparamagnetic, the particles can be
operated magnetically by application of external magnetic field.
Further, the superparmagnetic particles, which have no residual
magnetization or coercivity, will not aggregate together
magnetically when external magnetic field is not applied.
[0030] The magnet-containing carrier employed in the present
invention may contain a simple magnetic substance or a composite of
two or more of the magnetic substances.
[0031] The magnet-containing carrier may contain the aforementioned
magnetic substance on a usual polymer compound or an inorganic
solid such as a resin, glass, ceramic, a metal, and a metal oxide
by mixing, vapor deposition, plating or the like method. However,
for immobilizing the biosubstance on the magnet-containing
material, the magnetic substance should be bared at least on a part
of the surface of the magnetic substance.
[0032] Examples of the constituting material of magnet-containing
carrier other than the magnetic substance includes organic polymers
produced by polymerization of a polymerizable monomer: the monomer
including styrenic polymerizable monomers such as styrene,
.alpha.-methylstyrene, .beta.-methylstyrene, o-methylstyrene,
m-methylstyrene, p-methylstyrene, 2,4-dimethylstyrene,
p-n-butylstyrene, p-t-butylstyrene, p-n-hexylstyrene,
p-n-octylstyrene, p-n-nonylstyrene, p-n-decylstyrene,
p-n-dodecylstyrene, p-methoxystyrene, and p-phenylstyrene; acrylic
polymerizable monomers such as methyl acrylate, ethyl acrylate,
n-propyl acrylate, isopropyl acrylate, n-butyl acrylate, isobutyl
acrylate, t-butyl acrylate, n-amyl acrylate, n-hexyl acrylate,
2-ethylhexyl acrylate, n-octyl acrylate, n-nonyl acrylate,
cyclohexyl acrylate, benzyl acrylate, dimethylphosphatoethyl
acrylate, diethylphosphatoethyl acrylate, dibutylphosphatoethyl
acrylate, and 2-benzoyloxyethyl acrylate; methacrylic polymerizable
monomer such as methyl methacrylate, ethyl methacrylate, n-propyl
methacrylate, isopropyl methacrylate, n-butyl methacrylate,
isobutyl methacrylate, t-butyl methacrylate, n-amyl methacrylate,
n-hexyl methacrylate, 2-ethylhexyl methacrylate, n-octyl
methacrylate, n-nonyl methacrylate, diethylphosphatoethyl
methacrylate, and dibutylphosphatoethyl methacrylate;
methylene-aliphatic monocarboxylic acid esters; vinyl polymerizable
monomer such as vinyl esters such as vinyl acetate, vinyl
propionate, vinyl benzoate, vinyl butyrate, vinyl benzoate, and
vinyl formate; vinyl ethers such as vinyl methyl ether, vinyl ethyl
ether, and vinyl isobutyl ether; and vinyl ketones such as vinyl
methyl ketone, vinyl hexyl ketone, and vinyl isopropyl ketone.
[0033] Other examples of the constituting material of
magnet-containing carrier other than the magnetic substance are
inorganic solids, including clay minerals such as kaolinite,
bentonite, talc, and mica; metal oxides such as alumina, titanium
dioxide, and zinc oxide; insoluble inorganic salts such as silica
gel, hydroxyapatite, and calcium phosphate gel; metals such as
gold, silver, platinum, and copper; and semiconductor compounds
such as GaAs, GaP, and ZnS. The material is not limited
thereto.
[0034] The material other than the magnetic substance may be used
in combination of two or more thereof.
[0035] The shape of the carrier formed by use of the constituting
material in addition to the magnetic substance is preferably
particulate generally in consideration of application of the
produced magnetic substance-biosubstance complex structure.
However, in some application fields, the carrier may be in a shape
of a film of a plastic material such as polyethylene terephthalate
(PET), diacetate, triacetate, cellophane, celluloid, polycarbonate,
polyimide, polyvinyl chloride, polyvinylidene chloride,
polyacrylates, polyethylene, polypropylene, and polyesters; a
porous film of a polymer such as polyvinyl chloride, polyvinyl
alcohol, acetylcellulose, polycarbonate, nylon, polypropylene,
polyethylene, and Teflon; a wood plate; a glass plate; a silicon
substrate; a cloth formed from a material such as cotton, rayon,
acrylic fiber, silk, and polyester fiber; and a paper sheet such as
wood free paper, medium-quality paper, art paper, bond paper,
regenerated paper, baryta paper, cast-coated paper, corrugated
board paper, and resin-coated paper. Naturally the shape of the
carrier is not limited thereto. The material in a shape of a film
or sheet may have a smooth surface or a rough surface insofar as
the magnetic substance can be held thereon.
<Biosubstance>
[0036] The term "biosubstance", which is to be immobilized on the
carrier containing the magnet in the magnetic
substance-biosubstance complex structure of the present invention
includes nucleic acids, proteins, carbohydrates, lipids, and
complexes thereof, specifically including a biomolelcule selected
from the group consisting of nucleic acid, protein, carbohydrate,
and lipid, more specifically including at least one selected from
the group consisting of DNA, RNA, aptamer, gene, chromosome, cell
membrane, viruse, antigen, antibody, lectin, hapten, hormone,
receptor, enzyme and peptide. Any material containing the above
biosubstance can be employed in the present invention. Further, the
bacteria or cells thereof producing the above "biosubstance" can be
employed as the objective biological material in the present
invention. In the production of the magnetic substance-biosubstance
complex structure by use of the spacer containing an amino acid
sequence for linking to the magnetic substance, especially in a
state of fusion body with the peptide structure, by immobilization
on the carrier containing the magnetic substance as described
later, the biosubstance contains preferably a peptide chain capable
of fusion with the peptide structure, in particular a protein.
<Amino Acid Sequence-Containing Peptide Structure Capable of
Linking to Magnetic Substance>
[0037] The amino acid sequence capable of linking to the magnetic
substance in the present invention is the one selected by screening
of a random peptide library, or designed reasonably in
consideration of the chemical properties of the magnetic
substance.
[0038] The random peptide library utilizable in screening for
selection of the amino acid sequence capable of linking with the
magnetic substance in the present invention includes a random
synthetic peptide libraries obtained by chemical synthesis of
random peptide in a soluble form; solid-phase immobilized peptide
libraries synthesized on resin beads; peptide libraries
biosynthesized in a ribosome cell-free system containing a
chemically synthesized random DNA sequence, such as a phage display
peptide library prepared by linking a random synthetic gene with an
E-end side gene of surface protein of M13 type phage (e.g., gene
III protein); and random peptide libraries obtained in a similar
method by fusion of bacterial membrane protein, Omp A (Francisco et
al.: 1993, PNAS, 90, 10444-10448, or Pistor and Hoborn, 1989,
Klin.Wochenschr., 66, 110-116), PAL (Fuchs et al.: 1991,
Bio/Technology, 9, 1369-1372), Lamb (Charbit et al.: 1988, Gene,
70, 181-189, and Bradbury et al.: 1993, Bio/Technology, 1565-1568),
Fimbrin (Hedegaard and Klemm: 1989, Gene, 85, 115-124, and Hofnung:
1991, Methods Cell Biol., 34, 77-105), or a .beta.-region of IgA
protease (Klauser et al.: 1990, EMBO J., 9, 1991-1999).
[0039] Typical examples of the method of screening the amino acid
sequences capable of linking to the magnetic substance employ
chemically synthesized peptide library, or phage display peptide
libraries.
[0040] When a chemically synthesized peptide library is utilized,
the peptide library is brought into contact with the magnetic
substance, the peptide not linkable to the magnetic substance is
removed, then the peptide having been linked to the magnetic
substance is recovered, and the amino acid sequence thereof is
determined by an Edman degradation method or a like method.
[0041] Otherwise, when a phage display peptide library is utilized,
the library is brought into contact with the surface of a
particulate magnetic substance immobilized on a column or a plate
or with the surface of a plate-shaped magnetic substance,
non-linked phage is washed off, thereafter the remaining linked
phage is eluted with an acid or the like, the eluted phage solution
is neutralized, and the phage is allowed to infect Escherichia coli
to amplify the phage. By repeating this selection operation several
times, plural clones capable of linking to the objective magnetic
substance are concentrated. Then to obtain single clone, the
Escherichia coli is infected with the clones and is allowed to form
colonies on a culture plate. The respective single colonies are
cultivated in a liquid culture medium. The phage in the supernatant
of the culture is precipitated and purified by use of polyethylene
glycol or the like. Analysis of the base sequence of the random
region of the monoclone phage gives the peptide structure capable
of linking with the magnetic substance.
[0042] The selective screening of the peptide capable of linking to
the magnetic substance by use of a phage display peptide library
includes an operation of concentration of the phage capable of
linking stronger to the magnetic substance, a so-called panning
operation. Therefore, this method enables selection of a promising
peptide with higher reliability, and is applicable suitably in the
present invention. The display random peptide library can be
constructed, for example, by connecting a random synthesized gene
with an N-end side gene of the surface protein of M13 type phage
(e.g., gene III protein). The construction examples are disclosed
by Scott, J K. and Smith, G P: Science, vol. 249, 386, 1990; and
Cwirla, S E et al.: Proc. Natl. Acad. Sci. USA, vol. 87, 6378,
1990, and so forth. The size of the random synthesized gene is not
limited, provided that the peptide can be stably expressed. In
order that the prepared random peptide library includes all random
sequence and has capability of linking to the magnetic substance,
the size corresponds preferably to 6-40 amino acids (corresponding
to the molecular weight of about 600-4000), more preferably 7-18
amino acids. The phage capable of linking to an objective magnetic
substance is selected in the following procedure. The magnetic
substance is fixed on a column or plate, the above library is
brought into contact with the magnetic substance, non-linked phage
is washed off, thereafter the remaining linked phage is eluted with
an acid or the like, the eluted phage solution is neutralized with
a buffer solution, and the phage is allowed to infect Escherichia
coli to amplify the phage. By repeating the selection operation
several times, plural clones capable of linking to the objective
magnetic substance are concentrated. Then to obtain a monoclone,
the clones are allowed to infect the Escherichia coli and are
allowed to form colonies on a culture plate. The respective single
colonies are cultivated in a liquid culture medium. The phage in
the supernatant of the culture is precipitated and purified by use
of polyethylene glycol or the like. Analysis of the base sequence
of the random region of the monoclone phage gives the peptide
structure (amino acid sequence) capable of linking to the magnetic
substance.
[0043] The peptide library having a random amino acid sequence can
also be prepared by use of a chemically synthesized peptide,
similarly as in the above-mentioned method employing a phage. The
process of preparation of a chemically synthesized peptide library
includes a process employing beads (Lam, K S et al.: Nature, 354,
82, 1991), a liquid-phase focusing process (Houghton, R A et al.:
Nature, 354, 84, 1991), a microplate process (Fodor, S P A et al.:
Science, 251, 767, 1991), and so forth. Any of these processes is
useful for the screening in the present invention.
[0044] In the case where two or more kinds of peptides capable of
linking to the magnetic substance have been obtained, at least one
selected from the peptides, or a peptide having an amino acid
sequence prepared by combining all or a part of the amino acid
sequences of the peptides in series may be used as the peptide
capable of linking to a magnetic substance. In combining two of the
amino acid sequences, a suitable linker sequence is preferably
inserted between the combined two amino acid sequences. The linker
sequence is preferably contains about 3 to about 400 amino acid
units. More preferably, the linker sequence will not suppress the
function of the biosubstance (e.g., a PHA synthase) in the formed
complex, and will not prevent the linking reaction of the objective
biosubstance complex with the magnetic substance with any of the
linked two amino acid sequences. The amino acid sequence capable of
linking to the magnetic substance utilized in the present invention
may be an amino acid sequence reasonably designed in consideration
of the chemical properties of the magnetic substance as well as the
one decided by screening of a random peptide library.
<Deposition and Immobilization of Biosubstance>
[0045] The biosubstance is immobilized on a magnetic substance
through a spacer having a peptide structure. The spacer forms a
complex with the biosubstance and contains an amino acid sequence
capable of linking to the magnetic substance in the present
invention.
[0046] Specifically, for deposition and immobilization of the
biosubstance on the magnetic substance, a biosubstance-spacer
complex is firstly prepared by linking the biosubstance with a
spacer having a peptide structure containing an amino acid sequence
capable of linking with the magnetic substance, and then the
resulting biosubstance-spacer complex is brought into contact with
the magnetic substance in an aqueous medium to form a linkage of
the magnet with the peptide structure containing the amino acid
sequence capable of linking with the magnet.
[0047] The aqueous medium used in the deposition-immobilization
process should be selected not to cause retardation of the
immobilization reaction between the magnetic substance and the
peptide structure containing the amino acid sequence capable of
linking with the magnetic substance, and further to allow the
deposited and immobilized biosubstance to perform its function.
[0048] The aqueous medium to allow the biosubstance to perform its
function includes buffer solutions. The buffer solutions include
ordinary buffer solutions useful in biochemical reactions such as
acetate buffers, phosphate buffers, calcium phosphate buffers,
3-(N-morpholino)propanesulfonate (MOPS) buffers,
N-tris(hydroxymethyl)-methyl-3-aminopropanesulfonate (TAPS)
buffers, tris hydrochloric acid buffers, glycine buffers, and
2-(cyclohexylamino)-ethanesulfonate (CHES) buffers. The
concentration of the buffer solution for performance of the
function of the biosubstance is in a usual range from 5 mM to 1.0
M, preferably from 10 to 200 mM. The pH of the solution is in the
range from 5.5 to 9.0, preferably from 7.0 to 8.5, but may be
outside the above range depending on the optimum pH or pH-stability
of the applied biosubstance.
[0049] In the case where the magnetic substance is powdery, a
suitable kind of surfactant may be added thereto to keep the
powdery magnetic substance in a dispersion state in the aqueous
medium in the deposition, immobilization, and later steps within
the concentration range not to impair the function of the
biosubstance. The surfactant includes anionic surfactants such as
sodium oleate, sodium dodecylsulfonate, sodium dodecylsulfate,
sodium dodecyl-N-sarcosinate, sodium cholate, sodium deoxycholate,
and sodium taurodeoxycholate; cationic surfactants such as
cetyltrimethylammonium bromide, and dodecylpyridinium chloride;
amphoteric surfactants such as
3-[(cholamidopropyl)dimethylammonio]-1-propanesulfonic acid
(CHAPS),
3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic
acid (CHAPSO), palmitoyllysolecithin, and dodecyl-.beta.-alanine;
and nonionic surfactants such as octylglucoside,
octylthioglucoside, heptylthioglucoside, decanoyl-N-methylglucamide
(MEGA-10), polyoxyethylene dodecyl ether (Brij, Lubrol),
polyoxyethylene-isooctylphenyl ether (Triton X), polyoxyethylene
nonyl phenyl ether (Nonidet P-40, Triton N), polyoxyethylene fatty
acid ester (Span), and polyoxyethylene sorbitol ester (Tween).
[0050] For keeping the dispersion state of the powdery magnetic
substance in the aqueous medium, a suitable kind of auxiliary
solvent may be added within a concentration range not to retard the
deposition-immobilization step, and subsequent steps, and not to
impair the function of the biosubstance. The auxiliary solvent may
be one or more of the solvent selected from the group of linear
aliphatic hydrocarbons such as hexane; monohydric alcohols such as
methanol and ethanol; polyhydric alcohols such as glycerol; and
derivatives of fatty acid ethers and carboxylic acid esters.
[0051] The composition of the aqueous medium for mixing the
magnetic substance and the biosubstance complex is decided
preferably in consideration of the fact that, in the
deposition-immobilization steps, the magnetic substance and the
biosubstance complex, especially the peptide structure containing
the amino acid sequence capable of linking with the magnetic
substance contained in the spacer portion, vary their charge
polarity, charge quantity, and hydrophobicity depending on the pH
and salt concentration of the aqueous medium. For example, in the
case where the linking force depends on the ion adsorption between
the magnetic substance and the peptide structure containing the
amino acid sequence capable of linking with the magnetic substance,
the electric charge contributing the adsorption of the peptide
structure on the magnetic substance can be increased by lowering
the salt concentration, and the opposing charge can be increased by
changing the pH. On the other hand, in the case where the linking
between the magnetic substance and the peptide structure is caused
mainly by hydrophobic adsorption, the hydrophobicity of the both
can be increased by increasing the salt concentration. The
composition suitable for the adsorption can be decided by
investigating the charging state and the hydrophobicity of the
magnetic substance and the peptide structure by measurement by the
electrophoresis and the wetting angle.
<Biosubstance-Peptide Fusion Product>
[0052] The amino acid sequence of the peptide capable of linking
with the magnetic substance obtained by the aforementioned method
may be fused with a desired protein for preparation of the
biosubstance complex according to a conventional genetic
engineering technique, when the biosubstance is a protein. For
instance, the polypeptide capable of linking with the magnetic
substance may be linked to an amino end (--NH.sub.2) or a carboxyl
end (--COOH) of the aforementioned protein to express fusion
protein by gene recombination. In this fusion protein, in the
linking portion between the peptide portion capable of linking to
the magnetic substance and the objective protein, one or more units
of amino acids may be inserted as a linker sequence. The linker
sequence contains preferably about 3 to about 400 amino acid units.
The linker sequence may contain any amino acid. More preferably,
the linker sequence will not suppress the function of the protein,
and will not prevent the linking with the magnetic substance by
employing the peptide capable of linking with the magnetic
substance. Therefore, a protein-peptide fusion type biosubstance
complex can be obtained in which a spacer containing the amino acid
sequence of the peptide capable of linking the magnetic substance
and containing another amino acid sequence having the
aforementioned linker sequence as necessary.
[0053] In the magnetic substance-biosubstance complex structure of
the present invention, the protein as the biosubstance may be any
protein which can be immobilized according the above method. When a
fusion protein is prepared by fusion with the aforementioned
peptide, the gene sequence is preferably to be known, whereas when
the linking with the peptide capable of linking with the magnetic
substance is conducted chemically, the gene sequence need not be
known.
[0054] When the biosubstance is a protein having a known gene
sequence, the biosubstance-peptide structure fusion product can be
directly recombined and recovered as a fusion protein constituted
of the peptide structure and the protein, for example by using a
host bacterium strain like Escherichia coli.
[0055] In the case where the biosubstance is a protein having an
unknown gene sequence, a nucleic acid, or a carbohydrate, a complex
can be prepared by linking after the biosubstance and/or the spacer
containing the peptide structure is chemically modified or
transformed without impairing the function.
[0056] Specifically, one or both of the biosubstance and the spacer
containing the peptide structure is modified or transformed into
any of the combinations of groups of maleimido and sulfanyl (--SH),
succinimido and amino, isocyanato and amino, halogeno and hydroxyl,
halogeno and sulfanyl (--SH), epoxy and amino, and epoxy and
sulfanyl (--SH), and then a chemical bonding is formed between the
above functional groups.
[0057] In the case where the biosubstance is a lipid, a spacer
complex of a lipid-spacer containing a peptide structure can be
obtained by preparing a "spacer" by linking a "hydrophobic peptide
structure" containing plural amino acids having a free hydrophobic
group such as alanine, valine, leucine, isoleucine methionine,
tryptophan, phenylalanine, and proline to the magnet-linking
peptide structure, and bonding the hydrophobic peptide structure to
the lipid by hydrophobic bonding.
<Polyhydroxyalkanoate-Synthesizing Enzyme>
[0058] In the magnetic substance-biosubstance complex structure of
the present invention, an example of the protein employed as the
biosubstance immobilized on the magnetic substance is a
polyhydroxyalkanoate-synthesizing enzyme (hereinafter referred to
as a "PHA synthase").
[0059] The polyhydroxyalkanoate (hereinafter referred to as "PHA")
is an aliphatic polyester synthesized by PHA synthase of a
microorganism, and is a promising material for use for application
in biochemistry and medical treatment because the PHA is
biodegradable and biocompatible, and various functional groups can
be introduced to the side chains thereof. In recent years, complex
materials have come to be developed which have a PHA coating on a
surface of a core structure (Japanese Patent Application Laid-Open
Nos. 2002-327046, 2003-11312, etc.). Such complex materials are
useful in various application fields since the complex have both
the functions of the core structure and the properties of the PHA
coating combinedly (Japanese Patent Application Laid-Open Nos.
2003-12957, 2003-15168, 2003-12984, 2003-15359, 2003-26506, and
2003-26493).
[0060] One embodiment of the magnetic-biosubstance complex
structure is a magnet-PHA synthase complex type structure which
contains a PHA synthase immobilized on a magnetic
substance-containing carrier. In copresence of this structure with
a PHA monomer unit precursor, a magnetic complex material can be
produced which is constituted of the magnet-PHA synthase complex
structure coated with PHA synthesized by the PHA synthase. The
magnetic composite material coated with PHA is promising in various
industrial fields including biochemical and medical fields.
[0061] The PHA synthase itself as the biosubstance to be
immobilized on a magnetic substance may be the one produced by a
microorganism selected suitably from microorganisms capable of
producing the PHA synthase, or by a transformant having a PHA
synthase gene of microorganism introduced by a transformant.
[0062] The microorganisms which produce the PHA synthase include
Aeromonas sp., Alcaligenes sp., Chromatium sp., Comamonas sp.,
Methylobacterium sp., Paracocuus sp., and Pseudomonas sp., and
further include the microorganism isolated by the inventor of the
present invention such as Burkholderia cepacia KKO1, Ralstonia
eutropha TB64, and Alcaligenes sp. TL2. Of these microorganisms,
KKO1 strain, TB64 strain, and TL2 strain are deposited with deposit
numbers respectively of FERM BP-4235, FERM BP-6933, and FERM
BP-6913 and deposited date of respectively Mar. 11, 1992, Nov. 9,
1999 and Oct. 12, 1999, to International Patent Organism Depositary
in National Institute of Advanced Industrial Science and Technology
(independent administrative cooperation), AIST Tsukuba Central 6,
1-1, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken 305-8566 Japan,
which is the international depositary authority according to
"Budapest Treaty on the International Recognition of the Deposit of
Microorganisms for the Purpose of Patent Procedure".
[0063] Further, PHA synthases are useful which are derived from a
Pseudomonas microorganism such as Pseudomonas oleoborans,
Pseudomonas resinoborans, Pseudomonas sp. 61-3, Pseudomonas putida
KT2442, and Pseudomonas aeruginosa; the microorganisms isolated by
the inventors of the present invention such as Pseudomonas putida
P91, Pseudomonas cichorii H45, Pseudomonas cichorii YN.sup.2, and
Pseudomonas jessenii P161; and Burkholderia bacteria such as
Burkholderia sp. OK3, FERM P-17370 disclosed in Japanese Patent
Laid-Open No. 2001-78753, and Burkholderia sp. OK4, FERM P-17371
disclosed in Japanese Patent Application Laid-Open No. 2001-69968.
Further, PHA synthases are useful which are derived from the
microorganisms such as Aeromonas sp. and Comamonas sp. producing
mcl-PHA or unusual-PHA.
[0064] P91 strain (Deposit No. FERM BP-7373), H45 strain (Deposit
No. FERM BP-7374), YN2 strain (Deposit No. FERM BP-7375), and P161
strain (Deposit No. FERM BP-7376), these four types of strains are
deposited on Nov. 20, 2002 at International Patent Organism
Depositary in National Institute of Advanced Industrial Science and
Technology (independent administrative cooperation), AIST Tsukuba
Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken 305-8566
Japan, which is the international depositary authority according to
"Budapest Treaty on the International Recognition of the Deposit of
Microorganisms for the Purpose of Patent Procedure". The bacteria
which are deposited domestically to International Patent Organism
Depositary in National Institute of Advanced Industrial Science and
Technology are tagged respectively with the deposit numbers.
[0065] The intended PHA synthase can be produced by recombination
by use of a transformant having PHA synthase gene derived from the
aforementioned PHA-producing bacterium. Conventional methods can be
employed for the cloning of the PHA synthase gene derived from a
PHA-producing bacterium, construction of an expression vector for
recombination production, and preparation of a transformant
employing the expression vector.
[0066] The isolation and purification of the PHA synthase can be
conducted by any method, insofar as the enzymatic activity of the
PHA synthase is retained. For example, a mass of a PHA
synthase-producing microorganism is crushed by a French press or a
supersonic crusher or with lysozyme or a surfactant, the crude
enzyme solution obtained by centrifugation or a deposit derived
form the crude enzyme solution by salting out with ammonium sulfate
is purified by a purification means such as affinity
chromatography, cation or anion exchange chromatography, gel
filtration, or combination thereof to obtain purified enzyme
protein. The gene recombinant protein can be purified more simply
by binding a "tag" like a histidine residue to express a fusion
protein and linking it through the tag with an affinitive resin.
From the fusion protein bonded to the affinitive resin, the
objective enzyme protein can be isolated in various methods such as
cleavage by protease like thrombin and blood coagulation factor Xa,
lowering of pH, and addition of imidazole as a bonding competitive
agent in a high concentration. Otherwise, when the tag contains
"intein" by use of pTYB1 (produced by New England Biolab Co.) as
the expression factor, the system is brought to a reductive
condition to cleave the --S--S-bond. The fusion proteins which can
be purified by affinity chromatography includes glutathione
S-transferase (GST), chitin-bonded domain (CBD), maltose-bonded
protein (MBP), and thioredoxin (TRX), in addition to the histidine
tag. The GST fusion protein can be purified by GST-affinitive
resin.
EXAMPLES
[0067] The present invention is described below more specifically
by reference to Examples. The Examples below show best embodiments
of the present invention without limiting the technical range of
the present invention.
Reference Example 1
Preparation of Magnetic Particles
[0068] An aqueous solution containing ferrous hydroxide was
prepared by adding, to an aqueous ferrous sulfate solution, a
sodium hydroxide solution in an amount of 1.0-1.1 equivalent to the
ferrous ion. Air was blown into this aqueous solution by keeping
the pH of the aqueous solution at about 8 to cause oxidation
reaction at 80-90.degree. C. to obtain a liquid slurry for
formation of seed crystals.
[0069] To this liquid slurry, was added an aqueous ferrous sulfate
solution in an amount of 0.9-1.2 equivalent to the above added
alkali (the sodium of the above added sodium hydroxide), and air is
blown therein to proceed an oxidation reaction by keeping the pH at
about 8. The formed magnetic iron oxide particles after the
oxidation reaction was collected by filtration, washed, and dried.
The particulate iron oxide in an aggregating state was crushed to
obtain magnetic particles (1) having an average particle size of
0.10 .mu.m.
Example 1
Formation of Peptide Structure Capable of Linking to Magnetic
substance
1. Preparation of Liquid Suspension of Magnetic Particles
[0070] To 5 mg of magnetic particle (1) prepared in Reference
Example 1, was added 1 mL of a TBS buffer (50 mM tris-HCl (pH 7.5),
150 mM NaCl) to form a suspension. The suspension was centrifuged
at 10,000 rpm (9300 g) for 5 minutes, and the supernatant was
eliminated. The precipitate was suspended in 1 mL of acetone. The
suspension was again centrifuged and the supernatant was eliminated
under the same conditions as above. Further to the precipitate, was
added 1 mL of a TBS-0.1T buffer (50 mM tris-HCl (pH 7.5), 150 mM
NaCl, 0.1% Tween-20) to form a suspension. The suspension was
centrifuged under the above conditions, and the supernatant was
eliminated. This operation was repeated two more times. The
obtained precipitate was suspended in 1 mL of a TBS-0.1T buffer. To
10 .mu.L of the suspension, was added 990 .mu.L of the TBS-0.1T
buffer to prepare a magnetic particle liquid suspension for
screening of the phage display peptide library.
2. Biopanning the Phage Display Peptide Library
[0071] To the above magnetic particle liquid suspension, were added
4.times.10.sup.10 pfu of Ph.D.-12 phage display library (New
England Biolabs, Inc.) and 100 .mu.L of a TBS-0.1T buffer. The
mixture was incubating for 30 minutes at room temperature
(25.degree. C.). Then the mixture was centrifuged at 10,000 (9300
g) for 5 minutes, and the supernatant was eliminated. The resulting
precipitate was suspended in 1 mL of a TBS-0.1T buffer, the
suspension was centrifuged at 10,000 rpm (9300 g) for 5 minutes,
and the supernatant was eliminated. This washing treatment for
elimination of the non-linked phage was repeated additionally 9
times. Thereafter the phage linked to the magnetic particles was
recovered by a buffer of pH 2.2 (0.2 M glycine-HCl (pH 2.2), 1
mg/mL BSA). The recovered phage was allowed to infect Escherichia
coli ER2537 for amplification.
[0072] This phage having been fractionated and amplified by the
primary screening was subjected to secondary and successive
screening. In the secondary and successive screening, the amount of
the added phage was 2.times.10.sup.11 pfu, and the buffer for the
washing was a TBS-0.5T buffer (50 mM tris-HCl (pH 7.5), 150 mM
NaCl, 0.5% Tween-20)
3. Sequencing of the DNA
[0073] After the above multi-step screening, a portion of the
finally amplified phage was taken and subjected to cloning. From
each of the isolated 35 clones, ssDNA was prepared respectively,
and the base sequence in the random region in the peptide library
was determined. From the coded base sequence in the random region,
the corresponding amino acid sequences shown by SEQ ID NO:15 to SEQ
ID NO:30 were selected as the amino acid sequence of the peptide
capable of linking to the magnetic particles.
Example 2
Evaluation of Linkage of Magnet-Linking Peptide Structure to
Magnetic substance
[0074] A 5 .mu.g portion of the magnetic fine particles prepared in
Reference Example 1 was suspended in 100 .mu.L of a TBS-0.1T buffer
(50 mM tris-HCl (pH 7.5), 150 mM NaCl, 0.1% Tween 20). Thereto was
added 10 .mu.L of a solution containing 2.times.10 pfu of the phage
having a peptide of the amino acid sequence SEQ ID NO:15. The
mixture was agitated at room temperature (25.degree. C.) for 30
minutes to cause linking reaction. Further it was suspended in
TBS-0.5T buffer (50 mM tris-HCl (pH 7.5), 150 mM NaCl, 0.5% Tween
20), and was recovered by magnetic force. This treatment was
repeated 10 times to release and wash off the non-adsorbed and
non-specifically adsorbed phage.
[0075] According to the method described in Nature, 405, 665-668
(2000), the recovered magnetic fine particles containing
specifically adsorbed phage were allowed to react with an anti-fd
phage antibody-biotin complex (produced by Sigma Co.) and
streptavidin-tetramethylrhodamine, and was recovered by magnetic
force and was washed. This treatment was repeated three times in
total. The magnetic fine particles were again dispersed in the
aforementioned TBS-0.1T buffer.
[0076] The resulting liquid dispersion was observed by a
incident-light microscope in a normal mode and in a fluorescence
mode (excitation with green light by use of a filter). Thereby, the
magnetic fine particles in the dispersion were observed to emit
orange fluorescence caused by the rhodamine fluorochrome, which
shows the presence of streptavidin-tetramethylrhodamine bonding to
the anti-fd phage antibody-biotin complex.
[0077] For comparison, in place of the phage presenting the
magnet-linking peptide selected by the above screening, the same
amount of phage extracted arbitrarily from a random phage library
was treated and observed for fluorescence in the same manner as
above. Consequently, no fluorescence caused by the rhodamine
fluorochrome was observed with the observed magnetic fine
particles.
[0078] Further the linking experiments were conducted in the same
manner by employing a phage presenting a magnet-linking peptide
having an amino acid sequence of any of SEQ ID NO:16 to SEQ ID
NO:30. In any of the experiments, the orange fluorescence caused by
the rhodamine fluorochrome was observed to be emitted from the
magnetic fine particles.
[0079] The above results show that through the peptide having the
amino acid sequence selected by the screening in Example 1, the M13
phage presenting the peptide was immobilized by specific linkage on
the magnetic fine particles.
Reference Example 2
Preparation of Transformant Capable of Producing PHA synthase, and
Production of PHA synthase
[0080] A transformant capable of producing a PHA synthase was
prepared in a manner as below.
[0081] A Pseudomonas cichorii YN2 strain (hereinafter also referred
to as "YN2") was cultivated in 100 mL of an LB medium (1%
polypeptone, 0.5% yeast extract, 0.5% sodium chloride, pH 7.4) at
30.degree. C. overnight. Then chromosomal DNA was isolated and
recovered according to a Marmar's method. The obtained chromosomal
DNA was completely cleaved by a restriction enzyme HindIII. pUC18
was employed as the cloning vector, which was cleaved by
restriction enzyme HindIII. After dephosphorylation treatment of
the terminal (Molecular Cloning, 1, 572, (1989); published by Cold
Spring Harbor Laboratory), the complete decomposition fragments of
chromosomal DNA by HindIII was linked and inserted into the
cleavage site (cloning site) of the vector by use of a DNA ligation
kit Ver. II (Takara Shuzo Co.). A DNA library of YN2 strain was
prepared by transformation of Escherichia coli HB101 by use of the
plasmid vector incorporating the above chromosomal DNA
fragment.
[0082] Next, a colony hybridization probe was prepared for
selection of DNA fragments containing PHA synthase chromosome
derived from the YN2 strain. Oligonucleotide comprising the base
sequences of SEQ ID NO:5 or SEQ ID NO: 6 was synthesized (Amasham
Pharmacia Biotech Co.). With these oligonucleotides as the primer,
PCR amplification was conducted on a chromosomal DNA as the
template. The DNA fragment formed by the PCR amplification was used
as a probe for colony hybridization. The labeling of the probe was
conducted by use of a commercial labeling enzyme system
AlkPhosDirect (Amasham Pharmacia Biotech Co.). With the obtained
labeled probe, an Escherichia coli strain having recombinant
plasmid containing a PHA synthase gene was selected from the
chromosomal DNA library of the YN2 strain by colony hybridization.
From the selected bacteria strain, a DNA fragment containing PHA
synthase gene was obtained by recovery of the plasmid by an alkali
method.
[0083] The obtained gene DNA fragment was treated for recombination
to vector pBBR122 (Mo Bi Tec) containing a large replication region
in the host cell, not belonging to any of IncP, IncQ, and IncW
which are incompatible. This recombinant plasmid was introduced
into Pseudomonas cichorii YN2 ml (defective in PHA-synthesizing
ability) by electroporation to cause transformation. As the result,
the YN2 ml strain restored the PHA-synthesizing ability and became
complementary. Thereby the selected gene DNA fragment was confirmed
to contain a PHA synthase gene region which is capable of
translation into a PHA synthase in the YN2 ml.
[0084] The base sequence of the DNA fragment containing the PHA
synthase region was determined by a Sanger method. Thereby it was
confirmed that the determined base sequence contained the base
sequences SEQ ID NO:2 and SEQ ID NO:4 which codes respectively a
peptide chain. As described below, the two proteins containing
respectively the peptide chains coded as the above two base
sequences have the enzymatic activity. Therefore, the base
sequences SEQ ID NO:2 and SEQ ID NO:4 were confirmed to be a gene
for coding a PHA synthase respectively. That is, the base sequence
SEQ ID NO:2 codes the amino acid sequence represented by SEQ ID
NO:1, and the base sequence SEQ ID NO:4 codes the amino acid
sequence represented by SEQ ID NO:3. Each of the two proteins
having the above amino acid sequence has the PHA-synthesizing
ability.
[0085] The PHA synthase gene having the base sequence represented
by SEQ ID NO:2 was subjected to PCR by employing the chromosomal
DNA as the template to reproduce the entire length of the PHA
synthase gene.
[0086] An oligonucleotide (SEQ ID NO:7) was designed and
synthesized which has a base sequence of the upstream side of the
initiation codon for the base sequence DEQ ID NO:2, and another
oligonucleotide (SEQ ID NO:8) was also designed and synthesized
which has a base sequence of the downstream side of the termination
codon therefor (Amasham Pharmacia Biotech Co.). By employing these
nucleotides as the primers, PCR was conducted to amplify the entire
length of the PHA synthase gene (LA-PCR kit: Takara Shuzo Co.).
[0087] In the same manner, the PHA synthase gene having the base
sequence represented by SEQ ID NO:4 was subjected to PCR by
employing the chromosomal DNA as the template to reproduce the
entire length of the PHA synthase gene. An oligonucleotide (SEQ ID
NO:9) was designed and synthesized which has a base sequence of the
upstream side of the initiation codon for the base sequence DEQ ID
NO:4, and another oligonucleotide (SEQ ID NO:10) was also designed
and synthesized which has a base sequence of the downstream side of
the termination codon therefor (Amasham Pharmacia Biotech Co.). By
employing these nucleotides as the primers, PCR was conducted to
amplify the entire length of the PHA synthase gene (LA-PCR kit:
Takara Shuzo Co.).
[0088] The two kinds of the resulting PCR amplified fragments
comprising the entire length of the PHA synthase gene were cleaved
completely by the restriction enzyme HindIII. The expression vector
pTrc99A was also cleaved by the restriction enzyme HindIII, and was
treated for dephosphorylation (Molecular Cloning, vol. 1, 572,
1989, published by cold Spring Harbor Laboratory). The DNA fragment
from which unnecessary base sequences had been eliminated by
digestion by the restriction enzyme HindIII and which has the
complete length of the PHA synthase gene was ligated to the
cleavage site of this expression vector pTrc99A by use of the DNA
ligation kit Ver.II (Takara Shuzo Co.).
[0089] With the resulting recombinant plasmids, Escherichia coli
was transformed by a calcium chloride method. The obtained
recombinants were cultivated to amplify the recombinant plasmid.
The recombinant plasmids were recovered. The recombinant plasmid
holding the genetic DNA of SEQ ID NO:2 was named pYN2-C1
(originating from SEQ ID NO:2), and the recombinant plasmid holding
the genetic DNA of SEQ ID NO:4 was named pYN2-C2 (originating from
SEQ ID NO:4).
[0090] With the recombinant plasmids pYN2-C1 and pYN2-C2,
Escherichia coli HB101fB (fadB defective strain) was transformed by
a calcium chloride method to obtain a pYN2-C1 recombinant strain
and a pYN2-C2 recombinant strain, each holding the respective
recombinant plasmid.
[0091] The pYN2-C1 recombinant strain and the pYN2-C2 recombinant
strain were inoculated respectively into 200 mL of M9 cultures
containing 0.5% of yeast extract and 0.1% octanoic acid, and were
cultivated at 37.degree. C. with shaking at 125 stroke/min. After
24 hours of the cultivation, each bacteria mass was collected by
centrifugation to recover the respective plasmid DNAs in a
conventional manner.
[0092] For the plasmid pYN2-C1, an oligonucleotide (SEQ ID NO:11)
as the upstream primer, and an oligonucleotide (SEQ ID NO:12) as
the downstream primer were respectively designed and synthesized
(Amasham Pharmacia Biotech.). By use of these nucleotides as the
primers and the pYN2-C1 as the template, PCR was conducted to
amplify the entire length of the PHA synthase gene having
restriction sites of BamHI and SacI at the upstream side and
restriction sites of SpeI and XhoI at the downstream side (LA-PCR
kit, Takara Shuzo Co.).
[0093] In the same manner, for the plasmid pYN2-C2, an
oligonucleotide (SEQ ID NO:13) as the upstream primer, and an
oligonucleotide (SEQ ID NO:14) as the downstream primer were
respectively designed and synthesized (Amasham Pharmacia Biotech
Co.). By use of these nucleotides as the primers and the pYN2-C2 as
the template, PCR was conducted to amplify the entire length of the
PHA synthase gene having a restriction site of BamHI at the
upstream side and a restriction site of XhoI at the downstream side
(LA-PCR kit, Takara Shuzo Co.).
[0094] The purified PCR amplification products were respectively
digested by BamHI and XhoI, and were inserted into corresponding
sites of plasmid pGEX-6P-1 (Amasham Pharmacia Biotech. Co.). With
these vectors (pGEX-C1 and pGEX-C2), Escherichia coli (JM109) was
transformed to obtain an expression bacteria strain. The bacteria
strain was confirmed by determining the base sequence of the DNA
fragments formed by treatment of the plasmid prepared in a large
amount with BamHI and XhoI by means of Miniprep (Wizard Minipreps
DNA Purification Systems, produced by PROMEGA Co.). The obtained
bacteria strain was pre-cultivated overnight in 10 mL of an LB-Amp
culture. A 0.1 mL portion of the culture was added to 10 mL of an
LB-Amp culture, and cultivated at 37.degree. C. by shaking at 170
rpm for 3 hours. Thereto IPTG was added (final concentration: 1 mM)
and the cultivation was continued at 37.degree. C. for 4-12 hours.
Incidentally, in the aforementioned expression vectors (pGEX-C1 and
pGEX-C2), the PHA synthase gene is linked to the GST protein gene
in a plasmid pGEX-6P-1, and is expressed as a GST fusion
protein.
[0095] The Escherichia coli derived by addition of IPTG was
collected by centrifugation (8000.times.g, 2 minutes, 4.degree.
C.). A 1/10 portion thereof was suspended again in PBS. The
bacteria mass was crushed by freeze-thawing and sonication. Any
solid contaminant was eliminated by centrifugation (8000.times.g,
10 minutes, 4.degree. C.). After confirmation of the presence of
the objective expression protein in the supernatant by SDS-PAGE,
the GST fusion protein derived and expressed was purified by
Glutathione Sepharose 4B (Glutathione Sepharose 4B Beads: produced
by Amasham Pharmacia Biotech Co.).
[0096] Preliminarily, the glutathione-sepharose before use was
treated for suppression of nonspecific adsorption as follows. The
glutathione-sepharose was washed with an equal amount of PBS three
times (8000.times.g, one minute, 4.degree. C.), and treated with an
equal amount of 4% BSA-containing PBS at 4.degree. C. for one hour.
The treated glutathione-sepharose was washed with an equal amount
of PBS twice, and suspended again in half an amount of PBS. A 40
.mu.L portion of the pretreated glutathione-sepharose was added to
1 mL of the cell-free liquid extract, and stirred gently at
4.degree. C. Thereby, the fusion proteins GST-YN2-C1 and GST-YN-C2
were respectively adsorbed by the glutathione-sepharose.
[0097] After the adsorption, the glutathione-sepharose was
recovered by centrifugation (8000.times.g, one minute, 4.degree.
C.) and washed with 400 .mu.L of PBS three times. Thereto, 40 .mu.L
of 10 mM glutathione was added, and the mixture was stirred at
4.degree. C. for one hour to elute the adsorbed fusion protein. The
mixture was centrifuged (8000.times.g, two minutes, 4.degree. C.)
to recover the supernatant. The recovered supernatant was dialyzed
by use of PBS to purify the GST fusion protein. After the
purification, the protein was confirmed to show a single band in
SDS-PAGE analysis.
[0098] A 500 .mu.g portion of each of the GST fusion proteins was
digested by PreScission protease (Amasham Pharmacia Biotech Co.,
5U). The digested mixture was allowed to flow through a
glutathione-sepharose to remove the protease and the GST. The
flow-through fraction containing the PHA synthase was allowed to
flow though a Sephadex G200 column having been equilibrated with
PBS. Thus final purified products of recombinant expression
proteins YN2-C1 and YN2-C2 were obtained. The proteins were
confirmed to show respectively a single band in SDS-PAGE at 60.8
kDa, and 61.5 kDa.
[0099] The enzymatic activity of each of the purified PHA synthases
was measured by causing 3-hydroxyacyl CoA polymerization by the
enzyme to synthesize PHA. The activity is represented by the amount
of CoA released in the PHA synthesis reaction, taking the amount of
PHA synthase for releasing 1 .mu.mol of CoA per minute as one unit
(U). The protein concentration in the test sample was measured with
a MicroBCA Protein Determination Reagent Kit (produced by Pias
Chemical Co.). Table 1 shows the measured activities of the
respective purified enzymes. TABLE-US-00001 TABLE 1 Activities of
Polyhydroxyalkanoate-Synthesizing Enzyme Enzyme Activity Specific
activity YN2-C1 2.1 U/mL 4.1 U/mg-protein YN2-C2 1.5 U/mL 3.6
U/mg-protein
[0100] The respective purified enzyme solution was concentrated by
use of a biosubstance solution-concentrating agent (Mizubutorikun
AB-1100, produced by Atoh K.K.) to obtain a purified enzyme
solution having an activity of 10 U/mL.
Example 3
Preparation of Fusion Protein of Magnet-Linking Peptide and PHA
Synthase
[0101] An E. coli-expressing vector was constructed which expresses
a fusion protein of a magnet-linking peptide and a PHA synthase in
which a magnet-linking amino acid sequence SEQ ID NO:15 is fused
through a linker sequence SEQ ID NO:38 with an N-terminal of PHA
synthase amino acid sequence in a procedure shown below. The DNA
for coding the magnet-linking sequence and the linker sequence
portion is prepared as a double-stranded synthesizing
oligonucleotide, and is ligated to a suitable restricted cleavage
site (BamHI and SacI) of plasmid pGEX-C1 plasmid for expressing a
fusion protein GST-YN2-C1. In this operation, according to the
instruction given by the maker, two synthesized oligonucleotides O1
(SEQ ID NO: 31) and O2 (SEQ ID NO:32) were phosphorylated by use of
T4 polynucleotide-kinase (produced by Gibco Co.). Subsequently, the
obtained products were cooled slowly to room temperature. The
resulting double-stranded DNA fragments were used directly for the
cloning.
[0102] The plasmid pGEX-C1 was digested by BamHI and SacI, and
thereto the above double-stranded DNA fragments were inserted. With
this vector, Escherichia coli (JM109) was transformed into an
expression bacteria. The bacteria strain was confirmed by
sequencing the base sequence of the insert with pGEX 5' Sequencing
Primer (Amasham Pharmacia Biotech Co.) by employing as the template
a plasmid DNA prepared by use of Miniprep (Wizard Minipreps DNA
Purification Systems, produced by PROMEGA Co.). The obtained
bacteria was cultivated preliminarily in 10 mL of an LB-Amp culture
medium. A 0.1 mL of this liquid culture was added to 10 mL of an
LB-Amp culture medium, and cultivated at 37.degree. C. with shaking
at 170 rpm for 3 hours. Thereto IPTG was added (final
concentration: 1 mM), and the cultivation was continued at
37.degree. C. for 4-12 hours. In the resulting expression vector,
GST fusion protein was coded for the linkage in the order of the
fusion partner protein GST, the magnetic-linking sequence and the
linker sequence, and the PHA synthase YN2-C1.
[0103] The Escherichia coli derived by addition of IPTG was
collected by centrifugation (8000.times.g, 2 minutes, 4.degree.
C.). A 1/10 portion thereof was suspended again in a PBS. The
bacteria mass was crushed by freeze-thawing and sonication. Any
solid contaminant was eliminated by centrifugation (8000.times.g,
10 minutes, 4.degree. C.). After confirmation of the presence of
the objective expression protein in the supernatant by SDS-PAGE,
the GST fusion protein derived and expressed was purified with
Glutathione Sepharose 4B (Glutathione Sepharose 4B Beads: produced
by Amasham Pharmacia Biotech Co.).
[0104] Preliminarily, the glutathione-sepharose before use was
treated for suppression of nonspecific adsorption as follows. The
glutathione-sepharose was washed with an equal amount of PBS three
times (8000.times.g, one minute, 4.degree. C.), and treated with an
equal amount of 4% BSA-containing PBS at 4.degree. C. for one hour.
The treated glutathione-sepharose was washed with an equal amount
of PBS twice, and suspended again in half an amount of PBS.
[0105] A 40 .mu.L portion of the pretreated glutathione-sepharose
was added to 1 mL of the cell-free extract, and stirred gently at
4.degree. C. Thereby, the fusion protein GST-(magnet-linking
peptide+linker peptide)-YN2-C1 was adsorbed by the
glutathione-sepharose.
[0106] After the adsorption, the glutathione-sepharose was
recovered by centrifugation (8000.times.g, one minute, 4.degree.
C.) and washed with 400 .mu.L portions of PBS three times. Thereto,
40 .mu.L of 10 mM glutathione was added, and the mixture was
stirred at 4.degree. C. for one hour to elute the adsorbed GST
fusion protein. The mixture was centrifuged (8000.times.g, two
minutes, 4.degree. C.) to recover the supernatant. The recovered
supernatant was dialyzed by use of PBS to purify the GST fusion
protein. After the purification, the protein was confirmed to show
a single band in SDS-PAGE analysis.
[0107] A 500 .mu.g portion of the purified GST fusion protein was
digested by PreScission protease (Amasham Pharmacia Biotech Co.
5U). The digested mixture was allowed to flow through a
glutathione-sepharose to remove the protease and the GST. The
flow-through fraction was further allowed to flow though a Sephadex
G200 column having been equilibrated with PBS. The obtained final
purified product was a recombinant expression protein YN2-C1(Fe)15
of a type of a complex of a magnet-linking peptide PHA synthase.
The protein was confirmed to show a single band in SDS-PAGE at 61.9
kDa.
[0108] The enzymatic activity of the purified enzyme was measured
in the same manner as in Example 2. The protein concentration in
the test sample was measured with a MicroBCA Protein Determination
Reagent kit (produced by Pias Chemical Co.). The enzyme activity
was found to be 1.9 U/mL, and the specific activity was 4.0 U/mg
protein. The purified enzyme solution was concentrated by use of a
biosubstance solution-concentrating agent (Mizubutorikun AB-1100,
produced by Atoh K.K.) to obtain a purified enzyme solution having
an activity of 10 U/mL.
[0109] In this Example, BamHI and SacI were used as the restriction
enzyme. In the same manner, by use of Spec I and XhoI, an
expression vector of Escherichia coli can be constructed which
expresses a complex of a magnet-linking peptide and PHA synthase by
employing SEQ ID NO:33 and SEQ ID NO:34. Similarly by using
suitable sequence of synthesizing oligonucleotide, vectors having
an amino acid sequence selected from sequence 16-30 can be
constructed.
Example 4
Evaluation of Ability of Fusion Protein of Magnet-Linking Peptide
and HPA-Synthesizing Enzyme for Linking with Magnetic Substance
[0110] Fine particulate magnetic substance was suspended at a
concentration of 0.5% (w/v) in a TBS buffer containing 0.1%
Tween-20. A 10 mL portion of this liquid suspension was placed in a
centrifuging teflon tube. Thereto were added the enzyme protein
YN2-C1(Fe)15 of a type of the fusion protein, prepared in Example
3, of a magnet-linking peptide and a PHA synthase in an amount
corresponding to 0.5 U. The mixture was shaken at room temperature
for 30 minutes.
[0111] From the suspension after the reaction, the magnetic
particles were recovered as precipitate by magnetic force to
separate the supernatant containing the enzyme not linking to the
magnetic particles.
[0112] The recovered magnetic particles were suspended again in a
TBS buffer containing 0.1% Tween-20, and recovered by magnetic
force for washing. The washing operation was repeated.
[0113] After the washing, the enzyme activity of the recovered
magnetic substance suspension was measured.
[0114] The enzyme protein YN2-C1 prepared in Reference Example 2
was tested in the same manner as above.
[0115] Table 2 shows the measured enzyme activities. TABLE-US-00002
TABLE 2 Ability of PHA synthase to Link to Ferrite Enzyme Activity
(U) YN2-C1(Fe)15 0.12 YN2-C1 0.01
[0116] Fusion proteins of the magnet-linking peptide and the PHA
synthase (YN2-C1(Fe)16 to YN2-C1(Fe)30) were prepared also by
employing 15 magnet-linking sequences, SEQ ID NO:16 to SEQ ID
NO:30, and were evaluated for ability of linking to the magnetic
substance by measuring the PHA synthase activity for linking to the
magnetic substance. Table 3 shows the results. TABLE-US-00003 TABLE
3 Ability of PHA synthase to Link to Ferrite Enzyme Activity (U)
YN2-C1(Fe)16 0.11 YN2-C1(Fe)17 0.10 YN2-C1(Fe)18 0.12 YN2-C1(Fe)19
0.09 YN2-C1(Fe)20 0.09 YN2-C1(Fe)21 0.09 YN2-C1(Fe)22 0.12
YN2-C1(Fe)23 0.10 YN2-C1(Fe)24 0.11 YN2-C1(Fe)25 0.10 YN2-C1(Fe)26
0.12 YN2-C1(Fe)27 0.12 YN2-C1(Fe)28 0.12 YN2-C1(Fe)29 0.11
YN2-C1(Fe)30 0.09 YN2-C1 0.01
[0117] The magnetic substances brought into contact with one of the
enzymes, YN2-C1(Fe)16 to YN2-C1(Fe)30, which are fused with a
magnet-linking sequence had high enzyme activity in comparison with
the control brought into contact with the enzyme, YN2-C1, which
contains no magnet-linking sequence. This means that the enzyme in
a state of a complex of magnet-linking peptide and PHA synthase can
be immobilized on the magnetic substance effectively.
Example 5
Evaluation of Linking Ability of Joined Two-Peptide to Magnetic
Substance
[0118] An E. coli expression vector for expressing a complex was
constructed: the complex being constituted by combining two amino
acid sequences, SEQ ID NO:15 and SEQ ID NO:16, capable of linking
to a magnetic substance, through a linker sequence SEQ ID NO:39 in
this order in series to form a sequence SEQ ID NO:35, and further
by combining this sequence through a linker sequence GS to an
N-terminal of a PHA synthase. The construction was conducted
specifically as below. The DNA for coding the amino acid sequence
to be joined to the N-terminal of the PHA synthase was formed by
phosphorylating two synthetic oligonucleotides, SEQ ID NO:36 and
SEQ ID NO:37, respectively with a T4 polynucleotide kinase
(produced by Gibco Co.), mixing the products in equimolar amounts,
heating the mixture at 80.degree. C. for 5 minutes, and cooling it
slowly to room temperature to form a double-stranded DNA fragment.
The formed double-stranded DNA fragment was inserted into
BamHI/SacI site of the plasmid pGEX, and with this vector,
Escherichia coli (JM109) was transformed into a bacteria strain for
expression in the same manner as Example 3. The expression protein
YN-C1(Fe)95 obtained by fusing the amino acid sequence SEQ ID NO:35
to the N-terminal was purified in the same manner as in Example 3
to obtain a purified enzyme solution having activity of 10 U/mL.
The ability of linking to the magnetic substance was evaluated in
the same manner as in Example 4. Table 4 shows the evaluation
result. TABLE-US-00004 TABLE 4 Ability of
Polyhydroxyalkanoate-Synthesizing Enzyme to Link to Ferrite Enzyme
Activity (U) YN2-C1(Fe)95 0.11 YN2-C1 0.01
[0119] The magnetic substances brought into contact with the
enzymes, YN2-C1(Fe).sub.95, which are fused with a magnet-linking
sequence had high enzyme activity in comparison with the control
brought into contact with the enzyme, YN2-C1, which does not
contain the magnet-linking sequence. This means that the enzyme can
be immobilized on the magnetic substance effectively in a form of a
complex of a magnet-linking peptide and PHA synthase (aaal
47-YN2-C1(cb): YN2-C1(Fe).sub.95).
Sequence Listing Free Text
<210>5
<223> Primer for PCR multiplication
<210> 6
<223> Primer for PCR multiplication
<210> 7
<223> Primer for PCR multiplication
<210> 8
<223> Primer for RCR multiplication
<210> 9
<223> Primer for PCR multiplication
<210> 10
<223> Primer for PCR multiplication
<210> 11
<223> Primer for PCR multiplication
<210> 12
<223> Primer for PCR multiplication
<210> 13
<223> Primer for PCR multiplication
<210> 14
<223> Primer for PCR multiplication
<210> 15
<223> Ferrite-binding peptide
<210> 16
<223> Ferrite-binding peptide
<210> 17
<223> Ferrite-binding peptide
<210> 18
<223> Ferrite-binding peptide
<210> 19
<223> Ferrite-binding peptide
<210> 20
<223> Ferrite-binding peptide
<210> 21
<223> Ferrite-binding peptide
<210> 22
<223> Ferrite-binding peptide
<210> 23
<223> Ferrite-binding peptide
<210> 24
<223> Ferrite-binding peptide
<210> 25
<223> Ferrite-binding peptide
<210> 26
<223> Ferrite-binding peptide
<210> 27
<223> Ferrite-binding peptide
<210> 28
<223> Ferrite-binding peptide
<210> 29
<223> Ferrite-binding peptide
<210> 30
<223> Ferrite-binding peptide
<210> 31
<223> Primer for PCR multiplication
<210> 32
<223> Primer for PCR multiplication
<210> 33
<223> Primer for PCR multiplication
<210> 34
<223> Primer for PCR multiplication
<210> 35
<223> Ferrite-binding peptide
<210> 36
<223> Primer for PCR multiplication
<210> 37
<223> Primer for PCR multiplication
<210> 38
<223> linker sequence
<210> 39
<223> Linker
Sequence CWU 1
1
39 1 559 PRT Pseudomonas cichorii YN2; FERM BP-7375 1 Met Ser Asn
Lys Ser Asn Asp Glu Leu Lys Tyr Gln Ala Ser Glu Asn 1 5 10 15 Thr
Leu Gly Leu Asn Pro Val Val Gly Leu Arg Gly Lys Asp Leu Leu 20 25
30 Ala Ser Ala Arg Met Val Leu Arg Gln Ala Ile Lys Gln Pro Val His
35 40 45 Ser Val Lys His Val Ala His Phe Gly Leu Glu Leu Lys Asn
Val Leu 50 55 60 Leu Gly Lys Ser Gly Leu Gln Pro Thr Ser Asp Asp
Arg Arg Phe Ala 65 70 75 80 Asp Pro Ala Trp Ser Gln Asn Pro Leu Tyr
Lys Arg Tyr Leu Gln Thr 85 90 95 Tyr Leu Ala Trp Arg Lys Glu Leu
His Asp Trp Ile Asp Glu Ser Asn 100 105 110 Leu Ala Pro Lys Asp Val
Ala Arg Gly His Phe Val Ile Asn Leu Met 115 120 125 Thr Glu Ala Met
Ala Pro Thr Asn Thr Ala Ala Asn Pro Ala Ala Val 130 135 140 Lys Arg
Phe Phe Glu Thr Gly Gly Lys Ser Leu Leu Asp Gly Leu Ser 145 150 155
160 His Leu Ala Lys Asp Leu Val His Asn Gly Gly Met Pro Ser Gln Val
165 170 175 Asn Met Gly Ala Phe Glu Val Gly Lys Ser Leu Gly Val Thr
Glu Gly 180 185 190 Ala Val Val Phe Arg Asn Asp Val Leu Glu Leu Ile
Gln Tyr Lys Pro 195 200 205 Thr Thr Glu Gln Val Tyr Glu Arg Pro Leu
Leu Val Val Pro Pro Gln 210 215 220 Ile Asn Lys Phe Tyr Val Phe Asp
Leu Ser Pro Asp Lys Ser Leu Ala 225 230 235 240 Arg Phe Cys Leu Arg
Asn Asn Val Gln Thr Phe Ile Val Ser Trp Arg 245 250 255 Asn Pro Thr
Lys Glu Gln Arg Glu Trp Gly Leu Ser Thr Tyr Ile Glu 260 265 270 Ala
Leu Lys Glu Ala Val Asp Val Val Thr Ala Ile Thr Gly Ser Lys 275 280
285 Asp Val Asn Met Leu Gly Ala Cys Ser Gly Gly Ile Thr Cys Thr Ala
290 295 300 Leu Leu Gly His Tyr Ala Ala Ile Gly Glu Asn Lys Val Asn
Ala Leu 305 310 315 320 Thr Leu Leu Val Ser Val Leu Asp Thr Thr Leu
Asp Ser Asp Val Ala 325 330 335 Leu Phe Val Asn Glu Gln Thr Leu Glu
Ala Ala Lys Arg His Ser Tyr 340 345 350 Gln Ala Gly Val Leu Glu Gly
Arg Asp Met Ala Lys Val Phe Ala Trp 355 360 365 Met Arg Pro Asn Asp
Leu Ile Trp Asn Tyr Trp Val Asn Asn Tyr Leu 370 375 380 Leu Gly Asn
Glu Pro Pro Val Phe Asp Ile Leu Phe Trp Asn Asn Asp 385 390 395 400
Thr Thr Arg Leu Pro Ala Ala Phe His Gly Asp Leu Ile Glu Leu Phe 405
410 415 Lys Asn Asn Pro Leu Ile Arg Pro Asn Ala Leu Glu Val Cys Gly
Thr 420 425 430 Pro Ile Asp Leu Lys Gln Val Thr Ala Asp Ile Phe Ser
Leu Ala Gly 435 440 445 Thr Asn Asp His Ile Thr Pro Trp Lys Ser Cys
Tyr Lys Ser Ala Gln 450 455 460 Leu Phe Gly Gly Asn Val Glu Phe Val
Leu Ser Ser Ser Gly His Ile 465 470 475 480 Gln Ser Ile Leu Asn Pro
Pro Gly Asn Pro Lys Ser Arg Tyr Met Thr 485 490 495 Ser Thr Glu Val
Ala Glu Asn Ala Asp Glu Trp Gln Ala Asn Ala Thr 500 505 510 Lys His
Thr Asp Ser Trp Trp Leu His Trp Gln Ala Trp Gln Ala Gln 515 520 525
Arg Ser Gly Glu Leu Lys Lys Ser Pro Thr Lys Leu Gly Ser Lys Ala 530
535 540 Tyr Pro Ala Gly Glu Ala Ala Pro Gly Thr Tyr Val His Glu Arg
545 550 555 2 1680 DNA Pseudomonas cichorii YN2; FERM BP-7375 2
atgagtaaca agagtaacga tgagttgaag tatcaagcct ctgaaaacac cttggggctt
60 aatcctgtcg ttgggctgcg tggaaaggat ctactggctt ctgctcgaat
ggtgcttagg 120 caggccatca agcaaccggt gcacagcgtc aaacatgtcg
cgcactttgg tcttgaactc 180 aagaacgtac tgctgggtaa atccgggctg
caaccgacca gcgatgaccg tcgcttcgcc 240 gatccggcct ggagccagaa
cccgctctat aaacgttatt tgcaaaccta cctggcgtgg 300 cgcaaggaac
tccacgactg gatcgatgaa agtaacctcg cccccaagga tgtggcgcgt 360
gggcacttcg tgatcaacct catgaccgaa gccatggcgc cgaccaacac cgcggccaac
420 ccggcggcag tcaaacgctt tttcgaaacc ggtggcaaaa gcctgctcga
cggcctctcg 480 cacctggcca aggatctggt acacaacggc ggcatgccga
gccaggtcaa catgggtgca 540 ttcgaggtcg gcaagagcct gggcgtgacc
gaaggcgcgg tggtgtttcg caacgatgtg 600 ctggaactga tccagtacaa
gccgaccacc gagcaggtat acgaacgccc gctgctggtg 660 gtgccgccgc
agatcaacaa gttctacgtt ttcgacctga gcccggacaa gagcctggcg 720
cggttctgcc tgcgcaacaa cgtgcaaacg ttcatcgtca gctggcgaaa tcccaccaag
780 gaacagcgag agtggggcct gtcgacctac atcgaagccc tcaaggaagc
ggttgatgtc 840 gttaccgcga tcaccggcag caaagacgtg aacatgctcg
gcgcctgctc cggcggcatc 900 acttgcaccg cgctgctggg ccattacgcg
gcgattggcg aaaacaaggt caacgccctg 960 accttgctgg tgagcgtgct
tgataccacc ctcgacagcg atgttgccct gttcgtcaat 1020 gaacagaccc
ttgaagccgc caagcgccac tcgtaccagg ccggcgtact ggaaggccgc 1080
gacatggcga aggtcttcgc ctggatgcgc cccaacgatc tgatctggaa ctactgggtc
1140 aacaattacc tgctaggcaa cgaaccgccg gtgttcgaca tcctgttctg
gaacaacgac 1200 accacacggt tgcccgcggc gttccacggc gacctgatcg
aactgttcaa aaataaccca 1260 ctgattcgcc cgaatgcact ggaagtgtgc
ggcaccccca tcgacctcaa gcaggtgacg 1320 gccgacatct tttccctggc
cggcaccaac gaccacatca ccccgtggaa gtcctgctac 1380 aagtcggcgc
aactgtttgg cggcaacgtt gaattcgtgc tgtcgagcag cgggcatatc 1440
cagagcatcc tgaacccgcc gggcaatccg aaatcgcgct acatgaccag caccgaagtg
1500 gcggaaaatg ccgatgaatg gcaagcgaat gccaccaagc ataccgattc
ctggtggctg 1560 cactggcagg cctggcaggc ccaacgctcg ggcgagctga
aaaagtcccc gacaaaactg 1620 ggcagcaagg cgtatccggc aggtgaagcg
gcgccaggca cgtacgtgca cgaacggtaa 1680 3 560 PRT Pseudomonas
cichorii YN2; FERM BP-7375 3 Met Arg Asp Lys Pro Ala Arg Glu Ser
Leu Pro Thr Pro Ala Lys Phe 1 5 10 15 Ile Asn Ala Gln Ser Ala Ile
Thr Gly Leu Arg Gly Arg Asp Leu Val 20 25 30 Ser Thr Leu Arg Ser
Val Ala Ala His Gly Leu Arg His Pro Val His 35 40 45 Thr Ala Arg
His Ala Leu Lys Leu Gly Gly Gln Leu Gly Arg Val Leu 50 55 60 Leu
Gly Asp Thr Leu His Pro Thr Asn Pro Gln Asp Arg Arg Phe Asp 65 70
75 80 Asp Pro Ala Trp Ser Leu Asn Pro Phe Tyr Arg Arg Ser Leu Gln
Ala 85 90 95 Tyr Leu Ser Trp Gln Lys Gln Val Lys Ser Trp Ile Asp
Glu Ser Asn 100 105 110 Met Ser Pro Asp Asp Arg Ala Arg Ala His Phe
Ala Phe Ala Leu Leu 115 120 125 Asn Asp Ala Val Ser Pro Ser Asn Ser
Leu Leu Asn Pro Leu Ala Ile 130 135 140 Lys Glu Ile Phe Asn Ser Gly
Gly Asn Ser Leu Val Arg Gly Ile Gly 145 150 155 160 His Leu Val Asp
Asp Leu Leu His Asn Asp Gly Leu Pro Arg Gln Val 165 170 175 Thr Arg
His Ala Phe Glu Val Gly Lys Thr Val Ala Thr Thr Thr Gly 180 185 190
Ala Val Val Phe Arg Asn Glu Leu Leu Glu Leu Ile Gln Tyr Lys Pro 195
200 205 Met Ser Glu Lys Gln Tyr Ser Lys Pro Leu Leu Val Val Pro Pro
Gln 210 215 220 Ile Asn Lys Tyr Tyr Ile Phe Asp Leu Ser Pro His Asn
Ser Phe Val 225 230 235 240 Gln Phe Ala Leu Lys Asn Gly Leu Gln Thr
Phe Val Ile Ser Trp Arg 245 250 255 Asn Pro Asp Val Arg His Arg Glu
Trp Gly Leu Ser Thr Tyr Val Glu 260 265 270 Ala Val Glu Glu Ala Met
Asn Val Cys Arg Ala Ile Thr Gly Ala Arg 275 280 285 Glu Val Asn Leu
Met Gly Ala Cys Ala Gly Gly Leu Thr Ile Ala Ala 290 295 300 Leu Gln
Gly His Leu Gln Ala Lys Arg Gln Leu Arg Arg Val Ser Ser 305 310 315
320 Ala Thr Tyr Leu Val Ser Leu Leu Asp Ser Gln Leu Asp Ser Pro Ala
325 330 335 Thr Leu Phe Ala Asp Glu Gln Thr Leu Glu Ala Ala Lys Arg
Arg Ser 340 345 350 Tyr Gln Lys Gly Val Leu Glu Gly Arg Asp Met Ala
Lys Val Phe Ala 355 360 365 Trp Met Arg Pro Asn Asp Leu Ile Trp Ser
Tyr Phe Val Asn Asn Tyr 370 375 380 Leu Met Gly Lys Glu Pro Pro Ala
Phe Asp Ile Leu Tyr Trp Asn Asn 385 390 395 400 Asp Asn Thr Arg Leu
Pro Ala Ala Leu His Gly Asp Leu Leu Asp Phe 405 410 415 Phe Lys His
Asn Pro Leu Ser His Pro Gly Gly Leu Glu Val Cys Gly 420 425 430 Thr
Pro Ile Asp Leu Gln Lys Val Thr Val Asp Ser Phe Ser Val Ala 435 440
445 Gly Ile Asn Asp His Ile Thr Pro Trp Asp Ala Val Tyr Arg Ser Thr
450 455 460 Leu Leu Leu Gly Gly Glu Arg Arg Phe Val Leu Ala Asn Ser
Gly His 465 470 475 480 Val Gln Ser Ile Leu Asn Pro Pro Asn Asn Pro
Lys Ala Asn Tyr Leu 485 490 495 Glu Gly Ala Lys Leu Ser Ser Asp Pro
Arg Ala Trp Tyr Tyr Asp Ala 500 505 510 Lys Pro Val Asp Gly Ser Trp
Trp Thr Gln Trp Leu Gly Trp Ile Gln 515 520 525 Glu Arg Ser Gly Ala
Gln Lys Glu Thr His Met Ala Leu Gly Asn Gln 530 535 540 Asn Tyr Pro
Pro Met Glu Ala Ala Pro Gly Thr Tyr Val Arg Val Arg 545 550 555 560
4 1683 DNA Pseudomonas cichorii YN2; FERM BP-7375 4 atgcgcgata
aacctgcgag ggagtcacta cccacccccg ccaagttcat caacgcacaa 60
agtgcgatta ccggcctgcg tggccgggat ctggtttcga ctttgcgcag tgtcgccgcc
120 catggcctgc gccaccccgt gcacaccgcg cgacacgcct tgaaactggg
tggtcaactg 180 ggacgcgtgt tgctgggcga caccctgcat cccaccaacc
cgcaagaccg tcgcttcgac 240 gatccggcgt ggagtctcaa tcccttttat
cgtcgcagcc tgcaggcgta cctgagctgg 300 cagaagcagg tcaagagctg
gatcgacgaa agcaacatga gcccggatga ccgcgcccgt 360 gcgcacttcg
cgttcgccct gctcaacgat gccgtgtcgc cgtccaacag cctgctcaat 420
ccgctggcga tcaaggaaat cttcaactcc ggcggcaaca gcctggtgcg cgggatcggc
480 catctggtcg atgacctctt gcacaacgat ggcttgcccc ggcaagtcac
caggcatgca 540 ttcgaggttg gcaagaccgt cgccaccacc accggcgccg
tggtgtttcg caacgagctg 600 ctggagctga tccaatacaa gccgatgagc
gaaaagcagt attccaaacc gctgctggtg 660 gtgccgccac agatcaacaa
gtactacatt tttgacctca gcccccataa cagcttcgtc 720 cagttcgcgc
tcaagaacgg cctgcaaacc ttcgtcatca gctggcgcaa tccggatgta 780
cgtcaccgcg aatggggcct gtcgacctac gtcgaagcgg tggaagaagc catgaatgtc
840 tgccgggcaa tcaccggcgc gcgcgaggtc aacctgatgg gcgcctgcgc
tggcgggctg 900 accattgctg ccctgcaggg ccacttgcaa gccaagcgac
agctgcgccg cgtctccagc 960 gcgacgtacc tggtgagcct gctcgacagc
caactggaca gcccggccac actcttcgcc 1020 gacgaacaga ccctggaggc
ggccaagcgc cgctcctacc agaaaggtgt gctggaaggc 1080 cgcgacatgg
ccaaggtttt cgcctggatg cgccccaacg atttgatctg gagctacttc 1140
gtcaacaatt acctgatggg caaggagccg ccggcgttcg acattctcta ctggaacaat
1200 gacaacacac gcctgccggc cgccctgcat ggtgacttgc tggacttctt
caagcacaac 1260 ccgctgagcc atccgggtgg cctggaagtg tgcggcaccc
cgatcgactt gcaaaaggtc 1320 accgtcgaca gtttcagcgt ggccggcatc
aacgatcaca tcacgccgtg ggacgcggtg 1380 tatcgctcaa ccctgttgct
cggtggcgag cgtcgctttg tcctggccaa cagcggtcat 1440 gtgcagagca
ttctcaaccc gccgaacaat ccgaaagcca actacctcga aggtgcaaaa 1500
ctaagcagcg accccagggc ctggtactac gacgccaagc ccgtcgacgg tagctggtgg
1560 acgcaatggc tgggctggat tcaggagcgc tcgggcgcgc aaaaagaaac
ccacatggcc 1620 ctcggcaatc agaattatcc accgatggag gcggcgcccg
ggacttacgt gcgcgtgcgc 1680 tga 1683 5 20 DNA Artificial Primer for
PCR multiplication 5 tgctggaact gatccagtac 20 6 23 DNA Artificial
Primer for PCR multiplication 6 gggttgagga tgctctggat gtg 23 7 29
DNA Artificial Primer for PCR multiplication 7 ggaccaagct
tctcgtctca gggcaatgg 29 8 29 DNA Artificial Primer for RCR
multiplication 8 cgagcaagct tgctcctaca ggtgaaggc 29 9 29 DNA
Artificial Primer for PCR multiplication 9 gtattaagct tgaagacgaa
ggagtgttg 29 10 30 DNA Artificial Primer for PCR multiplication 10
catccaagct tcttatgatc gggtcatgcc 30 11 45 DNA Artificial Primer for
PCR multiplication 11 agtggatcct ccgagctcag taacaagagt aacgatgagt
tgaag 45 12 45 DNA Artificial Primer for PCR multiplication 12
atactcgaga ctactagtcc gttcgtgcac gtacgtgcct ggcgc 45 13 45 DNA
Artificial Primer for PCR multiplication 13 agtggatcct ccgagctccg
cgataaacct gcgagggagt cacta 45 14 45 DNA Artificial Primer for PCR
multiplication 14 atactcgaga ctactagtgc gcacgcgcac gtaagtcccg ggcgc
45 15 12 PRT Artificial Ferrite-binding peptide 15 Met Pro Ser Trp
Arg Thr His His Val Ala Thr Pro 1 5 10 16 12 PRT Artificial
Ferrite-binding peptide 16 Met Gln Thr His His Thr Thr Val Thr Ser
Trp Thr 1 5 10 17 12 PRT Artificial Ferrite-binding peptide 17 Met
Leu Pro His Arg Pro Pro His Tyr Met Ser His 1 5 10 18 12 PRT
Artificial Ferrite-binding peptide 18 Met Leu Asn Pro Pro Gln Gly
His His His Met Gly 1 5 10 19 12 PRT Artificial Ferrite-binding
peptide 19 His Thr Met His Ala Trp Pro Pro Pro Ala Pro Phe 1 5 10
20 12 PRT Artificial Ferrite-binding peptide 20 His Ala His His Gln
Gln His Leu Lys Pro Gln Ser 1 5 10 21 12 PRT Artificial
Ferrite-binding peptide 21 Gly Leu Asp Ser Gly Pro Thr His Arg His
Met Phe 1 5 10 22 12 PRT Artificial Ferrite-binding peptide 22 Gly
Tyr Ala Ser Pro Lys Ala His Trp Ser Ser Gly 1 5 10 23 12 PRT
Artificial Ferrite-binding peptide 23 Ala Ser Arg Pro Met His Met
Pro His Ile Pro Ala 1 5 10 24 12 PRT Artificial Ferrite-binding
peptide 24 Ala Pro Gly Met Asn Ala Met Ala Ser Ile His His 1 5 10
25 12 PRT Artificial Ferrite-binding peptide 25 His Asn His Gln Phe
Gln Ala Ser Met His Pro Asp 1 5 10 26 12 PRT Artificial
Ferrite-binding peptide 26 Arg Ser Ile His His Asp Ser His Met Leu
Arg Gly 1 5 10 27 12 PRT Artificial Ferrite-binding peptide 27 Thr
His Ser Asn Ser Met Thr Arg Asn Thr Pro Met 1 5 10 28 12 PRT
Artificial Ferrite-binding peptide 28 Gly Leu Asp Ser Gly Pro Thr
His Arg His Met Phe 1 5 10 29 12 PRT Artificial Ferrite-binding
peptide 29 Asp Gly His Gln Pro Phe His Thr Leu Lys Pro Ala 1 5 10
30 12 PRT Artificial Ferrite-binding peptide 30 Gln Glu Ser His Gly
Gly Pro Pro Arg Ser Pro His 1 5 10 31 58 DNA Artificial Primer for
PCR multiplication 31 gatccatgcc gagttggagg actcatcatg ttgcgactcc
gggtggaggt tcggagct 58 32 50 DNA Artificial Primer for PCR
multiplication 32 ccgaacctcc acccggagtc gcaacatgat gagtcctcca
actcggcatg 50 33 54 DNA Artificial Primer for PCR multiplication 33
ctagtatgcc gagttggagg actcatcatg ttgcgactcc gggtggaggt tcgc 54 34
54 DNA Artificial Primer for PCR multiplication 34 tcgagcgaac
ctccacccgg agtcgcaaca tgatgagtcc tccaactcgg cata 54 35 32 PRT
Artificial Ferrite-binding peptide 35 Met Pro Ser Trp Arg Thr His
His Val Ala Thr Pro Gly Gly Gly Ser 1 5 10 15 Gly Gly Gly Ser Met
Gln Thr His His Thr Thr Val Thr Ser Trp Thr 20 25 30 36 106 DNA
Artificial Primer for PCR multiplication 36 gatccatgcc gagttggagg
actcatcatg ttgcgactcc gggcggcggc agcggcggcg 60 gcagcatgca
gacgcatcat actacggtga cttcgtggac tgagct 106 37 98 DNA Artificial
Primer for PCR multiplication 37 ccggctgatg acgaatatac ggcggccacc
accagttgct gccgccgccg ctgccgccgc 60 cgctcgccgg ccaccacact
ttccacgcat gcggccag 98 38 4 PRT Artificial linker sequence 38 Gly
Gly Gly Ser 1 39 8 PRT Artificial Linker 39 Gly Gly Gly Ser Gly Gly
Gly Ser 1 5
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