U.S. patent application number 14/880311 was filed with the patent office on 2016-04-21 for photocatalytic hydrogen production and polypeptides capable of same.
This patent application is currently assigned to RAMOT AT TEL-AVIV UNIVERSITY LTD.. The applicant listed for this patent is RAMOT AT TEL-AVIV UNIVERSITY LTD.. Invention is credited to Itai BENHAR, Ehud GAZIT, Nathan NELSON, Iftach YACOBY.
Application Number | 20160108434 14/880311 |
Document ID | / |
Family ID | 39766986 |
Filed Date | 2016-04-21 |
United States Patent
Application |
20160108434 |
Kind Code |
A1 |
YACOBY; Iftach ; et
al. |
April 21, 2016 |
PHOTOCATALYTIC HYDROGEN PRODUCTION AND POLYPEPTIDES CAPABLE OF
SAME
Abstract
An isolated polypeptide comprising a hydrogen generating enzyme
attached to a heterologous ferredoxin is disclosed, as well as
polynucleotides encoding same, nucleic acid constructs capable of
expressing same and cells expressing same. A method for generating
hydrogen using the isolated polypeptide is also disclosed.
Inventors: |
YACOBY; Iftach; (Kfar-Hess,
IL) ; GAZIT; Ehud; (Ramat-HaSharon, IL) ;
NELSON; Nathan; (Tel-Aviv, IL) ; BENHAR; Itai;
(Rehovot, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
RAMOT AT TEL-AVIV UNIVERSITY LTD. |
Tel-Aviv |
|
IL |
|
|
Assignee: |
RAMOT AT TEL-AVIV UNIVERSITY
LTD.
Tel-Aviv
IL
|
Family ID: |
39766986 |
Appl. No.: |
14/880311 |
Filed: |
October 12, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12670407 |
Jan 25, 2010 |
9181555 |
|
|
PCT/IL2008/001018 |
Jul 23, 2008 |
|
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14880311 |
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61064984 |
Apr 7, 2008 |
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60935015 |
Jul 23, 2007 |
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Current U.S.
Class: |
435/168 ;
435/188; 435/257.2; 435/292.1; 435/419; 536/23.2 |
Current CPC
Class: |
C12Y 112/98002 20130101;
C12M 47/10 20130101; C12M 21/18 20130101; C12N 9/96 20130101; C12M
31/10 20130101; C12N 9/0067 20130101; C12M 21/02 20130101; C12N
15/62 20130101; C12P 3/00 20130101 |
International
Class: |
C12P 3/00 20060101
C12P003/00; C12M 1/00 20060101 C12M001/00; C12M 1/40 20060101
C12M001/40; C12N 9/96 20060101 C12N009/96; C12N 9/02 20060101
C12N009/02 |
Claims
1. An isolated polypeptide comprising an algal Fe-only hydrogenase
attached to an algal ferredoxin, wherein the polypeptide is capable
of generating at least four time more hydrogen from electrons
donated thereto from methyl viologen than native hydrogenase.
2. The isolated polypeptide of claim 1, further comprising a
linker, capable of linking said algal Fe-only hydrogenase to said
plant ferredoxin.
3. The isolated polypeptide of claim 2, wherein said linker is a
peptide bond.
4. The isolated polypeptide of claim 2, wherein said linker
comprises a repeat sequence of glycine and serine.
5. An isolated polynucleotide encoding the polypeptide of claim
1.
6. A cell comprising the isolated polynucleotide of claim 5.
7. The cell of claim 6, being selected from the group consisting of
a cyanobacterial cell, an algal cell and a higher plant cell.
8. A method of generating hydrogen, the method comprising combining
the polypeptide of claim 1 with an electron donor so as to generate
an electron transfer chain, wherein said electron transfer chain is
configured such that said electron donor is capable of donating
electrons to the polypeptide of claim 1, thereby generating
hydrogen.
9. The method of claim 8, wherein the generating hydrogen is
effected under anaerobic conditions.
10. The method of claim 8, wherein said electron donor is selected
from the group consisting of a biomolecule, a chemical, water, an
electrode and a combination of the above.
11. The method of claim 10, wherein said biomolecule comprises
Photosystem I (PSI) or rhodopsin.
12. The method of claim 8, further comprising harvesting the
hydrogen following the generating.
13. The method of claim 8, wherein said combining is effected in a
cell-free system.
14. The method of claim 8, wherein said combining is effected in a
cellular system.
15. The method of claim 14, wherein said cellular system is
selected from the group consisting of a cyanobacteria, an algae and
a higher plant.
16. The method of claim 14, further comprising down-regulating an
expression of endogenous ferredoxin in said cellular system.
17. A system comprising the polypeptide of claim 1 and an electron
donor.
18. The system of claim 17, wherein said electron donor comprises
an agent selected from the group consisting of a biomolecule, a
chemical, water, an electrode and any combination of the above.
19. The system of claim 17, wherein said electron donor comprises
PS-1 or rhodopsin.
20. The system of claim 18, wherein said biomolecule is comprised
in particles.
21. The system of claim 19, being expressed in cells.
22. A bioreactor for producing hydrogen, comprising: a vessel
holding a hydrogen producing system, said system comprising a
suspension of the polypeptide of claim 1 and PSI; a light providing
apparatus comprising an optic fiber, said light providing apparatus
being configured to provide light of a selected spectrum to said
system; and a gas liquid separation membrane for separating gas
leaving the suspension from said suspension.
23. The bioreactor of claim 22, wherein said system comprises a
suspension of cells.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 12/670,407 filed on Jan. 25, 2010, which is a
National Phase of PCT Patent Application No. PCT/IL2008/001018
having International filing date of Jul. 23, 2008, which claims the
benefit of U.S. Provisional Patent Application Nos. 61/064,984,
filed on Apr. 7, 2008, and 60/935,015, filed on Jul. 23, 2007. The
contents of the above applications are all incorporated herein by
reference.
FIELD AND BACKGROUND OF THE INVENTION
[0002] The present invention relates to hydrogen production and,
more particularly, to polypeptides capable of same.
[0003] The development of a clean, sustainable and economically
viable energy supply for the future is one of the most urgent
challenges of our generation. Oil production is expected to peak in
the near future and economically viable oil reserves are expected
to be largely depleted by 2050. A viable hydrogen economy requires
clean, sustainable and economic ways of generating hydrogen.
Current hydrogen production depends almost entirely on the use of
non-renewable resources (i.e. steam reformation of natural gas,
coal gasification and nuclear power driven electrolysis of water).
Although these approaches are initially likely to drive a
transition towards a hydrogen economy, the hydrogen produced is
more expensive and contains less energy than the non-renewable
energy source from which it is derived. In addition, the use of
fossil fuels and nuclear power is unsustainable. Therefore, there
is a clear need to establish economically viable means of hydrogen
production.
[0004] A particularly desirable option is the production of
hydrogen using photosynthetic machinery, since the ultimate energy
source is solar energy. The twin hearts of the photosynthetic
machinery in plants, algae, and cyanobacteria are the two
photochemical reaction centers known as Photosystem I (PSI) and
Photosystem II (PSII). PSII drives the most highly oxidizing
reaction known to occur in biology, splitting water into oxygen,
protons and electrons. Oxygen is released into the atmosphere and
is responsible for maintaining aerobic life on Earth. The derived
electrons are passed along the photosynthetic electron transport
chain from PSII via Plastoquinone (PQ) to Cytochrome b.sub.6f (cyt
b.sub.6f) and Photosystem I (PSI). From PSI, most of the negative
redox potential is stabilized in the form of reduced ferredoxin
(Fd) that serves as an electron donor to
ferredoxin-NADP.sup.+-reductase (FNR) enzyme. Under normal
physiological conditions, Fd reduces NADP.sup.+to NADPH via the
Fd-FNR complex. In a parallel process (photophosphorylation),
protons are released into the thylakoid lumen where they generate a
proton gradient that is used to drive ATP production via ATP
synthase. NADPH and ATP are subsequently used to produce starch and
other biomolecules.
[0005] Some green algae and cyanobacteria have evolved the ability
to channel the protons and electrons stored in starch into hydrogen
production under anaerobic conditions by expressing a hydrogenase
enzyme. [Wunschiers, Stangier et al. 2001, Curr Microbiol 42(5):
353-60; Happe and Kaminski 2002, Eur J Biochem 269(3): 1022-32].
The hydrogenase enzyme is localized in the chloroplast stroma and
obtains electrons from ferredoxin or flavodoxin that is reduced by
Photosystem I and thus competes with FNR for the PSI generated
electrons (FIG. 1). However, oxygen is a powerful inhibitor of the
hydrogenase enzyme and thus, the generation of hydrogen in these
organisms is only transient and also inefficient.
[0006] Efforts to generate oxygen-tolerant algal hydrogenases have
not met with much success [Seibert et al. 2001, Strategies for
improving oxygen tolerance of algal hydrogen production.
Biohydrogen II. J. M. Miyake, T.; San Pietro, A., eds, Oxford, UK:
Pergamon 67-77]. McTavish et al [J Bacteriol 177(14): 3960-4, 1995]
have shown that site-directed mutagenesis of Azotobacter vinelandii
hydrogenase can render hydrogen production insensitive to oxygen
inhibition, but with a substantial (78%) loss of hydrogen evolution
activity.
[0007] Melis (U.S. Patent Application No. 2001/005343) teaches a
process in which the inhibition was lifted by temporally separating
the oxygen generating water splitting reaction, catalyzed by PSII,
from the oxygen sensitive hydrogen production catalyzed by the
chloroplast Hydrogenase (HydA). This separation was achieved by
culturing green algae first in the presence of sulfur to build
stores of an endogenous substrate and then in the absence of
sulfur. This led to inactivation of Photosystem II so that cellular
respiration led to anaerobiosis, the induction of hydrogenase, and
sustained hydrogen evolution in the light.
[0008] The Melis process is, however, subject to considerable
practical constraints. The actual rate of hydrogen gas accumulation
is at best 15 to 20% of the photosynthetic capacity of the cells
[Melis and Happe 2001, Plant Physiol. November; 127(3):740-8] and
suffers the inherent limitation that hydrogen production by sulfur
deprivation of the algae cannot be continued indefinitely. The
yield begins to level off and decline after about 40-70 hours of
sulfur deprivation, and after about 100 hours of sulfur deprivation
the algae need to revert to a phase of normal photosynthesis to
replenish endogenous substrates.
[0009] International Publication No. WO 03/067213 describes a
process for hydrogen production using Chlamydomonas reinhardtii
wherein the algae has been genetically modified to down regulate
expression of a sulfate permease, CrcpSulP, through insertion of an
antisense sequence. This is said to render obsolete prior art
sulfur deprivation techniques, as it obviates the need to
physically remove sulfur nutrients from growth media in order to
induce hydrogen production. The reduced sulfur uptake by the cell
using this technique not only results in a substantial lowering of
the levels of the major chloroplast proteins such as Rubisco, D1
and the LHCII, but also deprives the cell of sulfur for use in the
biosynthesis of other proteins.
[0010] Ihara et al (Ihara, Nakamoto et al. 2006; Ihara, Nishihara
et al. 2006) teach a fusion protein comprising membrane bound
[NiFe] hydrogenase (from the
.quadrature..quadrature.proteobacterium Raistonia eutropha H16) and
the peripheral PSI subunit PsaE of the cyanobacterium
Thermosynechococcus elongatus as a direct light-to-hydrogen
conversion system. The isolated hydrogenase-PSI isolated complex
displayed light-driven hydrogen production at a rate of [0.58
.mu.mol H.sub.2]/[mg chlorophyll] h in vitro. The inefficiency of
this system is thought to be derived from the mismatched ability of
the hydrogenase to accept electrons compared to the ability of PSI
to donate electrons.
[0011] Peters et al [Science, 282, 4 Dec., 1998], teach isolation
of an Fe-only hydrogenase from clostridium pasteurianum which
naturally comprises ferrodoxin-like structures. Although this
hydrogenase is potentially capable of directly generating hydrogen
under illuminated conditions, it can not accept electron from PSI
since it lacks the native plant structural docking site to do
so.
[0012] There is thus a widely recognized need for, and it would be
highly advantageous to have, a sustainable and efficient process
for photosynthetic hydrogen production devoid of the above
limitations.
SUMMARY OF THE INVENTION
[0013] According to one aspect of the present invention there is
provided an isolated polypeptide comprising a hydrogen generating
enzyme attached to a heterologous ferredoxin.
[0014] According to another aspect of the present invention there
is provided an isolated polynucleotide encoding a polypeptide
comprising a hydrogen generating enzyme attached to a heterologous
ferredoxin via a peptide bond.
[0015] According to yet another aspect of the present invention
there is provided a nucleic acid construct, comprising an isolated
polynucleotide encoding a polypeptide comprising a hydrogen
generating enzyme attached to a heterologous ferredoxin via a
peptide bond.
[0016] According to still another aspect of the present invention
there is provided a cell comprising a nucleic acid construct,
comprising an isolated polynucleotide encoding a polypeptide
comprising a hydrogen generating enzyme attached to a heterologous
ferredoxin via a peptide bond.
[0017] According to an additional aspect of the present invention
there is provided a method of generating hydrogen, the method
comprising combining an isolated polypeptide comprising a hydrogen
generating enzyme attached to a heterologous ferredoxin with an
electron donor so as to generate an electron transfer chain,
wherein the electron transfer chain is configured such that the
electron donor is capable of donating electrons to the polypeptide
thereby generating hydrogen.
[0018] According to yet an additional aspect of the present
invention there is provided a system comprising an isolated
polypeptide comprising a hydrogen generating enzyme attached to a
heterologous ferredoxin and an electron donor.
[0019] According to still an additional aspect of the present
invention there is provided a bioreactor for producing hydrogen,
comprising:
[0020] a vessel 321, holding a hydrogen producing system, the
system comprising a suspension of hydrogen generating enzyme
attached to a heterologous ferredoxin and PSI;
[0021] a light providing apparatus comprising an optic fiber, the
light providing apparatus being configured to provide light of a
selected spectrum to the system; and
[0022] a gas liquid separation membrane for separating gas leaving
the suspension from the suspension.
[0023] According to further features in the embodiments of the
invention described below, the hydrogen generating enzyme is a
hydrogenase.
[0024] According to still further features in the described
embodiments, the hydrogen generating enzyme is a nitrogenase.
[0025] According to still further features in the described
embodiments, the hydrogenase enzyme is selected from the group
consisting of an Fe only hydrogenase, a Ni--Fe hydrogenase and a
non-metal hydrogenase.
[0026] According to still further features in the described
embodiments, the polypeptide further comprises a linker, capable of
linking the hydrogen generating enzyme to the ferredoxin.
[0027] According to still further features in the described
embodiments, the linker is a covalent linker.
[0028] According to still further features in the described
embodiments, the linker is a non-covalent linker.
[0029] According to still further features in the described
embodiments, the covalent linker is a peptide bond.
[0030] According to still further features in the described
embodiments, the polypeptide is as set forth in SEQ ID NOs: 24 or
25.
[0031] According to still further features in the described
embodiments, the polynucleotide comprises a nucleic acid sequence
as set forth in SEQ ID NOs: 1-6.
[0032] According to still further features in the described
embodiments, wherein the nucleic acid construct further comprises a
cis-regulatory element.
[0033] According to still further features in the described
embodiments, the cis-regulatory element is a promoter.
[0034] According to still further features in the described
embodiments, the promoter is an inducible promoter.
[0035] According to still further features in the described
embodiments, the cell is a prokaryotic cell.
[0036] According to still further features in the described
embodiments, the cell is a eukaryotic cell.
[0037] According to still further features in the described
embodiments, the prokaryotic cell is a cyanobacteria cell.
[0038] According to still further features in the described
embodiments, the cell is an algae cell.
[0039] According to still further features in the described
embodiments, the eukaryotic cell is part of a higher plant.
[0040] According to still further features in the described
embodiments, the generating hydrogen is effected under anaerobic
conditions.
[0041] According to still further features in the described
embodiments, the electron donor is selected from the group
consisting of a biomolecule, a chemical, water, an electrode and a
combination of the above.
[0042] According to still further features in the described
embodiments, the electron donor comprises a biomolecule.
[0043] According to still further features in the described
embodiments, the biomolecule is light sensitive.
[0044] According to still further features in the described
embodiments, the light sensitive biomolecule comprises a
photocatalytic unit of a photosynthetic organism.
[0045] According to still further features in the described
embodiments, the photocatalytic unit comprises Photosystem I
(PSI).
[0046] According to still further features in the described
embodiments, a ratio of a polypeptide comprising a hydrogen
generating enzyme attached to a heterologous ferredoxin: PSI is
greater than 100:1.
[0047] According to still further features in the described
embodiments, the light sensitive biomolecule comprises
rhodopsin.
[0048] According to still further features in the described
embodiments, the biomolecule is immobilized to a solid support.
[0049] According to still further features in the described
embodiments, the chemical is selected from the group consisting of
dithiothreitol, ascorbic acid,
N,N,N',N'-tetramethyl-p-phenylendiamine (TMPD), 2,6-dichlorophenol
indophenol and a combination of any of the above.
[0050] According to still further features in the described
embodiments, the method further comprises illuminating the light
sensitive biomolecule following or concomitant with the
combining.
[0051] According to still further features in the described
embodiments, the method further comprises harvesting the hydrogen
following the generating.
[0052] According to still further features in the described
embodiments, the combining is effected in a cell-free system.
[0053] According to still further features in the described
embodiments, the cell-free system is selected from the group
consisting of polymeric particles, microcapsules liposomes,
microspheres, microemulsions, nano-plates, nanoparticles,
nanocapsules and nanospheres.
[0054] According to still further features in the described
embodiments, the combining is effected in a cellular system.
[0055] According to still further features in the described
embodiments, the cellular system is selected from the group
consisting of a cyanobacteria, an alga and a higher plant.
[0056] According to still further features in the described
embodiments, the method further comprises down-regulating an
expression of endogenous ferredoxin in the cellular system.
[0057] According to still further features in the described
embodiments, the biomolecule is comprised in particles.
[0058] According to still further features in the described
embodiments, the particles are selected from the group consisting
of polymeric particles, microcapsules liposomes, microspheres,
microemulsions, nanoparticles, nanocapsules, nano-plates and
nanospheres.
[0059] According to still further features in the described
embodiments, the biomolecule is encapsulated within the
particle.
[0060] According to still further features in the described
embodiments, the biomolecule is embedded within the particle.
[0061] According to still further features in the described
embodiments, the biomolecule is adsorbed on a surface of the
particle.
[0062] According to still further features in the described
embodiments, the system is expressed in cells.
[0063] According to still further features in the described
embodiments, the cells are selected from the group consisting of
cyanobacteria cells, algae cells and higher plant cells.
[0064] According to still further features in the described
embodiments, the system comprises a suspension of cells.
[0065] According to still further features in the described
embodiments, the system comprises a suspension of liposomes.
[0066] According to still further features in the described
embodiments, the spectrum is selected as not to damage the
system.
[0067] According to still further features in the described
embodiments, the spectrum is selected to include activating
wavelengths only.
[0068] The present invention successfully addresses the
shortcomings of the presently known configurations by providing
novel polypeptides capable of generating photocatalytically induced
hydrogen production.
[0069] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. In
case of conflict, the patent specification, including definitions,
will control. In addition, the materials, methods, and examples are
illustrative only and not intended to be limiting.
BRIEF DESCRIPTION OF THE DRAWINGS
[0070] The invention is herein described, by way of example only,
with reference to the accompanying drawings. With specific
reference now to the drawings in detail, it is stressed that the
particulars shown are by way of example and for purposes of
illustrative discussion of the preferred embodiments of the present
invention only, and are presented in the cause of providing what is
believed to be the most useful and readily understood description
of the principles and conceptual aspects of the invention. In this
regard, no attempt is made to show structural details of the
invention in more detail than is necessary for a fundamental
understanding of the invention, the description taken with the
drawings making apparent to those skilled in the art how the
several forms of the invention may be embodied in practice.
[0071] In the drawings:
[0072] FIG. 1 is a schematic diagram illustrating the overall
process of native light dependent hydrogen production in algae.
[0073] FIGS. 2A-B is a schematic diagram illustrating the position
of PSI in multilamellar (FIG. 2A) and unilamellar (FIG. 2B)
liposomes.
[0074] FIG. 3 is a schematic representation of a system for
producing hydrogen to an embodiment of the present invention. The
figure illustrates a system 10, illustrated comprising an electrode
12 in contact with the polypeptide of the present invention 14. The
polypeptide 14 may be attached to the electrode 12 for direct
bioelectrocatalysis using any method known in the art such as for
example the modification of electrode surfaces by redox mediators
or hydrophilic adsorption. Exemplary material that may be used for
generating electrode 12 is carbon covered with viologen substituted
poly(pyrrole), pyrolytic carbon paper (PCP) and packed graphite
columns (PGC). In order to generate hydrogen, the electrode 12 is
attached to an electrical source 16.
[0075] FIG. 4 is a schematic representation of an optic fiber
bioreactor according to an embodiment of the present invention.
FIG. 4 is a schematic illustration of a reactor 300 for producing
hydrogen according to an embodiment of the invention. Reactor 300
comprises a vessel 321, in which the hydrogen producing system
comprising PSI, and the ferredoxin unit of the polypeptide of the
present invention, are held in a suspension 322. The suspension 322
may also comprise other components such as sodium citrated and
TMPD. The suspension may include the hydrogen producing systems,
that is, the PSI and the ferredoxin unit, in any of the
above-mentioned ways, for instance, in liposomes or in cell
culture. Suspension 322 is constantly stirred with stirring blades
324 by rotor 325. The rotor and stirring blade are operated as not
to damage the cells or liposomes, but only homogenize them within
vessel 321. A temperature control 326 controls the temperature to
optimize the activity of the cells or liposomes, for instance,
37.degree. C. An optic fiber 323, provides light to the cells or
liposomes. Hydrogen produced by the hydrogen producing systems
bubbles out of the suspension, through a gas-liquid separation
membrane 328. From the gas side of membrane 328, the hydrogen is
optionally pumped with pump 329 to a hydrogen tank 330.
[0076] FIG. 5 is a photograph illustrating generation of chimeric
fusions of HydA1 and petF genes. 1HydFd-direct linkage of HydA1 and
petF; 2HydFd-short linker of four glycine and 1 serine between
HydA1 and petF; 3HydFd-medium linker of two repeats of: four
glycine and 1 serine between HydA1 and petF; 4HydFd-direct linkage
of C-terminus truncated HydA1 and N-terminus truncated petF;
5HydFd-direct linkage of C-terminus truncated HydA1 and petF;
6HydFd-direct linkage of HydA1 and N-terminus truncated petF.
[0077] FIGS. 6A-B are maps of exemplary expression constructs used
for the expression of the constructs of the present invention.
[0078] FIGS. 7A-B are photographs illustrating expression of the
polypeptides of the present invention. FIG. 7A--Western analysis
using the monoclonal anti StrpTagII (IBA.COPYRGT.). In addition,
the lower band of native HydA1 was used as internal control. It can
be seen that native HydA1 is expressed 10.times. more than fusion
proteins.
[0079] FIG. 7B--the 12% polyacrylamide gel shows the accessory
proteins HydG/F/E expression pattern which are expressed for all of
the chimeras as well as the native hydA1 protein.
[0080] FIG. 8 is a bar graph illustrating hydrogen generation from
a total cell extract prepared from E. coli cells that express
HydA1, Pet F and HydFd chimera. Dithionite was used as electron
donor. The experiment was performed in argon atmosphere. Light gray
bars: Hydrogen generation by the hydrogenase component alone, as
measured by addition of methyl violegen as an electron mediator.
Dark gray bars: Ferredoxin-mediated hydrogen production following
elimination of methyl viologen from the system. The displayed
values of hydrogen production were based on H.sub.2 gas production
measured by gas chromatography and corrected according to the
relative expression level of the proteins according to FIGS.
7A-B.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0081] The present invention is of a biological method of
generating hydrogen and polypeptides capable of catalyzing this
reaction.
[0082] Before explaining at least one embodiment of the invention
in detail, it is to be understood that the invention is not limited
in its application to the details set forth in the following
description or exemplified by the Examples. The invention is
capable of other embodiments or of being practiced or carried out
in various ways. Also, it is to be understood that the phraseology
and terminology employed herein is for the purpose of description
and should not be regarded as limiting.
[0083] Molecular hydrogen is a candidate for replacing or
supplementing fossil fuels and as a source of clean energy. Natural
biological production of hydrogen is based on the presence of
hydrogenase enzymes present in certain green algae and
photosynthetic bacteria which are capable of accepting electrons
from photosystem I (PSI) and conversion thereof into hydrogen gas.
The yield of molecular hydrogen from this process is limited
because the endogenous electron carriers donate their electrons to
destinations other than hydrogenase. For example, reduced electron
carriers, such as ferredoxin also donate electrons to
ferredoxin-NADP.sup.+-reductase (FNR) enzyme.
[0084] The present inventors have deduced that in order to increase
hydrogen production, electrons must be encouraged to shuttle
towards hydrogenase (or other hydrogen generating enzymes, such as
nitrogenase) at the expense of the competing processes. The present
inventors have contemplated a novel hydrogenase polypeptide which
is artificially linked to a heterologous ferredoxin. Such a
polypeptide would force the flow of electrons from an electron
donor such as photosystem I (PSI) directly to the hydrogenase at
the expense of FNR. This novel polypeptide may be expressed in
cellular or cell-free systems in order to generate hydrogen gas.
Furthermore, the present inventors have conceived that in order to
further up-regulate hydrogen production in photosynthetic
organisms, the competing process (i.e. the endogenous Fd-FNR
complex) is preferably down-regulated.
[0085] Whilst reducing the present invention to practice, the
present inventors have generated a number of hydrogenase
polypeptides, artificially linked to heterologous ferredoxins
(FIGS. 7A-B). Such polypeptides were shown to generate hydrogen
(FIG. 8) and may also comprise a reduced sensitivity to oxygen.
[0086] Thus, according to one aspect of the present invention,
there is provided an isolated polypeptide comprising a hydrogen
generating enzyme attached to a heterologous ferredoxin.
[0087] The phrase "hydrogen generating enzyme" refers to a protein
capable of catalyzing a reaction where at least one of the
end-products is hydrogen. According to one embodiment the hydrogen
generating enzyme is a hydrogenase.
[0088] As used herein, the phrase "hydrogenase enzyme" refers to an
amino acid sequence of a hydrogenase enzyme with the capability of
catalyzing hydrogen oxidation/reduction. Thus the present invention
contemplates full-length hydrogenase as well as active fragments
thereof. According to one embodiment, the hydrogenase enzyme is a
Fe only hydrogenase. According to another embodiment, the
hydrogenase is a Ni--Fe hydrogenase. According to yet another
embodiment, the hydrogenase is a non-metal hydrogenase. Exemplary
hydrogenase enzymes which may be used in accordance with the
present invention are set forth by EC 1.12.1.2, EC 1.12.1.3, EC
1.12.2.1, EC 1.12.7.2, EC 1.12.98.1, EC 1.12.99.6, EC 1.12.5.1, EC
1.12.98.2 and EC 1.12.98.3.
[0089] Other examples of hydrogenases that may be used according to
the teaching of the present invention are listed below in Table 1
together with their source organisms.
TABLE-US-00001 TABLE 1 Source Organism Protein accession number
Chlamydomonas reinhardtii AY055756 Desulfovibrio vulgaris
hydrogenase CA26266.1 Megasphaera elsdenii AF120457 Anabaena
variabilis CAA55878 Desulfovibrio Desulfuricans 1E3D_A Clostridium
Pasteurianum 1FEH_A Chlamydomonas reinhardtii AAR04931
[0090] According to another embodiment the hydrogen generating
enzyme is a nitrogenase enzyme.
[0091] As used herein, the phrase "nitrogenase enzyme" refers to an
amino acid sequence of a nitrogenase enzyme (EC 1.18.6.1) with the
capability of generating hydrogen as a byproduct in a nitrogen
fixation reaction. Thus the present invention contemplates
full-length nitrogenase as well as active fragments thereof.
Examples of nitrogenases that may be used according to the teaching
of the present invention are listed below in Table 2 together with
their source organisms.
TABLE-US-00002 TABLE 2 Source Organism Protein accession number
Azotobacter Vinelandii 1M1N_A Clostridium Pasteurianum 1MIO_A
Anabaena variabilis AAX82499
[0092] As mentioned the polypeptide of the present invention,
comprises a hydrogenase attached to a heterologous ferredoxin.
[0093] As used herein, the term "ferredoxin" refers to an amino
acid sequence of the iron sulfur protein that is capable of
mediating electron transfer to hydrogenase. Thus the present
invention contemplates full-length ferredoxin as well as active
fragments thereof. According to a preferred embodiment of this
aspect of the present invention, the ferredoxin is a plant-type
ferredoxin.
[0094] Exemplary ferredoxin polypeptides that may be used in
accordance with the present invention include, but are not limited
to cyanobacterial ferredoxins, algae ferredoxins and non
photosynthetic organism ferredoxins.
[0095] The qualifier "heterologous" when relating to the ferredoxin
indicates that the ferredoxin is not naturally associated with
(i.e. endogenous to) the hydrogenase of the present invention.
Thus, for example, the phrase "hydrogenase attached to a
heterologous ferredoxin" does not comprise the Fe-only hydrogenase
from clostridium pasteurianum.
[0096] The present invention envisages attachment of the
heterologous ferredoxin at any position to the hydrogen generating
enzyme so long as the hydrogen generating enzyme is capable of
generating hydrogen from electrons donated thereto from the
attached ferredoxin. The hydrogen generating enzyme and ferredoxin
may be linked via bonding at their carboxy (C) or amino (N)
termini, or via bonding to internal chemical groups such as
straight, branched or cyclic side chains, internal carbon or
nitrogen atoms, and the like. Methods of linking the hydrogen
generating enzyme to the ferredoxin are further described herein
below.
[0097] Amino acid sequences of exemplary polypeptides of the
present invention are set forth in SEQ ID NOs: 23-28.
[0098] The term "polypeptide" as used herein encompasses native
polypeptides (either degradation products, synthetically
synthesized polypeptides or recombinant polypeptides) and
peptidomimetics (typically, synthetically synthesized
polypeptides), as well as peptoids and semipeptoids which are
polypeptide analogs, which may have, for example, modifications
rendering the polypeptides more stable while in a body or more
capable of penetrating into cells. Such modifications include, but
are not limited to N terminus modification, C terminus
modification, polypeptide bond modification, including, but not
limited to, CH2-NH, CH2-S, CH2-S.dbd.O, O.dbd.C--NH, CH2-O,
CH2-CH2, S.dbd.C--NH, CH.dbd.CH or CF.dbd.CH, backbone
modifications, and residue modification. Methods for preparing
peptidomimetic compounds are well known in the art and are
specified, for example, in Quantitative Drug Design, C. A. Ramsden
Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which is
incorporated by reference as if fully set forth herein. Further
details in this respect are provided hereinunder. Polypeptide bonds
(--CO--NH--) within the polypeptide may be substituted, for
example, by N-methylated bonds (--N(CH3)-CO--), ester bonds
(--C(R)H--C--O--O--C(R)--N--), ketomethylen bonds (--CO--CH2-),
.alpha.-aza bonds (--NH--N(R)--CO--), wherein R is any alkyl, e.g.,
methyl, carba bonds (--CH2-NH--), hydroxyethylene bonds
(--CH(OH)--CH2-), thioamide bonds (--CS--NH--), olefinic double
bonds (--CH.dbd.CH--), retro amide bonds (--NH--CO--), polypeptide
derivatives (--N(R)--CH2-CO--), wherein R is the "normal" side
chain, naturally presented on the carbon atom.
[0099] These modifications can occur at any of the bonds along the
polypeptide chain and even at several (2-3) at the same time.
[0100] Natural aromatic amino acids, Trp, Tyr and Phe, may be
substituted for synthetic non-natural acid such as Phenylglycine,
TIC, naphthylelanine (Nol), ring-methylated derivatives of Phe,
halogenated derivatives of Phe or o-methyl-Tyr.
[0101] In addition to the above, the polypeptides of the present
invention may also include one or more modified amino acids or one
or more non-amino acid monomers (e.g. fatty acids, complex
carbohydrates etc).
[0102] As used herein in the specification and in the claims
section below the term "amino acid" or "amino acids" is understood
to include the 20 naturally occurring amino acids; those amino
acids often modified post-translationally in vivo, including, for
example, hydroxyproline, phosphoserine and phosphothreonine; and
other unusual amino acids including, but not limited to,
2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine,
nor-leucine and ornithine. Furthermore, the term "amino acid"
includes both D- and L-amino acids.
[0103] Tables 3 and 4 below list naturally occurring amino acids
(Table 3) and non-conventional or modified amino acids (Table 4)
which can be used with the present invention.
TABLE-US-00003 TABLE 3 Three-Letter Amino Acid Abbreviation
One-letter Symbol alanine Ala A Arginine Arg R Asparagine Asn N
Aspartic acid Asp D Cysteine Cys C Glutamine Gln Q Glutamic Acid
Glu E glycine Gly G Histidine His H isoleucine Iie I leucine Leu L
Lysine Lys K Methionine Met M phenylalanine Phe F Proline Pro P
Serine Ser S Threonine Thr T tryptophan Trp W tyrosine Tyr Y Valine
Val V Any amino acid as above Xaa X
TABLE-US-00004 TABLE 4 Non-conventional amino acid Code
Non-conventional amino acid Code .alpha.-aminobutyric acid Abu
L-N-methylalanine Nmala .alpha.-amino-.alpha.-methylbutyrate Mgabu
L-N-methylarginine Nmarg aminocyclopropane- Cpro
L-N-methylasparagine Nmasn carboxylate L-N-methylaspartic acid
Nmasp aminoisobutyric acid Aib L-N-methylcysteine Nmcys
aminonorbornyl- Norb L-N-methylglutamine Nmgin carboxylate
L-N-methylglutamic acid Nmglu cyclohexylalanine Chexa
L-N-methylhistidine Nmhis cyclopentylalanine Cpen
L-N-methylisolleucine Nmile D-alanine Dal L-N-methylleucine Nmleu
D-arginine Darg L-N-methyllysine Nmlys D-aspartic acid Dasp
L-N-methylmethionine Nmmet D-cysteine Dcys L-N-methylnorleucine
Nmnle D-glutamine Dgln L-N-methylnorvaline Nmnva D-glutamic acid
Dglu L-N-methylornithine Nmorn D-histidine Dhis
L-N-methylphenylalanine Nmphe D-isoleucine Dile L-N-methylproline
Nmpro D-leucine Dleu L-N-methylserine Nmser D-lysine Dlys
L-N-methylthreonine Nmthr D-methionine Dmet L-N-methyltryptophan
Nmtrp D-ornithine Dorn L-N-methyltyrosine Nmtyr D-phenylalanine
Dphe L-N-methylvaline Nmval D-proline Dpro L-N-methylethylglycine
Nmetg D-serine Dser L-N-methyl-t-butylglycine Nmtbug D-threonine
Dthr L-norleucine Nle D-tryptophan Dtrp L-norvaline Nva D-tyrosine
Dtyr .alpha.-methyl-aminoisobutyrate Maib D-valine Dval
.alpha.-methyl-.gamma.-aminobutyrate Mgabu D-.alpha.-methylalanine
Dmala .alpha. ethylcyclohexylalanine Mchexa
D-.alpha.-methylarginine Dmarg .alpha.-methylcyclopentylalanine
Mcpen D-.alpha.-methylasparagine Dmasn
.alpha.-methyl-.alpha.-napthylalanine Manap
D-.alpha.-methylaspartate Dmasp .alpha.-methylpenicillamine Mpen
D-.alpha.-methylcysteine Dmcys N-(4-aminobutyl)glycine Nglu
D-.alpha.-methylglutamine Dmgln N-(2-aminoethyl)glycine Naeg
D-.alpha.-methylhistidine Dmhis N-(3-aminopropyl)glycine Norn
D-.alpha.-methylisoleucine Dmile N-amino-.alpha.-methylbutyrate
Nmaabu D-.alpha.-methylleucine Dmleu .alpha.-napthylalanine Anap
D-.alpha.-methyllysine Dmlys N-benzylglycine Nphe
D-.alpha.-methylmethionine Dmmet N-(2-carbamylethyl)glycine Ngln
D-.alpha.-methylornithine Dmorn N-(carbamylmethyl)glycine Nasn
D-.alpha.-methylphenylalanine Dmphe N-(2-carboxyethyl)glycine Nglu
D-.alpha.-methylproline Dmpro N-(carboxymethyl)glycine Nasp
D-.alpha.-methylserine Dmser N-cyclobutylglycine Ncbut
D-.alpha.-methylthreonine Dmthr N-cycloheptylglycine Nchep
D-.alpha.-methyltryptophan Dmtrp N-cyclohexylglycine Nchex
D-.alpha.-methyltyrosine Dmty N-cyclodecylglycine Ncdec
D-.alpha.-methylvaline Dmval N-cyclododeclglycine Ncdod
D-.alpha.-methylalnine Dnmala N-cyclooctylglycine Ncoct
D-.alpha.-methylarginine Dnmarg N-cyclopropylglycine Ncpro
D-.alpha.-methylasparagine Dnmasn N-cycloundecylglycine Ncund
D-.alpha.-methylasparatate Dnmasp N-(2,2-diphenylethyl)glycine Nbhm
D-.alpha.-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycine Nbhe
D-N-methylleucine Dnmleu N-(3-indolylyethyl) glycine Nhtrp
D-N-methyllysine Dnmlys N-methyl-.gamma.-aminobutyrate Nmgabu
N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dnmmet
D-N-methylornithine Dnmorn N-methylcyclopentylalanine Nmcpen
N-methylglycine Nala D-N-methylphenylalanine Dnmphe
N-methylaminoisobutyrate Nmaib D-N-methylproline Dnmpro
N-(1-methylpropyl)glycine Nile D-N-methylserine Dnmser
N-(2-methylpropyl)glycine Nile D-N-methylserine Dnmser
N-(2-methylpropyl)glycine Nleu D-N-methylthreonine Dnmthr
D-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine Nva
D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap
D-N-methylvaline Dnmval N-methylpenicillamine Nmpen
.gamma.-aminobutyric acid Gabu N-(p-hydroxyphenyl)glycine Nhtyr
L-t-butylglycine Tbug N-(thiomethyl)glycine Ncys L-ethylglycine Etg
penicillamine Pen L-homophenylalanine Hphe L-.alpha.-methylalanine
Mala L-.alpha.-methylarginine Marg L-.alpha.-methylasparagine Masn
L-.alpha.-methylaspartate Masp L-.alpha.-methyl-t-butylglycine
Mtbug L-.alpha.-methylcysteine Mcys L-methylethylglycine Metg
L-.alpha. thylglutamine Mgln L-.alpha.-methylglutamate Mglu
L-.alpha.-methylhistidine Mhis L-.alpha.-methylhomo phenylalanine
Mhphe L-.alpha.-methylisoleucine Mile N-(2-methylthioethyl)glycine
Nmet D-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine Narg
D-N-methylglutamate Dnmglu N-(1-hydroxyethyl)glycine Nthr
D-N-methylhistidine Dnmhis N-(hydroxyethyl)glycine Nser
D-N-methylisoleucine Dnmile N-(imidazolylethyl)glycine Nhis
D-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine Nhtrp
D-N-methyllysine Dnmlys N-methyl-.gamma.-aminobutyrate Nmgabu
N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dnmmet
D-N-methylornithine Dnmorn N-methylcyclopentylalanine Nmcpen
N-methylglycine Nala D-N-methylphenylalanine Dnmphe
N-methylaminoisobutyrate Nmaib D-N-methylproline Dnmpro
N-(1-methylpropyl)glycine Nile D-N-methylserine Dnmser
N-(2-methylpropyl)glycine Nleu D-N-methylthreonine Dnmthr
D-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine Nval
D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap
D-N-methylvaline Dnmval N-methylpenicillamine Nmpen
.gamma.-aminobutyric acid Gabu N-(p-hydroxyphenyl)glycine Nhtyr
L-t-butylglycine Tbug N-(thiomethyl)glycine Ncys L-ethylglycine Etg
penicillamine Pen L-homophenylalanine Hphe L-.alpha.-methylalanine
Mala L-.alpha.-methylarginine Marg L-.alpha.-methylasparagine Masn
L-.alpha.-methylaspartate Masp L-.alpha.-methyl-t-butylglycine
Mtbug L-.alpha.-methylcysteine Mcys L-methylethylglycine Metg
L-.alpha.-methylglutamine Mgln L-.alpha.-methylglutamate Mglu
L-.alpha. ethylhistidine Mhis L-.alpha.-methylhomophenylalanine
Mhphe L-.alpha. thylisoleucine Mile N-(2-methylthioethyl)glycine
Nmet L-.alpha.-methylleucine Mleu L-.alpha.-methyllysine Mlys
L-.alpha.-methylmethionine Mmet L-.alpha.-methylnorleucine Mnle
L-.alpha.-methylnorvaline Mnva L-.alpha.-methylornithine Morn
L-.alpha.-methylphenylalanine Mphe L-.alpha.-methylproline Mpro
L-.alpha.-methylserine mser L-.alpha.-methylthreonine Mthr
L-.alpha. ethylvaline Mtrp L-.alpha.-methyltyrosine Mtyr
L-.alpha.-methylleucine Mval L-N-methylhomophenylalanine Nmhphe
Nnbhm N-(N-(2,2-diphenylethyl) N-(N-(3,3-diphenylpropyl)
carbamylmethyl-glycine Nnbhm carbamylmethyl(1)glycine Nnbhe
1-carboxy-1-(2,2-diphenyl Nmbc ethylamino)cyclopropane
[0104] The polypeptides of the present invention are preferably
utilized in a linear form, although it will be appreciated that in
cases where cyclization does not severely interfere with
polypeptide characteristics (e.g. electron transfer), cyclic forms
of the polypeptide can also be utilized.
[0105] The polypeptide of present invention can be synthesized
biochemically. Alternatively, the polypeptide of present invention
can be generated using recombinant techniques in order to generate
a fusion protein wherein the hydrogen generating enzyme amino acid
sequence is attached to the ferredoxin amino acid sequence via a
peptide bond or a substituted peptide bond as further described
herein above. It will be appreciated that the attachment of the
hydrogen generating enzyme to the ferredoxin may also be effected
following the independent synthesis (either biochemically, or using
recombinant techniques) of hydrogenase (or nitrogenase) and
ferredoxin. It addition, the hydrogen generating enzyme and/or
ferredoxin may also be isolated from their natural environment and
subsequently linked. Each alternative method will be further
described herein below.
[0106] Biochemical Synthesis
[0107] Standard solid phase techniques may be used to biochemically
synthesize the polypeptides of the present invention. These methods
include exclusive solid phase synthesis, partial solid phase
synthesis methods, fragment condensation, classical solution
synthesis. These methods are preferably used when the polypeptide
cannot be produced by recombinant techniques (i.e., not encoded by
a nucleic acid sequence) and therefore involves different
chemistry.
[0108] Solid phase polypeptide synthesis procedures are well known
in the art and further described by John Morrow Stewart and Janis
Dillaha Young, Solid Phase Polypeptide Syntheses (2nd Ed., Pierce
Chemical Company, 1984).
[0109] Synthetic polypeptides can be purified by preparative high
performance liquid chromatography [Creighton T. (1983) Proteins,
structures and molecular principles. WH Freeman and Co. N.Y.] and
the composition of which can be confirmed via amino acid
sequencing.
[0110] Recombinant Techniques
[0111] Recombinant techniques are preferably used to generate the
polypeptides of the present invention since these techniques are
better suited for generation of relatively long polypeptides (e.g.,
longer than 20 amino acids) and large amounts thereof, as long as
no modified amino acids are included in the sequence. As mentioned,
recombinant techniques may be used to generate the hydrogen
generating enzyme and ferredoxin independently or alternatively to
generate a fusion protein where the hydrogen generating enzyme is
attached to the ferredoxin via a peptide bond.
[0112] Such recombinant techniques are described by Bitter et al.,
(1987) Methods in Enzymol. 153:516-544, Studier et al. (1990)
Methods in Enzymol. 185:60-89, Brisson et al. (1984) Nature
310:511-514, Takamatsu et al. (1987) EMBO J. 6:307-311, Coruzzi et
al. (1984) EMBO J. 3:1671-1680 and Brogli et al., (1984) Science
224:838-843, Gurley et al. (1986) Mol. Cell. Biol. 6:559-565 and
Weissbach & Weissbach, 1988, Methods for Plant Molecular
Biology, Academic Press, NY, Section VIII, pp 421-463.
[0113] To produce the polypeptides of the present invention using
recombinant technology, a polynucleotide encoding the polypeptides
of the present invention is ligated into a nucleic acid expression
vector, which comprises the polynucleotide sequence under the
transcriptional control of a cis-regulatory sequence (e.g.,
promoter sequence) suitable for directing constitutive, tissue
specific or inducible transcription of the polypeptides of the
present invention in the host cells.
[0114] Thus, the present invention contemplates isolated
polynucleotides encoding the fusion protein of the present
invention.
[0115] The phrase "an isolated polynucleotide" refers to a single
or double stranded nucleic acid sequence which is isolated and
provided in the form of an RNA sequence, a complementary
polynucleotide sequence (cDNA), a genomic polynucleotide sequence
and/or a composite polynucleotide sequences (e.g., a combination of
the above).
[0116] As used herein the phrase "complementary polynucleotide
sequence" refers to a sequence, which results from reverse
transcription of messenger RNA using a reverse transcriptase or any
other RNA dependent DNA polymerase. Such a sequence can be
subsequently amplified in vivo or in vitro using a DNA dependent
DNA polymerase.
[0117] As used herein the phrase "genomic polynucleotide sequence"
refers to a sequence derived (isolated) from a chromosome and thus
it represents a contiguous portion of a chromosome.
[0118] As used herein the phrase "composite polynucleotide
sequence" refers to a sequence, which is at least partially
complementary and at least partially genomic. A composite sequence
can include some exon sequences required to encode the polypeptide
of the present invention, as well as some intronic sequences
interposing therebetween. The intronic sequences can be of any
source, including of other genes, and typically will include
conserved splicing signal sequences. Such intronic sequences may
further include cis acting expression regulatory elements.
[0119] Exemplary nucleic acid sequences of the polynucleotides of
the present invention are set forth in SEQ ID NOs: 1-6.
[0120] As mentioned hereinabove, polynucleotide sequences of the
present invention are inserted into expression vectors (i.e., a
nucleic acid construct) to enable expression of the recombinant
polypeptide. The expression vector of the present invention
includes additional sequences which render this vector suitable for
replication and integration in prokaryotes, eukaryotes, or
preferably both (e.g., shuttle vectors). Typical cloning vectors
contain transcription and translation initiation sequences (e.g.,
promoters, enhances) and transcription and translation terminators
(e.g., polyadenylation signals).
[0121] A variety of prokaryotic or eukaryotic cells can be used as
host-expression systems to express the polypeptides of the present
invention. These include, but are not limited to, microorganisms,
such as bacteria transformed with a recombinant bacteriophage DNA,
plasmid DNA or cosmid DNA expression vector containing the
polypeptide coding sequence; yeast transformed with recombinant
yeast expression vectors containing the polypeptide coding
sequence; plant cell systems infected with recombinant virus
expression vectors or transformed with recombinant plasmid
expression vectors, such as Ti plasmid, containing the polypeptide
coding sequence.
[0122] According to one embodiment of this aspect of the present
invention, the polynucleotides of the present invention are
expressed in a photosynthetic organism. (e.g. higher plant, alga,
cyanobacteria) which endogenously express PSI and/or PSII.
Advantages thereof are discussed herein below.
[0123] Examples of constitutive plant promoters include, but are
not limited to CaMV35S and CaMV19S promoters, tobacco mosaic virus
(TMV), FMV34S promoter, sugarcane bacilliform badnavirus promoter,
CsVMV promoter, Arabidpsis ACT2/ACT8 actin promoter, Arabidpsis
ubiquitin UBQ 1 promoter, barley leaf thionin BTH6 promoter, and
rice actin promoter.
[0124] An inducible promoter is a promoter induced by a specific
stimulus such as stress conditions comprising, for example, light,
temperature, chemicals, drought, high salinity, osmotic shock,
oxidant conditions or in case of pathogenicity. Examples of
inducible promoters include, but are not limited to, the
light-inducible promoter derived from the pea rbcS gene, the
promoter from the alfalfa rbcS gene, the promoters DRE, MYC and MYB
active in drought; the promoters INT, INPS, prxEa, Ha hsp17.7G4 and
RD21 active in high salinity and osmotic stress, and the promoters
hsr2O3J and str246C active in pathogenic stress.
[0125] These constructs can be introduced into plant cells using Ti
plasmid, Ri plasmid, plant viral vectors, direct DNA
transformation, microinjection, electroporation, Biolistics (gene
gun) and other techniques well known to the skilled artisan. See,
for example, Weissbach & Weissbach [Methods for Plant Molecular
Biology, Academic Press, NY, Section VIII, pp 421-463 (1988)].
Other expression systems such as insects and mammalian host cell
systems, which are well known in the art, can also be used by the
present invention.
[0126] It will be appreciated that other than containing the
necessary elements for the transcription and translation of the
inserted coding sequence (encoding the polypeptide), the expression
construct of the present invention can also include sequences
engineered to optimize stability, production, purification, yield
or activity of the expressed polypeptide.
[0127] Various methods can be used to introduce the expression
vector of the present invention into the host cell system. Such
methods are generally described in Sambrook et al., Molecular
Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New
York (1989, 1992), in Ausubel et al., Current Protocols in
Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989),
Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich.
(1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich.
(1995), Vectors: A Survey of Molecular Cloning Vectors and Their
Uses, Butterworths, Boston Mass. (1988) and Gilboa et at.
[Biotechniques 4 (6): 504-512, 1986] and include, for example,
stable or transient transfection, lipofection, electroporation and
infection with recombinant viral vectors. In addition, see U.S.
Pat. Nos. 5,464,764 and 5,487,992 for positive-negative selection
methods.
[0128] Transformed cells are cultured under effective conditions,
which allow for the expression of high amounts of recombinant
polypeptide. Effective culture conditions include, but are not
limited to, effective media, bioreactor, temperature, pH and oxygen
conditions that permit protein production. An effective medium
refers to any medium in which a cell is cultured to produce the
recombinant polypeptide of the present invention. Such a medium
typically includes an aqueous solution having assimilable carbon,
nitrogen and phosphate sources, and appropriate salts, minerals,
metals and other nutrients, such as vitamins. Cells of the present
invention can be cultured in conventional fermentation bioreactors,
shake flasks, test tubes, microtiter dishes and petri plates.
Culturing can be carried out at a temperature, pH and oxygen
content appropriate for a recombinant cell. In addition, cells of
the current invention can be cultured under field conditions such
as open ponds, covered ponds, plastic bags (see for example "A Look
Back at the U.S. Department of Energy's Aquatic Species
Program--Biodiesel from Algae, July 1998, U.S. Department of
Energy's Office of Fuels Development, incorporated herein by
reference). Such culturing conditions are within the expertise of
one of ordinary skill in the art.
[0129] Depending on the vector and host system used for production,
resultant polypeptides of the present invention may either remain
within the recombinant cell, secreted into the fermentation medium,
secreted into a space between two cellular membranes, such as the
periplasmic space in E. coli; or retained on the outer surface of a
cell or viral membrane.
[0130] It will be appreciated that if the polypeptide of the
present invention is to be used in a cell free system, following a
predetermined time in culture, recovery of the recombinant
polypeptide is effected.
[0131] The phrase "recovering the recombinant polypeptide" used
herein refers to collecting the whole fermentation medium
containing the polypeptide and need not imply additional steps of
separation or purification.
[0132] Thus, polypeptides of the present invention can be purified
using a variety of standard protein purification techniques, such
as, but not limited to, salting out (as in ammonium sulfate
precipitation), affinity chromatography, ion exchange
chromatography, filtration, electrophoresis, hydrophobic
interaction chromatography, gel filtration chromatography, reverse
phase chromatography, concanavalin A chromatography,
chromatofocusing and differential solubilization.
[0133] To facilitate recovery, the expressed coding sequence can be
engineered to encode the polypeptide of the present invention and
fused cleavable moiety. Such a fusion protein can be designed so
that the polypeptide can be readily isolated by affinity
chromatography; e.g., by immobilization on a column specific for
the cleavable moiety. Where a cleavage site is engineered between
the polypeptide and the cleavable moiety, the polypeptide can be
released from the chromatographic column by treatment with an
appropriate enzyme or agent that specifically cleaves the fusion
protein at this site [e.g., see Booth et al., Immunol. Lett.
19:65-70 (1988); and Gardella et al., J. Biol. Chem.
265:15854-15859 (1990)].
[0134] The polypeptide of the present invention is preferably
retrieved in "substantially pure" form.
[0135] As used herein, the phrase "substantially pure" refers to a
purity that allows for the effective use of the protein in the
applications described herein.
[0136] In addition to being synthesizable in host cells, the
polypeptide of the present invention can also be synthesized using
in vitro expression systems. These methods are well known in the
art and the components of the system are commercially
available.
[0137] Site-Directed Linkage of Hydrogenase to Ferredoxin
[0138] Non-natural amino acids may be added to specific places
within a recombinant protein followed by chemical conjugation at
these specific positions [Chin J W, Cropp T A, Anderson J C,
Mukherji M, Zhang Z, Schultz P G. Science. 2003 Aug. 15;
301(5635):964-7; Dieterich D C, Link A J, Graumann J, Tirrell D A,
Schuman E M. Proc Natl Acad Sci USA. 2006 Jun. 20;
103(25):9482-7].
[0139] Non-Recombinant Linkage of Hydrogenase to Ferredoxin
[0140] As mentioned, the hydrogen generating enzyme and the
ferredoxin may be generated (e.g. synthesized) or isolated
independently and chemically linked one to the other via a covalent
(e.g. peptide) or non-covalent linker either directly or via
bonding to an intervening linker element, such as a linker peptide
or other chemical moiety, such as an organic polymer.
[0141] Exemplary chemical crosslinking methods for conjugating the
hydrogen generating enzyme with ferredoxin are described herein
below:
[0142] Thiol-Amine Crosslinking:
[0143] In this scheme, the amine group of the hydrogen generating
enzyme is indirectly conjugated to a thiol group on the ferredoxin
or vica versa, usually by a two- or three-step reaction sequence.
The high reactivity of thiols and their relative rarity in most
polypeptides make thiol groups ideal targets for controlled
chemical crosslinking. Thiol groups may be introduced into one of
the two polypeptides using one of several thiolation methods
including SPDP. The thiol-containing biomolecule is then reacted
with an amine-containing biomolecule using a heterobifunctional
crosslinking reagent.
[0144] Amine--Amine Crosslinking:
[0145] Conjugation of the hydrogen generating enzyme with
ferredoxin can be accomplished by methods known to those skilled in
the art using amine-amine crosslinkers including, but not limited
to glutaraldehyde, bis(imido esters), bis(succinimidyl esters),
diisocyanates and diacid chlorides.
[0146] Carbodiimide Conjugation:
[0147] Conjugation of the hydrogen generating enzyme with
ferredoxin can be accomplished by methods known to those skilled in
the art using a dehydrating agent such as a carbodiimide. Most
preferably the carbodiimide is used in the presence of 4-dimethyl
aminopyridine. As is well known to those skilled in the art,
carbodiimide conjugation can be used to form a covalent bond
between a carboxyl group of one polypeptide and an hydroxyl group
of a second polypeptide (resulting in the formation of an ester
bond), or an amino group of a second polypeptide (resulting in the
formation of an amide bond) or a sulfhydryl group of a second
polypeptide (resulting in the formation of a thioester bond).
[0148] Likewise, carbodiimide coupling can be used to form
analogous covalent bonds between a carbon group of a first
polypeptide and an hydroxyl, amino or sulfhydryl group of a second
polypeptide. See, generally, J. March, Advanced Organic Chemistry:
Reaction's, Mechanism, and Structure, pp. 349-50 & 372-74 (3d
ed.), 1985. By means of illustration, and not limitation, the
hydrogen generating enzyme may be conjugated to the ferredoxin via
a covalent bond using a carbodiimide, such as
dicyclohexylcarbodiimide or EDC
(1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide Hydrochloride. See
generally, the methods of conjugation by B. Neises et al. (1978,
Angew Chem., Int. Ed. Engl. 17:522; A. Hassner et al. (1978,
Tetrahedron Lett. 4475); E. P. Boden et al. (1986, J. Org. Chem.
50:2394) and L. J. Mathias (1979, Synthesis 561).
[0149] Crosslinking of Cysteine Residues
[0150] The hydrogen generating enzyme and ferredoxin of the present
invention may be linked during a programmed cross linking by the
addition of cysteine residues within the sequence of one of the
proteins or both. Gentle reduction and oxidation, would then allow
the formation of a viable, disulfide linked hydrogenase-ferredoxin
complex.
[0151] As mentioned, the fusion polypeptide of the present
invention is artificially engineered such that the hydrogen
generating enzyme comprised within is in an optimal environment for
receiving electrons and therefore for generating hydrogen.
[0152] Thus, according to another aspect of the present invention,
there is provided a method of generating hydrogen. The method
comprises combining the fusion polypeptide of the present invention
(i.e. the polypeptide comprising the hydrogen generating enzyme and
ferredoxin) with an electron donor so as to generate an electron
transfer chain.
[0153] The phrase "electron donor" as used herein, refers to any
biological or non-biological component that is capable of donating
electrons. Thus, according to one embodiment of this aspect of the
present invention, the electron donor is an electrode. Thus the
present invention contemplates a system 10, illustrated herein in
FIG. 3 comprising an electrode 12 in contact with the polypeptide
of the present invention 14. The polypeptide 14 may be attached to
the electrode 12 for direct bioelectrocatalysis using any method
known in the art such as for example the modification of electrode
surfaces by redox mediators or hydrophilic adsorption. Exemplary
material that may be used for generating electrode 12 is carbon
covered with viologen substituted poly(pyrrole), pyrolytic carbon
paper (PCP) and packed graphite columns (PGC). In order to generate
hydrogen, the electrode 12 is attached to an electrical source
16.
[0154] According to another embodiment of this aspect of the
present invention the electron donor comprises a light-sensitive
biomolecule in combination with an electron source.
[0155] The term "biomolecule" as used herein refers to a molecule
that is or can be produced by a living system as well as structures
derived from such molecules. Biomolecules include, for example,
proteins, glycoproteins, carbohydrates, lipids, glycolipids, fatty
acids, steroids, purines, pyrimidines, and derivatives, analogs,
and/or combinations thereof.
[0156] According to a preferred embodiment of this aspect of the
present invention, the biomolecular electron donor comprises a
photocatalytic unit of a photosynthetic organism.
[0157] As used herein, the phrase "photocatalytic unit" refers to a
complex of at least one polypeptide and other small molecules (e.g.
chlorophyll and pigment molecules), which when integrated together
work as a functional unit converting light energy to chemical
energy. The photocatalytic units of the present invention are
present in photosynthetic organisms (i.e. organisms that convert
light energy into chemical energy). Examples of photosynthetic
organisms include, but are not limited to green plants,
cyanobacteria, red algae, purple and green bacteria.
[0158] Thus examples of photocatalytic units which can be used in
accordance with this aspect of the present invention include
biological photocatalytic units such as PS I and PS II, bacterial
light-sensitive proteins such as bacteriorhodopsin, photocatalytic
microorganisms, pigments (e.g., proflavine and rhodopsin), organic
dyes and algae. Preferably, the photocatalytic unit of the present
invention is photosystem I (PS I).
[0159] PSI is a protein-chlorophyll complex, present in green
plants and cyanobacteria, that is part of the photosynthetic
machinery within the thylakoid membrane. It is ellipsoidal in shape
and has dimensions of about 9 by 15 nanometers. The PS I complex
typically comprises chlorophyll molecules which serve as antennae
which absorb photons and transfer the photon energy to P700, where
this energy is captured and utilized to drive photochemical
reactions. In addition to the P700 and the antenna chlorophylls,
the PSI complex contains a number of electron acceptors. An
electron released from P700 is transferred to a terminal acceptor
at the reducing end of PSI through intermediate acceptors, and the
electron is transported across the thylakoid membrane.
[0160] Examples of PSI polypeptides are listed below in Table 5
together with their source organisms.
TABLE-US-00005 TABLE 5 Source Organism Protein accession number
Amphidinium carterae CAC34545 Juniperus chinensis CAC87929 Cedrus
libani CAC87143 Spathiphyllum sp. SM328 CAC87924 Persea americana
CAC87920 Zamia pumila CAC87935 Ophioglossum petiolatum CAC87936
Taxus brevifolia CAC87934 Afrocarpus gracilior CAC87933 Pinus
parviflora CAC87932 Picea spinulosa CAC87931 Phyllocladus
trichomanoides CAC87930 Serenoa repens CAC87923 Saururus cernuus
CAC87922 Platanus racemosa CAC87921 Pachysandra terminalis CAC87919
Nymphaea sp. cv. Paul Harriot CAC87918 Nuphar lutea CAC87917
Nelumbo nucifera CAC87916 Acer palmatum CAD23045 Cupressus
arizonica CAC87928 Cryptomeria japonica CAC87927 Abies alba]
CAC87926 Gnetum gnemon CAC87925 Magnolia grandiflora CAC87915
Liquidambar styraciflua CAC87914 Lilium brownii CAC87913 Isomeris
arborea CAC87912 Fagus grandifolia CAC87911 Eupomatia laurina
CAC87910 Enkianthus chinensis CAC87909 Coptis laciniata CAC87908
Chloranthus spicatus CAC87907 Calycanthus occidentalis CAC87906
Austrobaileya scandens] CAC87905 Amborella trichopoda CAC87904
Acorus calamus CAC87142
[0161] The photosystem I complex may be in the native cellular
membrane along with photosystem II and the rest of the
photosynthetic electron transport chain, or it can be provided in a
detergent-solubilized form. Methods for isolating native membranes
from photosynthetic organisms are known in the art and a preferred
method is provided in the publication of (Murata 1982, Plant Cell
Physiol 23: 533-9). Purified thylakoids may be quantitated and
expressed as a particular amount of chlorophyll. Methods for
quantitating chlorophyll are known for example as set forth by
(Arnon 1949, Plant Physiol 24(1): 1-15). Methods for obtaining
isolated photosystem I in a detergent solubilized form is also
known and an exemplary method is disclosed by (Evans, Sihra et al.
1977, Biochem J 162(1): 75-85).
[0162] According to one embodiment, the light sensitive biomolecule
is immobilized on a solid support. WIPO PCT Application
WO2006090381, incorporated herein by reference, teaches
immobilization of PS-I on a solid supports such as metal surfaces
by genetic manipulation thereof.
[0163] As mentioned hereinabove, in order for light sensitive
biomolecules to act as electron donors they typically act in
combination with an electron source. Exemplary electron sources
that may be used in combination with PSI include sodium dithionite
(e.g. at about 5 mM), dithiothreitol (e.g. at about 50 mM) and a
combination of dithiothreitol plus ascorbic acid (e.g. at about 2
mM ascorbic acid). According to one embodiment PSI is used in
combination with PSII, where the latter serves as an intermediate,
passing electrons from the electron source to PSI. In this case,
the electron source may be water. Other electron sources that may
be used according to the teachings of the present invention
include, but are not limited to
N,N,N',N'-tetramethyl-p-phenylendiamine (TMPD) and
2,6-dichlorophenol indophenol.
[0164] As mentioned, the ferredoxin-hydrogenase (or
ferredoxin-nitrogenase) fusion protein of the present invention is
preferably not inhibited by the presence of dissolved oxygen (or at
least, comprise a reduced sensitivity to oxygen). Such fusion
proteins may survive in the atmosphere for approximately .about.300
seconds (IC.sub.50) in comparison to 1 sec of native Chlimydomonas
renhardtii hydrogenase. However, if the ferredoxin-hydrogenase
fusion protein of the present invention is inhibited by dissolved
oxygen, it may be necessary to remove oxygen from the reaction
mixture. The removal of oxygen can be performed in a number of
ways. For example, if dithionite or high concentrations of
dithiothreitol (e.g., 50 mM dithiothreitol) are used as electron
donors, these will react with dissolved oxygen to remove it. If
water is used as an electron donor in cellular systems, oxygen will
be produced by photosystem II and 5 mM glucose plus the
over-expression/or the external addition of glucose oxidase (3
.mu.g/ml, Sigma, St. Louis, Mo.) can be included in the reaction
mix to rapidly remove oxygen as it is produced.
[0165] As mentioned herein above, the method of the present
invention envisages combining the fusion protein of the present
invention with an electron donor in such a way so as to promote
electron transfer from the donor to the fusion protein.
[0166] As used herein, the term "combining" refers to any method
where the fusion protein and the electron donor are in close enough
proximity that electron transfer from the latter to the former
occurs. Thus, the term "combining" incorporates such methods as
co-expressing and co-solubilizing the fusion protein and electron
donor of the present invention.
[0167] According to one embodiment, the fusion protein and electron
donor are combined in a cellular system. Thus, for example the
fusion protein may be expressed in a photosynthetic organism where
PSI and optionally PSII are endogenously expressed.
[0168] Preferably, the amount of fusion protein is adjusted for
maximal optimization of the system. Thus, according to an
embodiment of this aspect of the present invention, a ratio of
fusion protein:PSI is greater than 100:1, more preferably greater
than 500:1 and even more preferably greater than 1000:1.
Preferably, the cellular system is forced to respirate under
unaerobic conditions so as to avoid the generation of oxygen.
[0169] In order for PSI to transfer electrons to the ferredoxin
unit of the polypeptide of the present invention, preferably PSI is
energized using light energy. The illuminating may proceed
following or concomitant with the expression of the fusion protein
of the present invention. A bioreactor may be used which excludes
high energy wavelength such as UV radiation, and enables the
entrance of the visible red which exclusively feeds and ignite the
PSI. An exemplary bioreactor that may be used in accordance with
the teachings of the present invention is illustrated in FIG.
3.
[0170] FIG. 4 is a schematic illustration of a reactor 300 for
producing hydrogen according to an embodiment of the invention.
Reactor 300 comprises a vessel 321, in which the hydrogen producing
system comprising PSI, and the ferredoxin unit of the polypeptide
of the present invention, are held in a suspension 322. The
suspension 322 may also comprise other components such as sodium
citrated and TMPD. The suspension may include the hydrogen
producing systems, that is, the PSI and the ferredoxin unit, in any
of the above-mentioned ways, for instance, in liposomes or in cell
culture. Suspension 322 is constantly stirred with stirring blades
324 by rotor 325. The rotor and stirring blade are operated as not
to damage the cells or liposomes, but only homogenize them within
vessel 321.
[0171] A temperature control 326 controls the temperature to
optimize the activity of the cells or liposomes, for instance,
37.degree. C.
[0172] An optic fiber 323, provides light to the cells or
liposomes. Preferably, the light provided by fiber 323 is free of
damaging wavelengths, such as UV. Optionally, the light is also
free of non-activating wavelengths, for instance, green light.
Hydrogen produced by the hydrogen producing systems bubbles out of
the suspension, through a gas-liquid separation membrane 328. From
the gas side of membrane 328, the hydrogen is optionally pumped
with pump 329 to a hydrogen tank 330.
[0173] As mentioned herein above, the endogenous electron transport
system in all photosynthetic organisms comprises donation of
electrons from ferredoxin to ferredoxin-NADP.sup.+-reductase (FNR).
In order to divert the flow of electrons away from this competing
enzyme, the present invention contemplates down-regulation
thereof.
[0174] The phrase "ferredoxin-NADP.sup.+-reductase" as used herein
refers to the enzyme as set forth by EC 1.18.1.2. present in
photosynthetic organisms.
[0175] Downregulation of FNR may be effected on the genomic level
(using classical genetic approaches) and/or the transcript level.
This may be achieved using a variety of molecules which interfere
with transcription and/or translation (e.g., antisense, siRNA,
Ribozyme, DNAzyme).
[0176] Following is a list of agents capable of downregulating
expression level of FNR.
[0177] One agent capable of downregulating a FNR is a small
interfering RNA (siRNA) molecule. RNA interference is a two step
process. The first step, which is termed as the initiation step,
input dsRNA is digested into 21-23 nucleotide (nt) small
interfering RNAs (siRNA), probably by the action of Dicer, a member
of the RNase III family of dsRNA-specific ribonucleases, which
processes (cleaves) dsRNA (introduced directly or via a transgene
or a virus) in an ATP-dependent manner. Successive cleavage events
degrade the RNA to 19-21 bp duplexes (siRNA), each with
2-nucleotide 3' overhangs [Hutvagner and Zamore Curr. Opin.
Genetics and Development 12:225-232 (2002); and Bernstein Nature
409:363-366 (2001)].
[0178] In the effector step, the siRNA duplexes bind to a nuclease
complex to from the RNA-induced silencing complex (RISC). An
ATP-dependent unwinding of the siRNA duplex is required for
activation of the RISC. The active RISC then targets the homologous
transcript by base pairing interactions and cleaves the mRNA into
12 nucleotide fragments from the 3' terminus of the siRNA
[Hutvagner and Zamore Curr. Opin. Genetics and Development
12:225-232 (2002); Hammond et al. (2001) Nat. Rev. Gen. 2:110-119
(2001); and Sharp Genes. Dev. 15:485-90 (2001)]. Although the
mechanism of cleavage is still to be elucidated, research indicates
that each RISC contains a single siRNA and an RNase [Hutvagner and
Zamore Curr. Opin. Genetics and Development 12:225-232 (2002)].
[0179] Because of the remarkable potency of RNAi, an amplification
step within the RNAi pathway has been suggested. Amplification
could occur by copying of the input dsRNAs which would generate
more siRNAs, or by replication of the siRNAs formed. Alternatively
or additionally, amplification could be effected by multiple
turnover events of the RISC [Hammond et al. Nat. Rev. Gen.
2:110-119 (2001), Sharp Genes. Dev. 15:485-90 (2001); Hutvagner and
Zamore Curr. Opin. Genetics and Development 12:225-232 (2002)]. For
more information on RNAi see the following reviews Tuschl
ChemBiochem. 2:239-245 (2001); Cullen Nat. Immunol. 3:597-599
(2002); and Brantl Biochem. Biophys. Act. 1575:15-25 (2002).
[0180] Synthesis of RNAi molecules suitable for use with the
present invention can be effected as follows. First, the FNR mRNA
sequence is scanned downstream of the AUG start codon for AA
dinucleotide sequences. Occurrence of each AA and the 3' adjacent
19 nucleotides is recorded as potential siRNA target sites.
Preferably, siRNA target sites are selected from the open reading
frame, as untranslated regions (UTRs) are richer in regulatory
protein binding sites. UTR-binding proteins and/or translation
initiation complexes may interfere with binding of the siRNA
endonuclease complex [Tuschl ChemBiochem. 2:239-245]. It will be
appreciated though, that siRNAs directed at untranslated regions
may also be effective, as demonstrated for GAPDH wherein siRNA
directed at the 5' UTR mediated about 90% decrease in cellular
GAPDH mRNA and completely abolished protein level
(www.ambion.com/techlib/tn/91/912.html).
[0181] Second, potential target sites are compared to an
appropriate genomic database (e.g., plant or bacteria etc.) using
any sequence alignment software, such as the BLAST software
available from the NCBI server (www.ncbi.nlm.nih.gov/BLAST/).
Putative target sites which exhibit significant homology to other
coding sequences are filtered out.
[0182] Qualifying target sequences are selected as template for
siRNA synthesis. Preferred sequences are those including low G/C
content as these have proven to be more effective in mediating gene
silencing as compared to those with G/C content higher than 55%.
Several target sites are preferably selected along the length of
the target gene for evaluation. For better evaluation of the
selected siRNAs, a negative control is preferably used in
conjunction. Negative control siRNA preferably include the same
nucleotide composition as the siRNAs but lack significant homology
to the genome. Thus, a scrambled nucleotide sequence of the siRNA
is preferably used, provided it does not display any significant
homology to any other gene.
[0183] RNAi has been successfully used in plants for
down-regulation of proteins--see for example Moritoh et al., Plant
and Cell Physiology 2005 46(5):699-715.
[0184] Another agent capable of downregulating FNR is a DNAzyme
molecule capable of specifically cleaving an mRNA transcript or DNA
sequence of the FNR. DNAzymes are single-stranded polynucleotides
which are capable of cleaving both single and double stranded
target sequences (Breaker, R. R. and Joyce, G. Chemistry and
Biology 1995; 2:655; Santoro, S. W. & Joyce, G. F. Proc. Natl,
Acad. Sci. USA 1997; 943:4262) A general model (the "10-23" model)
for the DNAzyme has been proposed. "10-23" DNAzymes have a
catalytic domain of 15 deoxyribonucleotides, flanked by two
substrate-recognition domains of seven to nine deoxyribonucleotides
each. This type of DNAzyme can effectively cleave its substrate RNA
at purine:pyrimidine junctions (Santoro, S. W. & Joyce, G. F.
Proc. Natl, Acad. Sci. USA 199; for rev of DNAzymes see Khachigian,
L M [Curr Opin Mol Ther 4:119-21 (2002)].
[0185] Examples of construction and amplification of synthetic,
engineered DNAzymes recognizing single and double-stranded target
cleavage sites have been disclosed in U.S. Pat. No. 6,326,174 to
Joyce et al.
[0186] Downregulation of a FNR can also be effected by using an
antisense polynucleotide capable of specifically hybridizing with
an mRNA transcript encoding the FNR. Design of antisense molecules
which can be used to efficiently downregulate a FNR must be
effected while considering two aspects important to the antisense
approach. The first aspect is delivery of the oligonucleotide into
the cytoplasm of the appropriate cells, while the second aspect is
design of an oligonucleotide which specifically binds the
designated mRNA within cells in a way which inhibits translation
thereof.
[0187] Algorithms for identifying those sequences with the highest
predicted binding affinity for their target mRNA based on a
thermodynamic cycle that accounts for the energetics of structural
alterations in both the target mRNA and the oligonucleotide are
also available [see, for example, Walton et al. Biotechnol Bioeng
65: 1-9 (1999)].
[0188] In addition, several approaches for designing and predicting
efficiency of specific oligonucleotides using an in vitro system
were also published (Matveeva et al., Nature Biotechnology 16:
1374-1375 (1998)].
[0189] For more information pertaining to the inhibition of gene
expression in plant cells by expression of antisense RNA, see for
example Joseph R. Ecker and Ronald W. Davis, PNAS, 1986, vol. 83,
no. 15, 5372-5376.
[0190] Thus, the current consensus is that recent developments in
the field of antisense technology which, as described above, have
led to the generation of highly accurate antisense design
algorithms and a wide variety of oligonucleotide delivery systems,
enable an ordinarily skilled artisan to design and implement
antisense approaches suitable for downregulating expression of
known sequences without having to resort to undue trial and error
experimentation.
[0191] Another agent capable of downregulating a FNR is a ribozyme
molecule capable of specifically cleaving an mRNA transcript
encoding FNR. Ribozymes are being increasingly used for the
sequence-specific inhibition of gene expression by the cleavage of
mRNAs encoding proteins of interest [Welch et al., Curr Opin
Biotechnol. 9:486-96 (1998)]. The possibility of designing
ribozymes to cleave any specific target RNA has rendered them
valuable tools in both basic research and therapeutic
applications.
[0192] Yet another agent capable of downregulating FNR would be any
molecule which binds to and/or cleaves FNR. Such molecules can be
FNR antagonists, or FNR inhibitory peptide.
[0193] It will be appreciated that a non-functional analogue of at
least a catalytic or binding portion of FNR can be also used as an
agent which downregulates FNR.
[0194] Another agent which can be used along with the present
invention to downregulate FNR is a molecule which prevents FNR
activation or substrate binding.
[0195] As mentioned, the fusion protein and electron donor may also
be combined in a non-cellular system.
[0196] In one embodiment the components of the present invention
are suspended in a buffered aqueous solution at a pH at which both
the photosynthetic components (e.g. PSI) and ferredoxin-hydrogenase
fusion protein are active (for example, in a solution of about 2 mM
to about 500 mM Tris-HCl, pH 8.0, preferably 30 mM Tris-HCl to 100
mM Tris-HCl, and preferably about 40 mM Tris-HCl), at a temperature
at which both the fusion protein of the present invention and the
photosynthetic components are active (generally about 10.degree. C.
to about 40.degree. C.), and with an appropriate electron
donor.
[0197] In one embodiment, the fusion protein of the present
invention and the electron donor are encapsulated in a carrier
system (i.e., encapsulating agent) of desired properties. In a
specific embodiment, the encapsulating agent is a liposome.
[0198] As used herein and as recognized in the art, the term
"liposome" refers to a synthetic (i.e., not naturally occurring)
structure composed of lipid bilayers, which enclose a volume.
Exemplary liposomes include, but are not limited to emulsions,
foams, micelles, insoluble monolayers, liquid crystals,
phospholipid dispersions, lamellar layers and the like. The
liposomes may be prepared by any of the known methods in the art
[Monkkonen, J. et al., 1994, J. Drug Target, 2:299-308; Monkkonen,
J. et al., 1993, Calcif. Tissue Int., 53:139-145; Lasic D D.,
Liposomes Technology Inc., Elsevier, 1993, 63-105. (chapter 3);
Winterhalter M, Lasic D D, Chem Phys Lipids, 1993 September;
64(1-3):35-43]. The liposomes may be positively charged, neutral,
or, negatively charged.
[0199] The liposomes may be a single lipid layer or may be
multilamellar. In the case of PS-I as the electron donor,
multilamellar vesicles may be advantageous. Alternatively, it may
advantageous to increase the surface area of the liposome and
adsorb the PS-I on the surface thereof. An exemplary liposomal
system for the polypeptides of the present invention includes PS-I
constructed within the lipid bilayer, and the fusion protein of the
present invention constructed in the enclosed volume as illustrated
in FIGS. 2A-B. Surfactant peptide micelles are also
contemplated.
[0200] In another embodiment, the PSI and fusion protein of the
present invention are embedded in a carrier (i.e., embedding agent)
of desired properties. In specific embodiments, the embedding agent
(or carrier) is a microparticle, nanoparticle, nanosphere,
microsphere, nano-plate, microcapsule, or nanocapsule [M. Donbrow
in: Microencapsulation and Nanoparticles in Medicine and Pharmacy,
CRC Press, Boca Raton, Fla., 347, 1991]. The term carrier includes
both polymeric and non-polymeric preparations. According to a
specific embodiment, the embedding agent is a nanoparticle. The
polypeptides of the present invention may be embedded in the
nanoparticle, dispersed uniformly or non-uniformly in the polymer
matrix, adsorbed on the surface, or in combination of any of these
forms. Polymers which may be used for fabricating the nanoparticles
include, but are not limited to, PLA (polylactic acid), and their
copolymers, polyanhydrides, polyalkyl-cyanoacrylates (such as
polyisobutylcyanoacrylate), polyethyleneglycols, polyethyleneoxides
and their derivatives, chitosan, albumin, gelatin and the like.
[0201] It will be appreciated that the fusion protein of the
present invention and the electron donor need not be encapsulated.
Thus, according to yet another embodiment, the fusion protein and
the electron donor of the present invention are free in
solution.
[0202] Hydrogen gas can be harvested from the system of the present
invention by direct or indirect biophotolysis:
[0203] Direct biophotolysis has been demonstrated under conditions
where the resulting oxygen and hydrogen are flushed from the system
using inert gas [Greenbaum 1988, Biophysical Journal 54:
365-368].
[0204] Indirect biophotolysis intends to circumvent the oxygen
sensitivity of the hydrogenases by temporally separating the
hydrogen-producing reactions from the oxygen evolving ones.
According to one embodiment plant cells (e.g. algae) may be grown
in open ponds to evolve oxygen and store carbohydrates. The plant
cells may then be harvested, and placed in an anaerobic reactor.
(For whole plants an anaerobic or a semi-anaerobic environment
would be created in green-houses by flushing nitrogen inside at
night only). Induction of expression of the fusion protein of the
present invention may then ensue concomitant with the inactivation
of Photosystem II. Illumination then oxidizes the stored
carbohydrate, lipid, and produces hydrogen, either directly or
after an anaerobic dark fermentation [Hallenbeck P C 2002,
International Journal of Hydrogen Energy 27: 1185-1193]. Since
ideally only hydrogen and carbon dioxide are produced in the
photobioreactor, gas handling is simpler and less hazardous.
[0205] As used herein the term "about" refers to .+-.10%.
[0206] Additional objects, advantages, and novel features of the
present invention will become apparent to one ordinarily skilled in
the art upon examination of the following examples, which are not
intended to be limiting. Additionally, each of the various
embodiments and aspects of the present invention as delineated
hereinabove and as claimed in the claims section below finds
experimental support in the following examples.
[0207] It is appreciated that certain features of the invention,
which are, for clarity, described in the context of separate
embodiments, may also be provided in combination in a single
embodiment. Conversely, various features of the invention, which
are, for brevity, described in the context of a single embodiment,
may also be provided separately or in any suitable subcombination
or as suitable in any other described embodiment of the invention.
Certain features described in the context of various embodiments
are not to be considered essential features of those embodiments,
unless the embodiment is inoperative without those elements.
[0208] Various embodiments and aspects of the present invention as
delineated hereinabove and as claimed in the claims section below
find experimental support in the following examples.
EXAMPLES
[0209] Reference is now made to the following examples, which
together with the above descriptions illustrate some embodiments of
the invention in a non limiting fashion.
[0210] Reference is now made to the following examples, which
together with the above descriptions, illustrate the invention in a
non limiting fashion.
[0211] Generally, the nomenclature used herein and the laboratory
procedures utilized in the present invention include molecular,
biochemical, microbiological and recombinant DNA techniques. Such
techniques are thoroughly explained in the literature. See, for
example, "Molecular Cloning: A laboratory Manual" Sambrook et al.,
(1989); "Current Protocols in Molecular Biology" Volumes I-Ill
Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in
Molecular Biology", John Wiley and Sons, Baltimore, Md. (1989);
Perbal, "A Practical Guide to Molecular Cloning", John Wiley &
Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific
American Books, New York; Birren et al. (eds) "Genome Analysis: A
Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory
Press, New York (1998); methodologies as set forth in U.S. Pat.
Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057;
"Cell Biology: A Laboratory Handbook", Volumes I-Ill Cellis, J. E.,
ed. (1994); "Culture of Animal Cells--A Manual of Basic Technique"
by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current
Protocols in Immunology" Volumes I-Ill Coligan J. E., ed. (1994);
Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition),
Appleton & Lange, Norwalk, Conn. (1994); Mishell and Shiigi
(eds), "Selected Methods in Cellular Immunology", W. H. Freeman and
Co., New York (1980); available immunoassays are extensively
described in the patent and scientific literature, see, for
example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;
3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;
3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and
5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984);
"Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds.
(1985); "Transcription and Translation" Hames, B. D., and Higgins
S. J., eds. (1984); "Animal Cell Culture" Freshney, R. I., ed.
(1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A
Practical Guide to Molecular Cloning" Perbal, B., (1984) and
"Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols:
A Guide To Methods And Applications", Academic Press, San Diego,
Calif. (1990); Marshak et al., "Strategies for Protein Purification
and Characterization--A Laboratory Course Manual" CSHL Press
(1996); all of which are incorporated by reference as if fully set
forth herein. Other general references are provided throughout this
document. The procedures therein are believed to be well known in
the art and are provided for the convenience of the reader. All the
information contained therein is incorporated herein by
reference.
Example 1
[0212] Cloning, Expression, Purification and Analysis of
Hydrogenase-Ferredoxin Recombinant Fusion Proteins
[0213] Materials and Methods
[0214] Cloning
[0215] A clone of ferredoxin (petF) was amplified from
Synechocystis pcc 6803 genome using the following primers:
TABLE-US-00006 Forward: (SEQ ID NO: 7) ATCTATGGCATCCTATACCG; and
Reverse to plant: (SEQ ID NO: 8)
TTATGCGGTGAGCTCTTCTTCTTTGTGGGTTTCAATG
[0216] In addition the C-terminus of the native cyanobacterial
ferredoxin was replaced with the higher plant ferredoxin C-terminus
using the "reverse to plant primer--SEQ ID NO: 8".
[0217] A HydA1 gene of Chlamydomonas reinhardtii was expressed
essentially as described in (King et al., 2006, J. Bacterilogy:
2163-2172).
[0218] The ferredoxin petF was fused to the C-terminus of the
target hydrogenase HydA1 according to the free space available in
the PSI ferredoxin binding site of plant PSI utilizing the
published structures of PSI, ferredoxin and hydrogenase [Peters et
al., 1998, Science, 282, 4 December; Nicolet et al., 1999,
Structure 7, 13-23; Asada et al., 2000, Biochim Biophys Acta 1490,
269-78; Ben-Shem et al., 2003, Nature 426, 630-5; Amunts et al.,
2007, Nature 447, 58-63].
[0219] Different linkers as well as several deletions were prepared
using the following primers:
TABLE-US-00007 Primary For (Hyd For'): (SEQ ID NO: 9) gatata
CATATGGGCTGG Primary Rev' (Fd Rev'): (SEQ ID NO: 10) accaga
CTCGAGttatgcggtgagctcttc
[0220] 1) Hyd A1 Cr Fd no linker for construction of SEQ ID NO: 1,
a direct fusion of HydA1 C-terminus to petF N-terminus.
TABLE-US-00008 Hyd-Fd Rev: (SEQ ID NO: 11)
GGTATAGGATGCCATTTTTTTTTCATCTTTTTCTTCCAC. Hyd-Fd For: (SEQ ID NO:
12) gtggaagaaaaagatgaaaaaaaaATGGCATCCTATACCG.
[0221] 2) Hyd A1 Cr Fd short linker for construction of SEQ ID NO:
2, a short linker of four glycine a single serine used to create a
fusion of HydA1 C-terminus to petF N-terminus.
TABLE-US-00009 Hyd-Fd Rev: (SEQ ID NO: 13)
ggatccgccgccaccTTTTTTTTCATCTTTTTCTTCCAC. Hyd-Fd For: (SEQ ID NO:
14) ggtggcggcggatccATGGCATCCTATACCG.
[0222] 3) Hyd A1 Cr Fd medium linker for construction of SEQ ID NO:
3, a medium linker of two repeat of the short linker (composited
from four glycine and a single serine) used to create a fusion of
HydA1 C-terminus to petF N-terminus.
TABLE-US-00010 Hyd-Fd Rev: (SEQ ID NO: 15)
ggagccgccgccgccggatcctcctcctccTTTTTTTTCATCTTTTT CTTCCAC. Hyd-Fd
For: (SEQ ID NO: 16)
ggaggaggaggatccggcggcggcggctccATGGCATCCTATACCG.
[0223] 4) Hyd A1 Cr C truncated Fd N truncated no linker for
construction of SEQ ID NO: 4, a direct fusion of truncated (11aa
were deleted at C-terminus) HydA1 C-terminus to truncated (30aa
were deleted at N-terminus) petF N-terminus.
TABLE-US-00011 Hyd-Fd Rev: (SEQ ID NO: 17)
gaggatataggtatcgtccacataatgggtatgcag. Hyd-Fd For: (SEQ ID NO: 18)
ctgcatacccattatgtggacgatacctatatcctc.
[0224] 5) Hyd A1 Cr C truncated Fd no linker for construction of
SEQ ID NO: 5, a direct fusion of truncated (11aa were deleted at
C-terminus) HydA1 C-terminus to petF N-terminus.
TABLE-US-00012 Hyd-Fd Rev: (SEQ ID NO: 19)
cggtataggatgccatcacataatgggtatgcag. Hyd-Fd For: (SEQ ID NO: 20)
ctgcatacccattatgtgatggcatcctataccg.
[0225] 6) Hyd A1 Cr Fd N truncated no linker for construction of
SEQ ID NO: 6, a direct fusion of HydA1 C-terminus to truncated
(30aa were deleted at N-terminus) petF N-terminus.
TABLE-US-00013 Hyd-Fd Rev: (SEQ ID NO: 21)
gaggatataggtatcgtcttttttttcatctttttcTTCCAC. Hyd-Fd For: (SEQ ID NO:
22) gtggaagaaaaagatgaaaaaaaagacgatacctatatcctc.
[0226] HydA1 and petF genes were assembled using two PCR steps. In
the first PCR, each gene HydA1 and petF were amplified separately
with the appropriate primers set, which include overlapping region
with the product of each other (at sense primers of petF and at
antisense primers of HydA1). In the next step the two products of
these PCRs were mixed at 1:1 proportion and amplified in a second
PCR reaction using only the edges primer set e.g sense primer of
hydA1 and antisense primer of petF. The assembly gene fusion
product was introduced into the pETDuet system and transformed
together with the HydF+G in pCDFduet as described essentially in
(King et al., 2006, J. Bacterilogy: 2163-2172).
[0227] Protein Expression
[0228] Ferredoxin-hydrogenase fusion proteins were expressed in E.
coli. (BL-21 DE3 Rosetta). Essentially, an overnight starter of 5
ml TB+ampicillin (Amp) (100 .quadrature.g/ml) 15 .mu.l+5
streptomycin (Sm) (50 .quadrature.g/ml) was washed twice with fresh
media (to remove traces of .beta.-lactamases). 2 ml of the washed
starter was diluted into 100 ml TB and 30 mg (300 .mu.l of 100
mg/ml stock solution) Amp and 5 mg Sm (100 .mu.l of 50 mg/ml stock
solution) was added. The bacteria were grown in 250 ml flask for
.about.2.5 hr at 37.degree. C.; 250 RPM to reach
O.D.sub.600=0.5-0.7. Next, Fe citrate 10 mg/100 ml was added and
bacteria were grown for an additional 10 minutes at 37.degree. C.;
250 RPM. Induction was initiated by the addition of IPTG 37.5
mg/100 ml. The bacteria were then aerobically grown for 1 hour at
30.degree. C.; 250 RPM, and then transferred to a 100 ml bottle.
Argon gas was added directly into the growing media to create
bubbles for an additional 6 hours at 30.degree. C. to maintain
anaerobic conditions.
[0229] Activity Assay
[0230] The activity assay was carried as described (King et al.,
2006, J. Bacteriol: 2163-2172) as follows: Activities of
hydrogenases alone was measured as the evolution of H.sub.2 gas
from reduced methyl viologen (MV). Activity assays of whole cells
extracts were performed with argon-flushed vials that contained an
anaerobically prepared whole-cell extract comprising reaction
buffer (50 mM potassium phosphate, pH 7; 10 mM MV; 20 mM sodium
dithionite; 6 mM NaOH; 0.2% Triton X-100) and 1 ml of anaerobically
grown and induced cells.
[0231] The fusion protein was analyzed for the ability to produce
hydrogen directly from dithionite as an electron donor using their
fused ferredoxin part as the electron mediator replacing the
function of the chemical mediator methyl viologen (native
hydrogenase can not accept electrons directly from dithionite).
[0232] Gas Chromatography
[0233] Hydrogen production was measured by gas chromatography using
Varian 3600 machine equipped with thermal conductivity detector
(TCD) and hydrogen column. Nitrogen was used as a carrier gas.
[0234] 100 .mu.l of gas sample was injected and the area of the
specific Hydrogen peak, eluted .about.1.25 minutes post injection
was calculated using the area of 5.15% hydrogen standard as
reference.
[0235] Results
[0236] The genes of petF and HydA1 was successfully amplified using
the primers and assembled together to form the chimera genes as can
be seen in FIG. 5.
[0237] Next these Hyd-Fd chimeras were cloned into pETDuet plasmids
that contained the HydE structural gene as previously described
(King et al., 2006, J. Bacteriol: 2163-2172) which is crucial for
recombinant expression of hydrogenases in E. coli. FIGS. 6A-B show
the two expression plasmids. FIG. 6A is a map of the pETDuet
plasmid which contains the Hyd-Fd chimeric genes between the
restriction sites NdeI and XhoI of Multiple cloning site II. In
addition, the plasmid contains the HydE structural gene. FIG. 6B is
a map of the plasmid CDFduet that contains the coding sequences for
the two other structural proteins HydF and HydG, both of which are
crucial for the recombinant expression of hydrogenase protein in E.
coli.
[0238] Following preparation of DNA constructs, the protein
expression pattern was tested by Western blot analysis using
monoclonal antibodies against the StrepTag II (IBA, Gottingen,
Germany) (which in our plasmids is located at the N-terminus of the
HydA1 protein or the Hyd-Fd chimera). All beside 6HydFd--SEQ ID NO:
6 (direct linkage of HydA1 and N-terminus truncated petF) were well
expressed (FIGS. 7A-B).
[0239] The hydrogen generated by the expressed chimeras was tested
sequentially. First, the hydrogenase component of each chimera was
tested separately using a well-known procedure by which dithionite
donates electrons to the chemical electron mediator methyl viologen
which feeds electrons directly to hydrogenase as described by (King
et al., 2006, J. Bacteriol: 2163-2172). Feredoxin electron
mediating ability was tested by elimination of the chemical
mediator methyl viologen. Under such conditions, hydrogen
production by native hydrogenase is negligible while chimera
protein produced at least 50% of full activity with the chemical
electron mediator methyl viologen. FIG. 8 illustrates hydrogen
generation by three selected chimera.
[0240] All the tested chimeras generated at least 4 fold more
hydrogen than the native hydrogenase.
Sequence CWU 1
1
2811677DNAArtificial sequencepETDuet Hyd E + A Cr Fd with no linker
1atgggctgga gccatccgca gtttgaaaaa agatctgaaa acctgtattt tcagggcgct
60gctcctgctg ctgaagcgcc gctgagccat gtgcagcagg cgctggcgga actggcgaaa
120ccgaaagatg atccgacccg taagcatgtg tgcgtgcagg tggcgccggc
ggtgcgtgtg 180gcgatcgcgg aaaccctggg cctggcgccg ggcgcgacca
ccccgaaaca gctggcggaa 240ggcctgcgtc gtctgggctt tgatgaagtg
ttcgataccc tgtttggcgc ggatctgacc 300atcatggaag aaggcagcga
actgctgcat cgtctgaccg aacatctgga agcgcatccg 360catagcgatg
aaccgctgcc gatgtttacc agctgctgcc cgggctggat tgcgatgctg
420gaaaaaagct atccggatct gattccgtat gtgagcagct gcaaaagccc
gcagatgatg 480ctggcggcga tggtgaaaag ctatctggcg gaaaaaaaag
gcattgcgcc gaaagatatg 540gtgatggtga gcattatgcc gtgcacccgt
aaacagagcg aagcggatcg tgattggttc 600tgcgtggatg cggatccgac
cctgcgtcag ctggatcatg tgattaccac cgtggaactg 660ggcaacattt
ttaaagaacg tggcattaac ctggcggaac tgccggaagg cgaatgggat
720aacccgatgg gcgtgggcag cggcgcgggc gtgctgttcg gcaccaccgg
cggcgtgatg 780gaagcggcgc tgcgtaccgc gtatgaactg tttaccggca
ccccgctgcc gcgtctgagc 840ctgagcgagg tgcgtggcat ggatggcatt
aaagagacca acattaccat ggtgccggcg 900ccgggcagca aatttgaaga
actgctgaaa catcgtgcgg cggcgcgtgc ggaagcggcg 960gcgcatggca
ccccgggccc gctggcgtgg gatggcggcg cgggctttac cagcgaagat
1020ggccgtggcg gcattaccct gcgtgtggcg gtggcgaacg gcctgggcaa
cgcgaaaaaa 1080ctgattacca aaatgcaggc gggcgaagcg aaatatgatt
ttgtggaaat tatggcgtgc 1140ccggcgggct gcgtgggcgg cggcggccag
ccgcgtagca ccgataaagc gatcacccag 1200aaacgtcagg cggcgctgta
taacctggat gagaagagca ccctgcgtcg tagccatgag 1260aacccgagca
ttcgtgaact gtatgatacc tatctgggcg aaccgctggg ccataaagcg
1320catgaactgc tgcataccca ttatgtggcg ggcggcgtgg aagaaaaaga
tgaaaaaaaa 1380atggcatcct ataccgttaa attgatcacc cccgatggtg
aaagttccat cgaatgctct 1440gacgatacct atatcctcga tgctgcggaa
gaagctggcc tagacctgcc ctattcctgc 1500cgtgctgggg cttgctccac
ctgtgccggt aagatcaccg ctggtagtgt tgaccaatcc 1560gatcagtctt
tcttggatga tgaccaaatt gaagctggtt atgttttgac ctgtgtagct
1620tatcccacct ccgattgcac cattgaaacc cacaaagaag aagagctcac cgcataa
167721691DNAArtificial sequencepETDuet Hyd E + A Cr Fd with a short
linker 2atgggctgga gccatccgca gtttgaaaaa agatctgaaa acctgtattt
tcagggcgct 60gctcctgctg ctgaagcgcc gctgagccat gtgcagcagg cgctggcgga
actggcgaaa 120ccgaaagatg atccgacccg taagcatgtt gcgtgcaggt
ggcgccggcg gtgcgtgtgg 180cgatcgcgga aaccctgggc ctggcgccgg
gcgcgaccac cccgaaacag ctggcggaag 240gcctgcgtcg tctgggcttt
gatgaagtgt tcgataccct gtttggcgcg gatctgacca 300tcatggaaga
aggcagcgaa ctgctgcatc gtctgaccga acatctggaa gcgcatccgc
360atagcgatga accgctgccg atgtttacca gctgctgccc gggctggatt
gcgatgctgg 420aaaaaagcta tccggatctg attccgtatg tgagcagctg
caaaagcccg cagatgatgc 480tggcggcgat ggtgaaaagc tatctggcgg
aaaaaaaagg cattgcgccg aaagatatgg 540tgatggtgag cattatgccg
tgcacccgta aacagagcga agcggatcgt gattggttct 600gcgtggatgc
ggatccgacc ctgcgtcagc tggatcatgt gattaccacc gtggaactgg
660gcaacatttt taaagaacgt ggcattaacc tggcggaact gccggaaggc
gaatgggata 720acccgatggg cgtgggcagc ggcgcgggcg tgctgttcgg
caccaccggc ggcgtgatgg 780aagcggcgct gcgtaccgcg tatgaactgt
ttaccggcac cccgctgccg cgtctgagcc 840tgagcgaggt gcgtggcatg
gatggcatta aagagaccaa cattaccatg gtgccggcgc 900cgggcagcaa
atttgaagaa ctgctgaaac atcgtgcggc ggcgcgtgcg gaagcggcgg
960cgcatggcac cccgggcccg ctggcgtggg atggcggcgc gggctttacc
agcgaagatg 1020gccgtggcgg cattaccctg cgtgtggcgg tggcgaacgg
cctgggcaac gcgaaaaaac 1080tgattaccaa aatgcaggcg ggcgaagcga
aatatgattt tgtggaaatt atggcgtgcc 1140cggcgggctg cgtgggcggc
ggcggccagc cgcgtagcac cgataaagcg atcacccaga 1200aacgtcaggc
ggcgctgtat aacctggatg agaagagcac cctgcgtcgt agccatgaga
1260acccgagcat tcgtgaactg tatgatacct atctgggcga accgctgggc
cataaagcgc 1320atgaactgct gcatacccat tatgtggcgg gcggcgtgga
agaaaaagat gaaaaaaaag 1380gtggcggcgg atccatggca tcctataccg
ttaaattgat cacccccgat ggtgaaagtt 1440ccatcgaatg ctctgacgat
acctatatcc tcgatgctgc ggaagaagct ggcctagacc 1500tgccctattc
ctgccgtgct ggggcttgct ccacctgtgc cggtaagatc accgctggta
1560gtgttgacca atccgatcag tctttcttgg atgatgacca aattgaagct
ggttatgttt 1620tgacctgtgt agcttatccc acctccgatt gcaccattga
aacccacaaa gaagaagagc 1680tcaccgcata a 169131707DNAArtificial
sequencepETDuet Hyd E + A Cr Fd with a medium linker 3atgggctgga
gccatccgca gtttgaaaaa agatctgaaa acctgtattt tcagggcgct 60gctcctgctg
ctgaagcgcc gctgagccat gtgcagcagg cgctggcgga actggcgaaa
120ccgaaagatg atccgacccg taagcatgtg tgcgtgcagg tggcgccggc
ggtgcgtgtg 180gcgatcgcgg aaaccctggg cctggcgccg ggcgcgacca
ccccgaaaca gctggcggaa 240ggcctgcgtc gtctgggctt tgatgaagtg
ttcgataccc tgtttggcgc ggatctgacc 300atcatggaag aaggcagcga
actgctgcat cgtctgaccg aacatctgga agcgcatccg 360catagcgatg
aaccgctgcc gatgtttacc agctgctgcc cgggctggat tgcgatgctg
420gaaaaaagct atccggatct gattccgtat gtgagcagct gcaaaagccc
gcagatgatg 480ctggcggcga tggtgaaaag ctatctggcg gaaaaaaaag
gcattgcgcc gaaagatatg 540gtgatggtga gcattatgcc gtgcacccgt
aaacagagcg aagcggatcg tgattggttc 600tgcgtggatg cggatccgac
cctgcgtcag ctggatcatg tgattaccac cgtggaactg 660ggcaacattt
ttaaagaacg tggcattaac ctggcggaac tgccggaagg cgaatgggat
720aacccgatgg gcgtgggcag cggcgcgggc gtgctgttcg gcaccaccgg
cggcgtgatg 780gaagcggcgc tgcgtaccgc gtatgaactg tttaccggca
ccccgctgcc gcgtctgagc 840ctgagcgagg tgcgtggcat ggatggcatt
aaagagacca acattaccat ggtgccggcg 900ccgggcagca aatttgaaga
actgctgaaa catcgtgcgg cggcgcgtgc ggaagcggcg 960gcgcatggca
ccccgggccc gctggcgtgg gatggcggcg cgggctttac cagcgaagat
1020ggccgtggcg gcattaccct gcgtgtggcg gtggcgaacg gcctgggcaa
cgcgaaaaaa 1080ctgattacca aaatgcaggc gggcgaagcg aaatatgatt
ttgtggaaat tatggcgtgc 1140ccggcgggct gcgtgggcgg cggcggccag
ccgcgtagca ccgataaagc gatcacccag 1200aaacgtcagg cggcgctgta
taacctggat gagaagagca ccctgcgtcg tagccatgag 1260aacccgagca
ttcgtgaact gtatgatacc tatctgggcg aaccgctggg ccataaagcg
1320catgaactgc tgcataccca ttatgtggcg ggcggcgtgg aagaaaaaga
tgaaaaaaaa 1380ggaggaggag gatccggcgg cggcggctcc atggcatcct
ataccgttaa attgatcacc 1440cccgatggtg aaagttccat cgaatgctct
gacgatacct atatcctcga tgctgcggaa 1500gaagctggcc tagacctgcc
ctattcctgc cgtgctgggg cttgctccac ctgtgccggt 1560aagatcaccg
ctggtagtgt tgaccaatcc gatcagtctt tcttggatga tgaccaaatt
1620gaagctggtt atgttttgac ctgtgtagct tatcccacct ccgattgcac
cattgaaacc 1680cacaaagaag aagagctcac cgcataa 170741584DNAArtificial
sequencepETDuet Hyd E + A Cr C' truncated Fd N' truncated and with
no linker 4atgggctgga gccatccgca gtttgaaaaa agatctgaaa acctgtattt
tcagggcgct 60gctcctgctg ctgaagcgcc gctgagccat gtgcagcagg cgctggcgga
actggcgaaa 120ccgaaagatg atccgacccg taagcatgtg tgcgtgcagg
tggcgccggc ggtgcgtgtg 180gcgatcgcgg aaaccctggg cctggcgccg
ggcgcgacca ccccgaaaca gctggcggaa 240ggcctgcgtc gtctgggctt
tgatgaagtg ttcgataccc tgtttggcgc ggatctgacc 300atcatggaag
aaggcagcga actgctgcat cgtctgaccg aacatctgga agcgcatccg
360catagcgatg aaccgctgcc gatgtttacc agctgctgcc cgggctggat
tgcgatgctg 420gaaaaaagct atccggatct gattccgtat gtgagcagct
gcaaaagccc gcagatgatg 480ctggcggcga tggtgaaaag ctatctggcg
gaaaaaaaag gcattgcgcc gaaagatatg 540gtgatggtga gcattatgcc
gtgcacccgt aaacagagcg aagcggatcg tgattggttc 600tgcgtggatg
cggatccgac cctgcgtcag ctggatcatg tgattaccac cgtggaactg
660ggcaacattt ttaaagaacg tggcattaac ctggcggaac tgccggaagg
cgaatgggat 720aacccgatgg gcgtgggcag cggcgcgggc gtgctgttcg
gcaccaccgg cggcgtgatg 780gaagcggcgc tgcgtaccgc gtatgaactg
tttaccggca ccccgctgcc gcgtctgagc 840ctgagcgagg tgcgtggcat
ggatggcatt aaagagacca acattaccat ggtgccggcg 900ccgggcagca
aatttgaaga actgctgaaa catcgtgcgg cggcgcgtgc ggaagcggcg
960gcgcatggca ccccgggccc gctggcgtgg gatggcggcg cgggctttac
cagcgaagat 1020ggccgtggcg gcattaccct gcgtgtggcg gtggcgaacg
gcctgggcaa cgcgaaaaaa 1080ctgattacca aaatgcaggc gggcgaagcg
aaatatgatt ttgtggaaat tatggcgtgc 1140ccggcgggct gcgtgggcgg
cggcggccag ccgcgtagca ccgataaagc gatcacccag 1200aaacgtcagg
cggcgctgta taacctggat gagaagagca ccctgcgtcg tagccatgag
1260aacccgagca ttcgtgaact gtatgatacc tatctgggcg aaccgctggg
ccataaagcg 1320catgaactgc tgcataccca ttatgtggac gatacctata
tcctcgatgc tgcggaagaa 1380gctggcctag acctgcccta ttcctgccgt
gctggggctt gctccacctg tgccggtaag 1440atcaccgctg gtagtgttga
ccaatccgat cagtctttct tggatgatga ccaaattgaa 1500gctggttatg
ttttgacctg tgtagcttat cccacctccg attgcaccat tgaaacccac
1560aaagaagaag agctcaccgc ataa 158451644DNAArtificial
sequencepETDuet Hyd E + A Cr C' truncated Fd with no linker
5atgggctgga gccatccgca gtttgaaaaa agatctgaaa acctgtattt tcagggcgct
60gctcctgctg ctgaagcgcc gctgagccat gtgcagcagg cgctggcgga actggcgaaa
120ccgaaagatg atccgacccg taagcatgtg tgcgtgcagg tggcgccggc
ggtgcgtgtg 180gcgatcgcgg aaaccctggg cctggcgccg ggcgcgacca
ccccgaaaca gctggcggaa 240ggcctgcgtc gtctgggctt tgatgaagtg
ttcgataccc tgtttggcgc ggatctgacc 300atcatggaag aaggcagcga
actgctgcat cgtctgaccg aacatctgga agcgcatccg 360catagcgatg
aaccgctgcc gatgtttacc agctgctgcc cgggctggat tgcgatgctg
420gaaaaaagct atccggatct gattccgtat gtgagcagct gcaaaagccc
gcagatgatg 480ctggcggcga tggtgaaaag ctatctggcg gaaaaaaaag
gcattgcgcc gaaagatatg 540gtgatggtga gcattatgcc gtgcacccgt
aaacagagcg aagcggatcg tgattggttc 600tgcgtggatg cggatccgac
cctgcgtcag ctggatcatg tgattaccac cgtggaactg 660ggcaacattt
ttaaagaacg tggcattaac ctggcggaac tgccggaagg cgaatgggat
720aacccgatgg gcgtgggcag cggcgcgggc gtgctgttcg gcaccaccgg
cggcgtgatg 780gaagcggcgc tgcgtaccgc gtatgaactg tttaccggca
ccccgctgcc gcgtctgagc 840ctgagcgagg tgcgtggcat ggatggcatt
aaagagacca acattaccat ggtgccggcg 900ccgggcagca aatttgaaga
actgctgaaa catcgtgcgg cggcgcgtgc ggaagcggcg 960gcgcatggca
ccccgggccc gctggcgtgg gatggcggcg cgggctttac cagcgaagat
1020ggccgtggcg gcattaccct gcgtgtggcg gtggcgaacg gcctgggcaa
cgcgaaaaaa 1080ctgattacca aaatgcaggc gggcgaagcg aaatatgatt
ttgtggaaat tatggcgtgc 1140ccggcgggct gcgtgggcgg cggcggccag
ccgcgtagca ccgataaagc gatcacccag 1200aaacgtcagg cggcgctgta
taacctggat gagaagagca ccctgcgtcg tagccatgag 1260aacccgagca
ttcgtgaact gtatgatacc tatctgggcg aaccgctggg ccataaagcg
1320catgaactgc tgcataccca ttatgtgatg gcatcctata ccgttaaatt
gatcaccccc 1380gatggtgaaa gttccatcga atgctctgac gatacctata
tcctcgatgc tgcggaagaa 1440gctggcctag acctgcccta ttcctgccgt
gctggggctt gctccacctg tgccggtaag 1500atcaccgctg gtagtgttga
ccaatccgat cagtctttct tggatgatga ccaaattgaa 1560gctggttatg
ttttgacctg tgtagcttat cccacctccg attgcaccat tgaaacccac
1620aaagaagaag agctcaccgc ataa 164461617DNAArtificial
sequencepETDuet Hyd E + A Cr Fd N' truncated 6atgggctgga gccatccgca
gtttgaaaaa agatctgaaa acctgtattt tcagggcgct 60gctcctgctg ctgaagcgcc
gctgagccat gtgcagcagg cgctggcgga actggcgaaa 120ccgaaagatg
atccgacccg taagcatgtg tgcgtgcagg tggcgccggc ggtgcgtgtg
180gcgatcgcgg aaaccctggg cctggcgccg ggcgcgacca ccccgaaaca
gctggcggaa 240ggcctgcgtc gtctgggctt tgatgaagtg ttcgataccc
tgtttggcgc ggatctgacc 300atcatggaag aaggcagcga actgctgcat
cgtctgaccg aacatctgga agcgcatccg 360catagcgatg aaccgctgcc
gatgtttacc agctgctgcc cgggctggat tgcgatgctg 420gaaaaaagct
atccggatct gattccgtat gtgagcagct gcaaaagccc gcagatgatg
480ctggcggcga tggtgaaaag ctatctggcg gaaaaaaaag gcattgcgcc
gaaagatatg 540gtgatggtga gcattatgcc gtgcacccgt aaacagagcg
aagcggatcg tgattggttc 600tgcgtggatg cggatccgac cctgcgtcag
ctggatcatg tgattaccac cgtggaactg 660ggcaacattt ttaaagaacg
tggcattaac ctggcggaac tgccggaagg cgaatgggat 720aacccgatgg
gcgtgggcag cggcgcgggc gtgctgttcg gcaccaccgg cggcgtgatg
780gaagcggcgc tgcgtaccgc gtatgaactg tttaccggca ccccgctgcc
gcgtctgagc 840ctgagcgagg tgcgtggcat ggatggcatt aaagagacca
acattaccat ggtgccggcg 900ccgggcagca aatttgaaga actgctgaaa
catcgtgcgg cggcgcgtgc ggaagcggcg 960gcgcatggca ccccgggccc
gctggcgtgg gatggcggcg cgggctttac cagcgaagat 1020ggccgtggcg
gcattaccct gcgtgtggcg gtggcgaacg gcctgggcaa cgcgaaaaaa
1080ctgattacca aaatgcaggc gggcgaagcg aaatatgatt ttgtggaaat
tatggcgtgc 1140ccggcgggct gcgtgggcgg cggcggccag ccgcgtagca
ccgataaagc gatcacccag 1200aaacgtcagg cggcgctgta taacctggat
gagaagagca ccctgcgtcg tagccatgag 1260aacccgagca ttcgtgaact
gtatgatacc tatctgggcg aaccgctggg ccataaagcg 1320catgaactgc
tgcataccca ttatgtggcg ggcggcgtgg aagaaaaaga tgaaaaaaaa
1380gacgatacct atatcctcga tgctgcggaa gaagctggcc tagacctgcc
ctattcctgc 1440cgtgctgggg cttgctccac ctgtgccggt aagatcaccg
ctggtagtgt tgaccaatcc 1500gatcagtctt tcttggatga tgaccaaatt
gaagctggtt atgttttgac ctgtgtagct 1560tatcccacct ccgattgcac
cattgaaacc cacaaagaag aagagctcac cgcataa 1617720DNAArtificial
sequenceSingle strand DNA oligonucleotide 7atctatggca tcctataccg
20837DNAArtificial sequenceSingle strand DNA oligonucleotide
8ttatgcggtg agctcttctt ctttgtgggt ttcaatg 37918DNAArtificial
sequenceSingle strand DNA oligonucleotide 9gatatacata tgggctgg
181030DNAArtificial sequenceSingle strand DNA oligonucleotide
10accagactcg agttatgcgg tgagctcttc 301139DNAArtificial
sequenceSingle strand DNA oligonucleotide 11ggtataggat gccatttttt
tttcatcttt ttcttccac 391240DNAArtificial sequenceSingle strand DNA
oligonucleotide 12gtggaagaaa aagatgaaaa aaaaatggca tcctataccg
401339DNAArtificial sequenceSingle strand DNA oligonucleotide
13ggatccgccg ccaccttttt tttcatcttt ttcttccac 391431DNAArtificial
sequenceSingle strand DNA oligonucleotide 14ggtggcggcg gatccatggc
atcctatacc g 311554DNAArtificial sequenceSingle strand DNA
oligonucleotide 15ggagccgccg ccgccggatc ctcctcctcc ttttttttca
tctttttctt ccac 541646DNAArtificial sequenceSingle strand DNA
oligonucleotide 16ggaggaggag gatccggcgg cggcggctcc atggcatcct
ataccg 461736DNAArtificial sequenceSingle strand DNA
oligonucleotide 17gaggatatag gtatcgtcca cataatgggt atgcag
361836DNAArtificial sequenceSingle strand DNA oligonucleotide
18ctgcataccc attatgtgga cgatacctat atcctc 361934DNAArtificial
sequenceSingle strand DNA oligonucleotide 19cggtatagga tgccatcaca
taatgggtat gcag 342034DNAArtificial sequenceSingle strand DNA
oligonucleotide 20ctgcataccc attatgtgat ggcatcctat accg
342142DNAArtificial sequenceSingle strand DNA oligonucleotide
21gaggatatag gtatcgtctt ttttttcatc tttttcttcc ac
422242DNAArtificial sequenceSingle strand DNA oligonucleotide
22gtggaagaaa aagatgaaaa aaaagacgat acctatatcc tc
4223558PRTArtificial sequence1HydFd a recombinant product of
pETDuet Hyd E + A Cr Fd with no linker 23Met Gly Trp Ser His Pro
Gln Phe Glu Lys Arg Ser Glu Asn Leu Tyr 1 5 10 15 Phe Gln Gly Ala
Ala Pro Ala Ala Glu Ala Pro Leu Ser His Val Gln 20 25 30 Gln Ala
Leu Ala Glu Leu Ala Lys Pro Lys Asp Asp Pro Thr Arg Lys 35 40 45
His Val Cys Val Gln Val Ala Pro Ala Val Arg Val Ala Ile Ala Glu 50
55 60 Thr Leu Gly Leu Ala Pro Gly Ala Thr Thr Pro Lys Gln Leu Ala
Glu 65 70 75 80 Gly Leu Arg Arg Leu Gly Phe Asp Glu Val Phe Asp Thr
Leu Phe Gly 85 90 95 Ala Asp Leu Thr Ile Met Glu Glu Gly Ser Glu
Leu Leu His Arg Leu 100 105 110 Thr Glu His Leu Glu Ala His Pro His
Ser Asp Glu Pro Leu Pro Met 115 120 125 Phe Thr Ser Cys Cys Pro Gly
Trp Ile Ala Met Leu Glu Lys Ser Tyr 130 135 140 Pro Asp Leu Ile Pro
Tyr Val Ser Ser Cys Lys Ser Pro Gln Met Met 145 150 155 160 Leu Ala
Ala Met Val Lys Ser Tyr Leu Ala Glu Lys Lys Gly Ile Ala 165 170 175
Pro Lys Asp Met Val Met Val Ser Ile Met Pro Cys Thr Arg Lys Gln 180
185 190 Ser Glu Ala Asp Arg Asp Trp Phe Cys Val Asp Ala Asp Pro Thr
Leu 195 200 205 Arg Gln Leu Asp His Val Ile Thr Thr Val Glu Leu Gly
Asn Ile Phe 210 215 220 Lys Glu Arg Gly Ile Asn Leu Ala Glu Leu Pro
Glu Gly Glu Trp Asp 225 230 235 240 Asn Pro Met Gly Val Gly Ser Gly
Ala Gly Val Leu Phe Gly Thr Thr 245 250 255 Gly Gly Val Met Glu Ala
Ala Leu Arg Thr Ala Tyr Glu Leu Phe Thr 260 265 270 Gly Thr Pro Leu
Pro Arg Leu Ser Leu Ser Glu Val Arg Gly Met Asp 275 280 285 Gly Ile
Lys Glu Thr Asn Ile Thr Met Val Pro Ala Pro Gly Ser Lys 290 295 300
Phe Glu Glu Leu Leu Lys His Arg Ala Ala Ala Arg Ala Glu Ala Ala 305
310 315 320 Ala His Gly Thr Pro Gly Pro Leu Ala Trp Asp Gly Gly Ala
Gly Phe 325 330 335 Thr Ser Glu Asp Gly Arg Gly Gly Ile Thr Leu Arg
Val Ala Val Ala 340 345 350 Asn Gly Leu Gly Asn Ala Lys Lys Leu Ile
Thr Lys Met Gln Ala Gly 355 360 365 Glu Ala Lys Tyr Asp Phe Val Glu
Ile Met Ala Cys Pro Ala Gly Cys 370 375 380 Val Gly Gly Gly Gly Gln
Pro Arg Ser Thr Asp Lys Ala Ile Thr Gln 385 390 395
400 Lys Arg Gln Ala Ala Leu Tyr Asn Leu Asp Glu Lys Ser Thr Leu Arg
405 410 415 Arg Ser His Glu Asn Pro Ser Ile Arg Glu Leu Tyr Asp Thr
Tyr Leu 420 425 430 Gly Glu Pro Leu Gly His Lys Ala His Glu Leu Leu
His Thr His Tyr 435 440 445 Val Ala Gly Gly Val Glu Glu Lys Asp Glu
Lys Lys Met Ala Ser Tyr 450 455 460 Thr Val Lys Leu Ile Thr Pro Asp
Gly Glu Ser Ser Ile Glu Cys Ser 465 470 475 480 Asp Asp Thr Tyr Ile
Leu Asp Ala Ala Glu Glu Ala Gly Leu Asp Leu 485 490 495 Pro Tyr Ser
Cys Arg Ala Gly Ala Cys Ser Thr Cys Ala Gly Lys Ile 500 505 510 Thr
Ala Gly Ser Val Asp Gln Ser Asp Gln Ser Phe Leu Asp Asp Asp 515 520
525 Gln Ile Glu Ala Gly Tyr Val Leu Thr Cys Val Ala Tyr Pro Thr Ser
530 535 540 Asp Cys Thr Ile Glu Thr His Lys Glu Glu Glu Leu Thr Ala
545 550 555 24563PRTArtificial sequence2HydFd a recombinant product
of pETDuet Hyd E + A Cr Fd with a short linker 24Met Gly Trp Ser
His Pro Gln Phe Glu Lys Arg Ser Glu Asn Leu Tyr 1 5 10 15 Phe Gln
Gly Ala Ala Pro Ala Ala Glu Ala Pro Leu Ser His Val Gln 20 25 30
Gln Ala Leu Ala Glu Leu Ala Lys Pro Lys Asp Asp Pro Thr Arg Lys 35
40 45 His Val Cys Val Gln Val Ala Pro Ala Val Arg Val Ala Ile Ala
Glu 50 55 60 Thr Leu Gly Leu Ala Pro Gly Ala Thr Thr Pro Lys Gln
Leu Ala Glu 65 70 75 80 Gly Leu Arg Arg Leu Gly Phe Asp Glu Val Phe
Asp Thr Leu Phe Gly 85 90 95 Ala Asp Leu Thr Ile Met Glu Glu Gly
Ser Glu Leu Leu His Arg Leu 100 105 110 Thr Glu His Leu Glu Ala His
Pro His Ser Asp Glu Pro Leu Pro Met 115 120 125 Phe Thr Ser Cys Cys
Pro Gly Trp Ile Ala Met Leu Glu Lys Ser Tyr 130 135 140 Pro Asp Leu
Ile Pro Tyr Val Ser Ser Cys Lys Ser Pro Gln Met Met 145 150 155 160
Leu Ala Ala Met Val Lys Ser Tyr Leu Ala Glu Lys Lys Gly Ile Ala 165
170 175 Pro Lys Asp Met Val Met Val Ser Ile Met Pro Cys Thr Arg Lys
Gln 180 185 190 Ser Glu Ala Asp Arg Asp Trp Phe Cys Val Asp Ala Asp
Pro Thr Leu 195 200 205 Arg Gln Leu Asp His Val Ile Thr Thr Val Glu
Leu Gly Asn Ile Phe 210 215 220 Lys Glu Arg Gly Ile Asn Leu Ala Glu
Leu Pro Glu Gly Glu Trp Asp 225 230 235 240 Asn Pro Met Gly Val Gly
Ser Gly Ala Gly Val Leu Phe Gly Thr Thr 245 250 255 Gly Gly Val Met
Glu Ala Ala Leu Arg Thr Ala Tyr Glu Leu Phe Thr 260 265 270 Gly Thr
Pro Leu Pro Arg Leu Ser Leu Ser Glu Val Arg Gly Met Asp 275 280 285
Gly Ile Lys Glu Thr Asn Ile Thr Met Val Pro Ala Pro Gly Ser Lys 290
295 300 Phe Glu Glu Leu Leu Lys His Arg Ala Ala Ala Arg Ala Glu Ala
Ala 305 310 315 320 Ala His Gly Thr Pro Gly Pro Leu Ala Trp Asp Gly
Gly Ala Gly Phe 325 330 335 Thr Ser Glu Asp Gly Arg Gly Gly Ile Thr
Leu Arg Val Ala Val Ala 340 345 350 Asn Gly Leu Gly Asn Ala Lys Lys
Leu Ile Thr Lys Met Gln Ala Gly 355 360 365 Glu Ala Lys Tyr Asp Phe
Val Glu Ile Met Ala Cys Pro Ala Gly Cys 370 375 380 Val Gly Gly Gly
Gly Gln Pro Arg Ser Thr Asp Lys Ala Ile Thr Gln 385 390 395 400 Lys
Arg Gln Ala Ala Leu Tyr Asn Leu Asp Glu Lys Ser Thr Leu Arg 405 410
415 Arg Ser His Glu Asn Pro Ser Ile Arg Glu Leu Tyr Asp Thr Tyr Leu
420 425 430 Gly Glu Pro Leu Gly His Lys Ala His Glu Leu Leu His Thr
His Tyr 435 440 445 Val Ala Gly Gly Val Glu Glu Lys Asp Glu Lys Lys
Gly Gly Gly Gly 450 455 460 Ser Met Ala Ser Tyr Thr Val Lys Leu Ile
Thr Pro Asp Gly Glu Ser 465 470 475 480 Ser Ile Glu Cys Ser Asp Asp
Thr Tyr Ile Leu Asp Ala Ala Glu Glu 485 490 495 Ala Gly Leu Asp Leu
Pro Tyr Ser Cys Arg Ala Gly Ala Cys Ser Thr 500 505 510 Cys Ala Gly
Lys Ile Thr Ala Gly Ser Val Asp Gln Ser Asp Gln Ser 515 520 525 Phe
Leu Asp Asp Asp Gln Ile Glu Ala Gly Tyr Val Leu Thr Cys Val 530 535
540 Ala Tyr Pro Thr Ser Asp Cys Thr Ile Glu Thr His Lys Glu Glu Glu
545 550 555 560 Leu Thr Ala 25568PRTArtificial sequence3HydFd a
recombinant product of pETDuet Hyd E + A Cr Fd with a medium linker
25Met Gly Trp Ser His Pro Gln Phe Glu Lys Arg Ser Glu Asn Leu Tyr 1
5 10 15 Phe Gln Gly Ala Ala Pro Ala Ala Glu Ala Pro Leu Ser His Val
Gln 20 25 30 Gln Ala Leu Ala Glu Leu Ala Lys Pro Lys Asp Asp Pro
Thr Arg Lys 35 40 45 His Val Cys Val Gln Val Ala Pro Ala Val Arg
Val Ala Ile Ala Glu 50 55 60 Thr Leu Gly Leu Ala Pro Gly Ala Thr
Thr Pro Lys Gln Leu Ala Glu 65 70 75 80 Gly Leu Arg Arg Leu Gly Phe
Asp Glu Val Phe Asp Thr Leu Phe Gly 85 90 95 Ala Asp Leu Thr Ile
Met Glu Glu Gly Ser Glu Leu Leu His Arg Leu 100 105 110 Thr Glu His
Leu Glu Ala His Pro His Ser Asp Glu Pro Leu Pro Met 115 120 125 Phe
Thr Ser Cys Cys Pro Gly Trp Ile Ala Met Leu Glu Lys Ser Tyr 130 135
140 Pro Asp Leu Ile Pro Tyr Val Ser Ser Cys Lys Ser Pro Gln Met Met
145 150 155 160 Leu Ala Ala Met Val Lys Ser Tyr Leu Ala Glu Lys Lys
Gly Ile Ala 165 170 175 Pro Lys Asp Met Val Met Val Ser Ile Met Pro
Cys Thr Arg Lys Gln 180 185 190 Ser Glu Ala Asp Arg Asp Trp Phe Cys
Val Asp Ala Asp Pro Thr Leu 195 200 205 Arg Gln Leu Asp His Val Ile
Thr Thr Val Glu Leu Gly Asn Ile Phe 210 215 220 Lys Glu Arg Gly Ile
Asn Leu Ala Glu Leu Pro Glu Gly Glu Trp Asp 225 230 235 240 Asn Pro
Met Gly Val Gly Ser Gly Ala Gly Val Leu Phe Gly Thr Thr 245 250 255
Gly Gly Val Met Glu Ala Ala Leu Arg Thr Ala Tyr Glu Leu Phe Thr 260
265 270 Gly Thr Pro Leu Pro Arg Leu Ser Leu Ser Glu Val Arg Gly Met
Asp 275 280 285 Gly Ile Lys Glu Thr Asn Ile Thr Met Val Pro Ala Pro
Gly Ser Lys 290 295 300 Phe Glu Glu Leu Leu Lys His Arg Ala Ala Ala
Arg Ala Glu Ala Ala 305 310 315 320 Ala His Gly Thr Pro Gly Pro Leu
Ala Trp Asp Gly Gly Ala Gly Phe 325 330 335 Thr Ser Glu Asp Gly Arg
Gly Gly Ile Thr Leu Arg Val Ala Val Ala 340 345 350 Asn Gly Leu Gly
Asn Ala Lys Lys Leu Ile Thr Lys Met Gln Ala Gly 355 360 365 Glu Ala
Lys Tyr Asp Phe Val Glu Ile Met Ala Cys Pro Ala Gly Cys 370 375 380
Val Gly Gly Gly Gly Gln Pro Arg Ser Thr Asp Lys Ala Ile Thr Gln 385
390 395 400 Lys Arg Gln Ala Ala Leu Tyr Asn Leu Asp Glu Lys Ser Thr
Leu Arg 405 410 415 Arg Ser His Glu Asn Pro Ser Ile Arg Glu Leu Tyr
Asp Thr Tyr Leu 420 425 430 Gly Glu Pro Leu Gly His Lys Ala His Glu
Leu Leu His Thr His Tyr 435 440 445 Val Ala Gly Gly Val Glu Glu Lys
Asp Glu Lys Lys Gly Gly Gly Gly 450 455 460 Ser Gly Gly Gly Gly Ser
Met Ala Ser Tyr Thr Val Lys Leu Ile Thr 465 470 475 480 Pro Asp Gly
Glu Ser Ser Ile Glu Cys Ser Asp Asp Thr Tyr Ile Leu 485 490 495 Asp
Ala Ala Glu Glu Ala Gly Leu Asp Leu Pro Tyr Ser Cys Arg Ala 500 505
510 Gly Ala Cys Ser Thr Cys Ala Gly Lys Ile Thr Ala Gly Ser Val Asp
515 520 525 Gln Ser Asp Gln Ser Phe Leu Asp Asp Asp Gln Ile Glu Ala
Gly Tyr 530 535 540 Val Leu Thr Cys Val Ala Tyr Pro Thr Ser Asp Cys
Thr Ile Glu Thr 545 550 555 560 His Lys Glu Glu Glu Leu Thr Ala 565
26527PRTArtificial sequence4HydFd a recombinant product of Hyd E +
A Cr C' truncated Fd N' truncated and with no linker 26Met Gly Trp
Ser His Pro Gln Phe Glu Lys Arg Ser Glu Asn Leu Tyr 1 5 10 15 Phe
Gln Gly Ala Ala Pro Ala Ala Glu Ala Pro Leu Ser His Val Gln 20 25
30 Gln Ala Leu Ala Glu Leu Ala Lys Pro Lys Asp Asp Pro Thr Arg Lys
35 40 45 His Val Cys Val Gln Val Ala Pro Ala Val Arg Val Ala Ile
Ala Glu 50 55 60 Thr Leu Gly Leu Ala Pro Gly Ala Thr Thr Pro Lys
Gln Leu Ala Glu 65 70 75 80 Gly Leu Arg Arg Leu Gly Phe Asp Glu Val
Phe Asp Thr Leu Phe Gly 85 90 95 Ala Asp Leu Thr Ile Met Glu Glu
Gly Ser Glu Leu Leu His Arg Leu 100 105 110 Thr Glu His Leu Glu Ala
His Pro His Ser Asp Glu Pro Leu Pro Met 115 120 125 Phe Thr Ser Cys
Cys Pro Gly Trp Ile Ala Met Leu Glu Lys Ser Tyr 130 135 140 Pro Asp
Leu Ile Pro Tyr Val Ser Ser Cys Lys Ser Pro Gln Met Met 145 150 155
160 Leu Ala Ala Met Val Lys Ser Tyr Leu Ala Glu Lys Lys Gly Ile Ala
165 170 175 Pro Lys Asp Met Val Met Val Ser Ile Met Pro Cys Thr Arg
Lys Gln 180 185 190 Ser Glu Ala Asp Arg Asp Trp Phe Cys Val Asp Ala
Asp Pro Thr Leu 195 200 205 Arg Gln Leu Asp His Val Ile Thr Thr Val
Glu Leu Gly Asn Ile Phe 210 215 220 Lys Glu Arg Gly Ile Asn Leu Ala
Glu Leu Pro Glu Gly Glu Trp Asp 225 230 235 240 Asn Pro Met Gly Val
Gly Ser Gly Ala Gly Val Leu Phe Gly Thr Thr 245 250 255 Gly Gly Val
Met Glu Ala Ala Leu Arg Thr Ala Tyr Glu Leu Phe Thr 260 265 270 Gly
Thr Pro Leu Pro Arg Leu Ser Leu Ser Glu Val Arg Gly Met Asp 275 280
285 Gly Ile Lys Glu Thr Asn Ile Thr Met Val Pro Ala Pro Gly Ser Lys
290 295 300 Phe Glu Glu Leu Leu Lys His Arg Ala Ala Ala Arg Ala Glu
Ala Ala 305 310 315 320 Ala His Gly Thr Pro Gly Pro Leu Ala Trp Asp
Gly Gly Ala Gly Phe 325 330 335 Thr Ser Glu Asp Gly Arg Gly Gly Ile
Thr Leu Arg Val Ala Val Ala 340 345 350 Asn Gly Leu Gly Asn Ala Lys
Lys Leu Ile Thr Lys Met Gln Ala Gly 355 360 365 Glu Ala Lys Tyr Asp
Phe Val Glu Ile Met Ala Cys Pro Ala Gly Cys 370 375 380 Val Gly Gly
Gly Gly Gln Pro Arg Ser Thr Asp Lys Ala Ile Thr Gln 385 390 395 400
Lys Arg Gln Ala Ala Leu Tyr Asn Leu Asp Glu Lys Ser Thr Leu Arg 405
410 415 Arg Ser His Glu Asn Pro Ser Ile Arg Glu Leu Tyr Asp Thr Tyr
Leu 420 425 430 Gly Glu Pro Leu Gly His Lys Ala His Glu Leu Leu His
Thr His Tyr 435 440 445 Val Asp Asp Thr Tyr Ile Leu Asp Ala Ala Glu
Glu Ala Gly Leu Asp 450 455 460 Leu Pro Tyr Ser Cys Arg Ala Gly Ala
Cys Ser Thr Cys Ala Gly Lys 465 470 475 480 Ile Thr Ala Gly Ser Val
Asp Gln Ser Asp Gln Ser Phe Leu Asp Asp 485 490 495 Asp Gln Ile Glu
Ala Gly Tyr Val Leu Thr Cys Val Ala Tyr Pro Thr 500 505 510 Ser Asp
Cys Thr Ile Glu Thr His Lys Glu Glu Glu Leu Thr Ala 515 520 525
27547PRTArtificial sequence5HydFd a recombinant product of pETDuet
Hyd E + A Cr C' truncated Fd and with no linker 27Met Gly Trp Ser
His Pro Gln Phe Glu Lys Arg Ser Glu Asn Leu Tyr 1 5 10 15 Phe Gln
Gly Ala Ala Pro Ala Ala Glu Ala Pro Leu Ser His Val Gln 20 25 30
Gln Ala Leu Ala Glu Leu Ala Lys Pro Lys Asp Asp Pro Thr Arg Lys 35
40 45 His Val Cys Val Gln Val Ala Pro Ala Val Arg Val Ala Ile Ala
Glu 50 55 60 Thr Leu Gly Leu Ala Pro Gly Ala Thr Thr Pro Lys Gln
Leu Ala Glu 65 70 75 80 Gly Leu Arg Arg Leu Gly Phe Asp Glu Val Phe
Asp Thr Leu Phe Gly 85 90 95 Ala Asp Leu Thr Ile Met Glu Glu Gly
Ser Glu Leu Leu His Arg Leu 100 105 110 Thr Glu His Leu Glu Ala His
Pro His Ser Asp Glu Pro Leu Pro Met 115 120 125 Phe Thr Ser Cys Cys
Pro Gly Trp Ile Ala Met Leu Glu Lys Ser Tyr 130 135 140 Pro Asp Leu
Ile Pro Tyr Val Ser Ser Cys Lys Ser Pro Gln Met Met 145 150 155 160
Leu Ala Ala Met Val Lys Ser Tyr Leu Ala Glu Lys Lys Gly Ile Ala 165
170 175 Pro Lys Asp Met Val Met Val Ser Ile Met Pro Cys Thr Arg Lys
Gln 180 185 190 Ser Glu Ala Asp Arg Asp Trp Phe Cys Val Asp Ala Asp
Pro Thr Leu 195 200 205 Arg Gln Leu Asp His Val Ile Thr Thr Val Glu
Leu Gly Asn Ile Phe 210 215 220 Lys Glu Arg Gly Ile Asn Leu Ala Glu
Leu Pro Glu Gly Glu Trp Asp 225 230 235 240 Asn Pro Met Gly Val Gly
Ser Gly Ala Gly Val Leu Phe Gly Thr Thr 245 250 255 Gly Gly Val Met
Glu Ala Ala Leu Arg Thr Ala Tyr Glu Leu Phe Thr 260 265 270 Gly Thr
Pro Leu Pro Arg Leu Ser Leu Ser Glu Val Arg Gly Met Asp 275 280 285
Gly Ile Lys Glu Thr Asn Ile Thr Met Val Pro Ala Pro Gly Ser Lys 290
295 300 Phe Glu Glu Leu Leu Lys His Arg Ala Ala Ala Arg Ala Glu Ala
Ala 305 310 315 320 Ala His Gly Thr Pro Gly Pro Leu Ala Trp Asp Gly
Gly Ala Gly Phe 325 330 335 Thr Ser Glu Asp Gly Arg Gly Gly Ile Thr
Leu Arg Val Ala Val Ala 340 345 350 Asn Gly Leu Gly Asn Ala Lys Lys
Leu Ile Thr Lys Met Gln Ala Gly 355 360 365 Glu Ala Lys Tyr Asp Phe
Val Glu Ile Met Ala Cys Pro Ala Gly Cys 370 375 380 Val Gly Gly Gly
Gly Gln Pro Arg Ser Thr Asp Lys Ala Ile Thr Gln 385 390 395 400 Lys
Arg Gln Ala Ala Leu Tyr Asn Leu Asp Glu Lys Ser Thr Leu Arg 405 410
415 Arg Ser His Glu Asn Pro Ser Ile Arg Glu Leu Tyr Asp Thr Tyr Leu
420 425
430 Gly Glu Pro Leu Gly His Lys Ala His Glu Leu Leu His Thr His Tyr
435 440 445 Val Met Ala Ser Tyr Thr Val Lys Leu Ile Thr Pro Asp Gly
Glu Ser 450 455 460 Ser Ile Glu Cys Ser Asp Asp Thr Tyr Ile Leu Asp
Ala Ala Glu Glu 465 470 475 480 Ala Gly Leu Asp Leu Pro Tyr Ser Cys
Arg Ala Gly Ala Cys Ser Thr 485 490 495 Cys Ala Gly Lys Ile Thr Ala
Gly Ser Val Asp Gln Ser Asp Gln Ser 500 505 510 Phe Leu Asp Asp Asp
Gln Ile Glu Ala Gly Tyr Val Leu Thr Cys Val 515 520 525 Ala Tyr Pro
Thr Ser Asp Cys Thr Ile Glu Thr His Lys Glu Glu Glu 530 535 540 Leu
Thr Ala 545 28538PRTArtificial sequence6HydFd a recombinant product
of pETDuet Hyd E + A Cr Fd N' truncated 28Met Gly Trp Ser His Pro
Gln Phe Glu Lys Arg Ser Glu Asn Leu Tyr 1 5 10 15 Phe Gln Gly Ala
Ala Pro Ala Ala Glu Ala Pro Leu Ser His Val Gln 20 25 30 Gln Ala
Leu Ala Glu Leu Ala Lys Pro Lys Asp Asp Pro Thr Arg Lys 35 40 45
His Val Cys Val Gln Val Ala Pro Ala Val Arg Val Ala Ile Ala Glu 50
55 60 Thr Leu Gly Leu Ala Pro Gly Ala Thr Thr Pro Lys Gln Leu Ala
Glu 65 70 75 80 Gly Leu Arg Arg Leu Gly Phe Asp Glu Val Phe Asp Thr
Leu Phe Gly 85 90 95 Ala Asp Leu Thr Ile Met Glu Glu Gly Ser Glu
Leu Leu His Arg Leu 100 105 110 Thr Glu His Leu Glu Ala His Pro His
Ser Asp Glu Pro Leu Pro Met 115 120 125 Phe Thr Ser Cys Cys Pro Gly
Trp Ile Ala Met Leu Glu Lys Ser Tyr 130 135 140 Pro Asp Leu Ile Pro
Tyr Val Ser Ser Cys Lys Ser Pro Gln Met Met 145 150 155 160 Leu Ala
Ala Met Val Lys Ser Tyr Leu Ala Glu Lys Lys Gly Ile Ala 165 170 175
Pro Lys Asp Met Val Met Val Ser Ile Met Pro Cys Thr Arg Lys Gln 180
185 190 Ser Glu Ala Asp Arg Asp Trp Phe Cys Val Asp Ala Asp Pro Thr
Leu 195 200 205 Arg Gln Leu Asp His Val Ile Thr Thr Val Glu Leu Gly
Asn Ile Phe 210 215 220 Lys Glu Arg Gly Ile Asn Leu Ala Glu Leu Pro
Glu Gly Glu Trp Asp 225 230 235 240 Asn Pro Met Gly Val Gly Ser Gly
Ala Gly Val Leu Phe Gly Thr Thr 245 250 255 Gly Gly Val Met Glu Ala
Ala Leu Arg Thr Ala Tyr Glu Leu Phe Thr 260 265 270 Gly Thr Pro Leu
Pro Arg Leu Ser Leu Ser Glu Val Arg Gly Met Asp 275 280 285 Gly Ile
Lys Glu Thr Asn Ile Thr Met Val Pro Ala Pro Gly Ser Lys 290 295 300
Phe Glu Glu Leu Leu Lys His Arg Ala Ala Ala Arg Ala Glu Ala Ala 305
310 315 320 Ala His Gly Thr Pro Gly Pro Leu Ala Trp Asp Gly Gly Ala
Gly Phe 325 330 335 Thr Ser Glu Asp Gly Arg Gly Gly Ile Thr Leu Arg
Val Ala Val Ala 340 345 350 Asn Gly Leu Gly Asn Ala Lys Lys Leu Ile
Thr Lys Met Gln Ala Gly 355 360 365 Glu Ala Lys Tyr Asp Phe Val Glu
Ile Met Ala Cys Pro Ala Gly Cys 370 375 380 Val Gly Gly Gly Gly Gln
Pro Arg Ser Thr Asp Lys Ala Ile Thr Gln 385 390 395 400 Lys Arg Gln
Ala Ala Leu Tyr Asn Leu Asp Glu Lys Ser Thr Leu Arg 405 410 415 Arg
Ser His Glu Asn Pro Ser Ile Arg Glu Leu Tyr Asp Thr Tyr Leu 420 425
430 Gly Glu Pro Leu Gly His Lys Ala His Glu Leu Leu His Thr His Tyr
435 440 445 Val Ala Gly Gly Val Glu Glu Lys Asp Glu Lys Lys Asp Asp
Thr Tyr 450 455 460 Ile Leu Asp Ala Ala Glu Glu Ala Gly Leu Asp Leu
Pro Tyr Ser Cys 465 470 475 480 Arg Ala Gly Ala Cys Ser Thr Cys Ala
Gly Lys Ile Thr Ala Gly Ser 485 490 495 Val Asp Gln Ser Asp Gln Ser
Phe Leu Asp Asp Asp Gln Ile Glu Ala 500 505 510 Gly Tyr Val Leu Thr
Cys Val Ala Tyr Pro Thr Ser Asp Cys Thr Ile 515 520 525 Glu Thr His
Lys Glu Glu Glu Leu Thr Ala 530 535
* * * * *