U.S. patent application number 11/177359 was filed with the patent office on 2006-05-04 for peptide sequence tags and method of using same.
Invention is credited to Douglas A. Campbell.
Application Number | 20060094058 11/177359 |
Document ID | / |
Family ID | 23209624 |
Filed Date | 2006-05-04 |
United States Patent
Application |
20060094058 |
Kind Code |
A1 |
Campbell; Douglas A. |
May 4, 2006 |
Peptide sequence tags and method of using same
Abstract
Peptide sequence tags are identified and used to produce a class
of global antibodies, which recognize all members of the GlnA
protein sub-family with uniform specificity, regardless of the
species of origin. The tags are used to create antibodies to the
major GlnA protein of the Glutamine Synthetase enzyme catalyzing of
carbon fixation. The antibodies have a range of applications as
diagnostic detection reagents for the major environmental process
of ammonia assimilation.
Inventors: |
Campbell; Douglas A.;
(Sackville, CA) |
Correspondence
Address: |
STIKEMAN ELLIOTT
1600-50 O'CONNOR STREET
OTTAWA
ON
KIP LS2
CA
|
Family ID: |
23209624 |
Appl. No.: |
11/177359 |
Filed: |
July 11, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10216810 |
Aug 13, 2002 |
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11177359 |
Jul 11, 2005 |
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60312042 |
Aug 13, 2001 |
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Current U.S.
Class: |
435/7.1 ;
435/70.21; 530/388.26 |
Current CPC
Class: |
G01N 33/6803 20130101;
G01N 33/6845 20130101; C07K 16/40 20130101; C07K 16/00 20130101;
G01N 33/68 20130101 |
Class at
Publication: |
435/007.1 ;
530/388.26; 435/070.21 |
International
Class: |
G01N 33/53 20060101
G01N033/53; C12P 21/04 20060101 C12P021/04; C07K 16/40 20060101
C07K016/40 |
Claims
1. A method for detecting the presence of a member of a GlnA
protein (glutamine synthetase) in a sample comprising of: (a)
identifying and obtaining a peptide sequence tag conserved for GlnA
protein, and exclusive to the GlnA protein, wherein said peptide
sequence tag is SEQ ID NO: 3 (b) utilizing the tag to elicit the
production of antibodies; and (c) using the antibodies to measure
the concentration of a member of the GlnA protein in a sample.
2. The peptide sequence tag SEQ ID NO: 3.
Description
[0001] This invention relates to peptide sequence tags used to
elicit antibodies, which can be used to detect defined families of
proteins.
[0002] Natural populations of phytoplankton include representatives
of numerous species of cyanobacteria, diatoms, green algae and
other groups. Nevertheless many of these species share core
biochemical pathways supporting primary productivity and elemental
cycling (Bryant, 1994; Falkowski & Raven, 1997). To assess the
gross capacity for key metabolic transformations in aquatic
habitats, and to track acclimatory changes in these capacities,
researchers require reagents to quantitatively detect all members
of a functional class of enzymes (Bouchard et al., 2002; Schofield
et al., 2002), for example the RbcL (RUBISCO) enzyme responsible
for carbon fixation in all photosynthetic organisms. Different
members of the organism population win contain somewhat different
versions of the RbcL enzyme, which nevertheless share similar core
properties and shared conserved regions (ncbi.nlm.nih.gov).
Conventional immunological detection uses an antibody raised
against one particular protein from one species, which will then
bind with variable affinity to other related proteins depending on
their antigenic similarity to the initial target molecule (Orellana
& Perry, 1992). This is problematic because change in
immunological signals could result from: a) changes in the level of
the targets b) changes in the population composition resulting in
shifts in the specific mix of target molecules present or c) a
combination of (a) and (b).
[0003] Conventional antibodies are raised against two classes of
protein targets; namely (a) purified or over-expressed protein from
a particular species, and (b) a peptide selected to match the
sequence of a region of a particular protein. Such antibodies are
generally raised against proteins from a model species, and show
variable cross-reactivity to related proteins from other species.
It is not practical to develop individual antibodies to detect each
protein of interest from each strain in a population, many of which
are poorly characterized or unknown, and even unculturable (Staley
& Reysenbach 2002). Most of the protein families of interest in
cyanobacteria and phytoplankton are also highly conserved in plants
so that the same detection system can meet needs for standard
antibodies in plant sciences.
[0004] Thus, a need exists for a set of peptide targets to elicit
production of a set of antibodies to detect key proteins involved,
inter alia, in photosynthetic activity. A system should be able to
evenly and universally recognize all members of a defined enzyme
family or subfamily based on shared characteristics.
[0005] The above defined need is met by the present invention which
provides a method for detecting the presence of members of the
target protein family in a sample comprising the steps of:
[0006] (a) identifying and obtaining a peptide sequence tag
conserved for all sequences of members of the target protein
family, and exclusive to the target protein family;
[0007] (b) assessing the tag for immunogenicity potential
[0008] (c) utilizing the tag to elicit the production of
antibodies; and
[0009] (d) using the antibodies to measure the concentration of
members of the target protein family in a sample.
[0010] The invention also provides a peptide sequence tag selected
from the group consisting of
[0011] SEQ ID NO: 1
[0012] SEQ ID NO: 2
[0013] SEQ ID NO: 3
[0014] SEQ ID NO: 4
[0015] SEQ ID NO: 5
[0016] SEQ ID NO: 7
[0017] SEQ ID NO: 8
[0018] SEQ ID NO: 9
[0019] SEQ ID NO: 10
[0020] SEQ ID NO: 11
[0021] SEQ ID NO: 13
[0022] SEQ ID NO: 14
[0023] SEQ ID NO: 15
[0024] In accordance with another aspect of the invention, the
invention provides a method for detecting the presence of a target
protein in a sample comprising the steps of:
[0025] (a) identifying and obtaining a peptide sequence tag
conserved for all members of and exclusive to a protein family;
[0026] (b) assessing the tag for immunogenicity; and
[0027] (c) synthesizing the tag provided it possesses a
predetermined level of immunogenicity.
[0028] (d) utilizing the tag to elicit the production antibodies;
and
[0029] (e) using the antibodies to provide an indication of protein
concentration in a sample.
[0030] According to yet another aspect, the invention provides a
method of using a peptide sequence tag for coupling to column
matrice materials for affinity purification of the global
antibodies produced according to the invention.
[0031] According to yet another aspect, there is provided a method
of developing characterized concentration standards for
quantitation of the concentration of target proteins in samples by
comparison to the concentration standards comprising the steps
of:
(a) coupling a defined molar quantity of protein carrier molecule
to a defined molar quantity of peptide sequence tag selected from
the group consisting of
SEQ ID NO: 1
SEQ ID NO: 2
SEQ ID NO: 3
SEQ ID NO: 4
SEQ ID NO: 5
SEQ ID NO: 7
SEQ ID NO: 8
SEQ ID NO: 9
SEQ ID NO: 10
SEQ ID NO: 11
SEQ ID NO: 13
SEQ ID NO: 14
SEQ ID NO: 15
[0032] (b) subjecting a known molar quantity of the coupled complex
from (a) to electrophoretic separation in parallel SDS-PAGE gel
lanes with protein extracts containing members of the target
protein family, followed by electrophoretic transfer to a
membrane
(c) immunodetection using a global antibody produced according to
the above-defined method of the coupled standard and any members of
the target protein family identified by the peptide sequence tag
and
(d) using an immunological signal from the known molar quantity of
coupled complex as a standard for measuring the molar quantity of
members of the target protein family present in the protein
extracts.
[0033] In a still further aspect, there is provided a method for
quantitation of members of the target protein families in multiple
samples using Enzyme-Linked ImmunoSorbent Assay kits using on
characterized global antibodies produced according to the
above-defined method and quantitation standards produced according
to the method defined in the preceding paragraph.
[0034] In another aspect, there is provided a method for eliciting
production of monoclonal, transgenic or synthetic antibodies using
peptide sequence tags produced according to the above defined
method and standard immunological protocols.
[0035] In a further aspect, there is provided a method for affinity
screening of libraries of reagents to detect specific reagent
binding to a peptide sequence tag produced according to the above
defined method and immobilized on a support matrice; and
[0036] testing reagents binding to the immobilized peptide sequence
tag for affinity binding to members of the target protein
families.
[0037] In a still further embodiment, there is provided a method to
use affinity binding using global antibodies produced according to
the above-defined method to capture target proteins from complex
mixtures for subsequent analyses of the specific sequences of
target proteins present in the mixture using mass spectroscopy.
[0038] In yet another embodiment, there is provided a method to use
affinity binding using global antibodies produced according to the
above defined method to capture and remove target proteins from
complex mixtures to lower interference with detection and analyses
of other less abundant protein classes in proteomics applications
such as two dimensional isoelectric focusing/SDS-PAGE and
subsequent mass spectroscopic analyses of protein sequences.
[0039] In general terms, the inventors have designed a set of
peptide targets or peptide sequence tags which elicit production of
a set of antibodies for detecting key proteins involved in
photosynthetic productivity. The inventors chose target protein
families based on scientific interest and wide applicability. They
have found and aligned sequences from public databases to detect
peptide sequence tags of 6-25 amino acids which are conserved in
all known members of the target protein family, and use
bioinformatic analyses to determine if the conserved peptides are
unique to the target protein family. The peptide sequence tags are
assessed for potential immunogenicity, feasibility of synthesis,
solubility and stability; avoiding sequences that are targets for
known or putative post-translational modification in proteins.
Selected peptide sequence tags are synthesized, coupled to a
carrier and used to elicit antibody production. The specificity and
titre of the antibodies were then listed. A set of antibodies
increases the utility of the system by allowing comprehensive
detection of key molecules in a sample, population or community.
The target protein families were selected based on their core roles
in the biosphere and their interest and importance for
environmental research, modelling, and monitoring.
[0040] Public sequence databases were scanned for all published
sequences of proteins in a given family (http://www.expasy.ch;
http://www.ncbi.nlm.nih.gov) The sequences of all published members
of each target protein family were aligned (Corpet, 1988). Peptide
sequence tags of 6-25 amino acids were selected whose sequences are
conserved in all known members of the target protein or sub-family.
The peptide sequence tags were tested to determine exclusivity to
the target protein family using short-peptide BLAST searches of
sequence databases (Altschul et al., 2001). The position of each
potential peptide sequence tag in a given protein family was
analyzed to ensure it is maintained in the mature proteins, and to
avoid regions of the proteins known or suspected to undergo
post-translational modifications such as glycosylation that could
interfere with later antibody recognition of the mature proteins.
The peptide sequence tags are screened for antigenic potential
using peptide property prediction algorithms, and to assess their
feasibility for synthesis, solubility and stability based on amino
acid composition. In summary the chosen peptide sequence tags are
conserved in all published members of the defined target protein
family or subfamily, do not align significantly with members of
other known protein families, and have acceptable predicted
antigenic and synthesis properties.
[0041] The selected peptide sequence tags are synthesized. The
peptide sequence tags are then coupled to appropriate immunogenic
carrier molecules, typically Keyhole Limpet Hemocyanin, and the
complexes are used to elicit production of IgY antibodies in hens.
The IgY fraction is separated from the eggs of the immunized hens
and the fraction is screened using Enzyme-Linked ImmunoSorbent
Assays (ELISA) for binding to the original peptide target. Each IgY
production run generates sufficient antibody for hundreds of
thousands of immunodetections. Additional hens can be immunized to
generate further comparable antibody preparations and for pooling
of antibody preparations from multiple hens.
[0042] Members of the target protein family are extracted from a
range of species, separated by SDS-polyacrylamide gel
electrophoresis, electrophoretically transferred to membranes and
immunoblotting is used to characterize the binding of the
antibodies to a range of members of the target protein family.
Antibody preparations with good target affinity but which show
non-specific binding to other proteins are subjected to affinity
purification followed by re-characterization to attempt to improve
specificity.
[0043] The novel antibodies can be applied to detect major proteins
in a range of species, including uncharacterized species, with
confidence that the detection affinity of the antibody is standard
for all denatured members of the target protein family. Therefore a
quantity standard protein from one species or a synthetic quantity
standard can be used for comparative quantitation of members of the
protein family from other species.
DETAILED DESCRIPTION OF INVENTION
[0044] Peptide sequence tags designed for eliciting production of
global antibodies binding all members of defined protein families
or subfamilies.
[0045] In the following, all peptides are written according to
convention from amino terminus to carboxy terminus using the
standard single letter amino acid code. All peptides are based on
alignments of protein sequences accessed through the NCBI
(ncbi.nlm.nih.gov) and SwissProt (expasy.ch) public databases.
Where present a lower-case "c" indicates a terminal cysteine not
present in the original protein family but added for chemical
coupling to the immunogenic carrier molecule, usually Keyhole
Limpet Hemocyanin. An upper case terminal "C" represents a cysteine
present in the original protein, but also used for chemical
coupling to the immunogenic carrier molecule.
[0046] 1. PsbA: EVMHERNAHN FPLDc (SEQ ID NO:1) Photosystem II is
the ultimate source of almost all biosynthetic reductant in the
biosphere. The PsbA (D1) protein of Photosystem II is rapidly
cycled under illumination in all oxygenic photobionts (Aro et al.,
1993). Disruptions of PsbA cycling or losses of PsbA pools are
central to loss of Photosystem II function and consequent
photoinhibition of photosynthesis in cyanobacteria, algae and
plants under a wide range of conditions including excess light, low
temperature and UV exposure (e.g. Bouchard et al., 2002; Campbell
et al., 1998). Tracking PsbA pools using the global PsbA antibody
elicited by the PsbA peptide sequence tag can show the functional
content of Photosystem II in a wide range of samples.
[0047] This PsbA peptide sequence tag is absolutely conserved in
the PsbA proteins from almost all known oxygenic photoautotrophs,
with only minor variants found in some liverworts. The global
antibody raised against this PsbA peptide sequence tag has to date
been demonstrated to specifically recognize the PsbA protein from a
wide range of species including plants, red algae, cyanobacteria,
green algal lichens and a mixed natural phytoplankton community.
For example the antibody is being applied to a biological
oceanography project to study UV acclimation in natural
phytoplankton at sites from the Arctic to the Antarctic (Bouchard
et al., 2002), and also to a study of seasonal acclimation in
lichens (Schofield et al., 2002)
[0048] 2. RbcL: CLRGGLDFTK DDENINS (SEQ ID NO:2) RbcL (RUBISCO) is
the catalytic subunit of the primary carbon dioxide fixation enzyme
in the biosphere and is present in all photobionts, along with many
other prokaryotic organisms that fix carbon through chemoautotropic
mechanisms. The kinetic properties of RbcL are well characterized
and the activity of RbcL limits total carbon dioxide uptake by many
communities (e.g. Badger & Andrews, 1987; von Cammerer &
Quick, 2001). The enzyme has a low turnover rate (low kcat) but
because the total flux of carbon fixation through the enzyme is
large in photosynthetic organisms, the enzyme accumulates to high
concentrations (e.g. 5-10% of extractable protein in
cyanobacteria). It is thus a major sink for nitrogen and protein
resources in photosynthetic organisms, and is indeed the most
abundant protein on earth and a major protein source in the human
diet, either directly through consumption of green plants or
through contributions to forage feed for animals. Quantitating RbcL
thus shows the total capacity for carbon uptake in a sample or
community. This RbcL peptide sequence tag is diagnostic of the Type
I sub-class of RUBISCO found in almost all oxygenic
photoautotrophic organisms with the exception of dinoflagellates
and the marine prochlorophyte Prochlorococcus. This RbcL peptide
sequence tag is absolutely conserved in all known sequences from
cyanobacteria, green algae, liverworts, mosses, conifers, eudicots,
and monocots. The RbcL peptide sequence tag is conserved perfectly
in some species, but shows minor variants in some species of ferns,
euglenoids, gamma-proteobacteria, beta-proteobacteria,
alpha-proteobacteria. It is present but imperfectly conserved in
red algae, diatoms, cryptomonads, haptophytes and brown algae. The
global antibody raised against this RbcL peptide sequence tag has
to date been demonstrated to specifically recognize the RbcL
protein from a wide range of species including cyanobacteria, green
algal lichens, various plants and a mixed phytoplankton community
dominated by diatoms.
IN THE DRAWING
[0049] The accompanying drawing shows the results of an immunoblot
chemiluminescent detection of RbcL protein in total protein
extracts from (a) an elm tree, (b) cyanobacterium (Synechococcus
sp. PCC 7942), (c) marsh grass (Spartina) and (d) mixed population
of marine phytoplankton from the Gulf of St. Lawrence, dominated by
diatoms.
[0050] Total denatured protein extracts from the four samples were
separated by SDS PAGE and electrophoretically transferred to
hydrophobic membrane. The membrane was washed with a 1:4000
dilution of the global RbcL IgY antibody fraction (non-affinity
purified) using standard immunoblotting procedures and solutions
(Ausubel et al., 1997). The Global RbcL antibody was then detected
using a commercial secondary goat anti-chicken IgY antibody
conjugated to a horse radish peroxidase enzyme label. Finally, the
areas with bound horse radish peroxidase were detected using ECL+
(Amersham Pharmacia) chemiluminescent.
[0051] The drawing illustrates the broad detection range and
examples of the three main utilities of the new global antibodies
raised against peptide sequence tags; namely (a) detection of a
major protein from organisms (elm and Spartina) which are
relatively uncharacterized at the molecular level but which are of
ecological interest, (b) detection of the same protein from a
widely studied model species, the cyanobacterium Synechcoccus, and
(c) detection of the same protein family from a mixed phytoplankton
community.
[0052] Application (c) is part of a study of natural phytoplankton
responses to changing UVB (Bouchard et al., 2002), where both the
absolute level of the target protein and the community structure
change under UVB exposure, necessitating an antibody with even
detection efficiencies for all members of the target protein
family.
[0053] 3. GlnA: cTNSYKRLVP G (SEQ ID NO:3) GlnA or glutamine
synthetase is the primary point for assimilation of inorganic
ammonia nitrogen into the biosphere. During nitrogen assimilation
all nitrogen sources are converted to ammonia, no matter what the
original source, and then assimilated predominately via the
activity of glutamine synthetase. Thus tracking levels of glutamine
synthetase shows the metabolic capacity of a sample or community
for total nitrogen assimilation.
[0054] This GlnA peptide sequence tag shows perfect to high
conservation in alpha, beta and gamma proteobacteria,
enterobacteria, most cyanobacteria, thermotogales, low GC gram+,
euryarchaeotes and crenarchaeotes. It shows moderate conservation
with: aquificales, high GC gram+ (Streptomyces) and Trichodesmium
thiebautii (a marine cyanobacteria).
[0055] The GlnA peptide sequence tag shows weak and sporadic
conservation with glutamine synthetase Type III (GlnN) and with
some glutaminyl-tRNA synthetases (glutamme-tRNA ligase) (GLNRS),
but antibodies raised against this peptide sequence tag are not
expected to detect these enzymes. This peptide sequence tag shows
no conservation with any eukaryotic GlnA, and therefore does not
react with glutamine synthetases from eukaryotic sources. The
global antibody raised against this GlnA peptide sequence tag has
to date been demonstrated to specifically recognize the GlnA
protein from several species of cyanobacteria.
[0056] 4. NifH: VESGGPEPGV GC (SEQ ID NO: 4) The NifH subunit is a
component of the unstable nitrogenase enzyme system responsible for
biological fixation of N.sub.2 to assimilable ammonia. Levels of
the NifH protein can be used to track the total potential metabolic
capacity for nitrogen fixation in any sample or community. This
NifH peptide sequence tag is perfectly or near-perfectly conserved
in NifH proteins from all known organisms including: alpha, gamma,
beta proteobacteria, enterobacteria, cyanobacteria, low GC gram+
bacteria, high GC gram+bacteria, euryarchaeotes.
[0057] 5. PsaA: CHFSWKMQSD VW (SEQ ID NO: 5) PsaA is a core subunit
of Photosystem I, a key complex involved in transduction of light
to chemical energy in all oxygenic photobionts. Photosystem I
participates in both linear and cyclic electron transport in
photoautotrophic organisms. The molar ratio between Photosystem II
and Photosystem I varies widely between taxa and under different
environmental conditions (Falkowski & Raven, 1997), and is an
important factor for inferring the acclimation state and
photosynthetic performance of an organism or a community. This PsaA
peptide sequence tag is specific to the sequence of the PsaA core
protein of Photosystem I from all known photoautotrophic organisms,
with the exception of a single amino acid mismatch at the third
position in the dinoflagellate Heterocapsa triquetra
[0058] 6. NirB: HWTGCPNSC (SEQ ID NO: 6) NirB or nitrite reductase
catalyzes the reduction of nitrite to ammonia, which is an
obligatory intermediary step in assimilation of inorganic nitrate
into the biosphere. Nitrate is the dominant source of inorganic
nitrogen supporting primary productivity in most ecosystems and
hence tracking NirB levels show the metabolic capacity for
assimilation of this key nitrogen source involved in
eutrophication, agricultural run-off and stimulation of algal
blooms including harmful (toxic) algal blooms.
[0059] 7. RbcL185: KPKLGLSc (SEQ ID NO: 7) This peptide sequence
tag is conserved in both Type I and Type II RbcL and hence can be
applied to raise antibodies that will recognize both classes of
RUBISCO enzyme, including the RUBISCO found in dinoflagellates and
the zooxanthellae symbionts of coral.
[0060] 8. RbcL185a: KPKLGLSGKN YGRc (SEQ ID NO: 8) This peptide
sequence tag is conserved in Type I RUBISCO and could be applied to
generate a second anti-RUBISCO antibody for use in ELISA sandwich
assays.
[0061] 9. RbcL115: DLFEEGSc (SEQ ID NO: 9) This peptide sequence
tag is conserved in Type I RUBISCO and could be applied to generate
a second anti-RUBISCO antibody for use in ELISA sandwich
assays.
[0062] 10. NarB: IFAEVGRRLG F (SEQ ID NO: 10) This peptide sequence
tag is specific to the nitrate reductase (NarB) enzyme from
cyanobacteria, a key enzyme in nitrate assimilation
[0063] 11. NifDMo: VSQSLGHHIA ND (SEQ ID NO: 11) This peptide
sequence tag is specific to the NifD subunit of the sub-set of
nitrogenases with an iron/molybdenum-based co-factor (as opposed to
iron/vanadium or pure iron cofactors).
[0064] 12. NifKMo: CTTCMAEVIG DDL (SEQ ID NO: 12) This peptide
sequence tag is specific to the NifK subunit of the sub-set of
nitrogenases with an iron/molybdenum-based co-factor (as opposed to
iron/vanadium or pure iron cofactors).
[0065] 13. NifKMo: CMAEVIGDDL (SEQ ID NO: 13) This peptide sequence
tag is an alternate target specific to the NifK subunit of the
sub-set of nitrogenases with an iron/molybdenum-based co-factor (as
opposed to iron/vanadium or pure iron cofactors).
[0066] 14. PsbA1: GRQWELc (SEQ ID NO: 14) This peptide sequence tag
is specific to cyanobacterial PsbA1, a form of PsbA expressed in
acclimated cyanobacteria, but not in eukaryotic photobionts (plants
and algae). Monitoring this protein can thus track the contribution
of acclimated cyanobacteria to Photosystem II light energy
conversion in a mixed community.
[0067] 15. PsbA2: GREWELc (SEQ ID NO: 15) This peptide sequence tag
is specific to cyanobacterial PsbA2, a form of PsbA expressed only
in cyanobacteria experiencing excitation stress or UVB stress (e.g.
Campbell et al., 1998). Monitoring this protein can thus track when
a cyanobacterial population is under excitation or UVB stress. It
is also specific to the sole constitutive form of PsbA in
eukaryotic photobionts (plants and algae).
[0068] 16. PsaB: FPCDGPGRGG TC (SEQ ID NO: 16) This peptide
sequence tag is specific to the PsaB core protein of Photosystem I,
a key complex involved in transduction of light to chemical energy
in all oxygenic photobionts.
REFERENCES
[0069] Altschul S et al. (2001) http://www.ncbi.nlm.nih.gov/BLAST
[0070] Aro E M et al. (1993) Biochim. Biophys. Acta 1143:113-134
[0071] Ausubel F et al. (1997) Short Protocols in Molecular
Biology, Wiley, New York. [0072] Badger M R, Andrews T J (1987)
Progress in Photosynthesis Research Vol. III. Martinus Nijhoff
Publishers, pp 601-609. [0073] Bouchard J N et al. (2002) American
Society of Photobiology, Quebec, Canada [0074] Bryant D (ed.)
(1994) The Molecular Biology of Cyanobacteria. Kluwer Academic.
[0075] Campbell D et al. (1998) Proceedings of the National Academy
of Sciences of the USA 95: 364-369. [0076] Corpet F (1988) Nucleic
Acids Research 16 (22): 10881-10890. [0077] http://www.expasy.ch
SwissProt public database of annotated protein sequences and
accompanying proteomic analysis tools. [0078] Falkowski P G &
Raven J A (1997) Aquatic Photosynthesis. Blackwell Science. [0079]
http://www.ncbi.nlm.nih.gov Searches for the target protein
families show a range of representatives from different taxonomic
groups, nonetheless sharing key conserved regions and core
biochemical functions. [0080] Orellana M V & Perry M J (1992)
Limnology & Oceanography 478-490 [0081] Schofield S C et al.
(2002) in revision. [0082] Staley J T & Reysenbach A-L (eds.)
(2002) Biodiversity of Microbial Life. Wiley-Liss. [0083] von
Caemmerer, S. & Quick, W. P. (2000) In Photosynthesis:
Physiology and Metabolism, (ed. R. C. Leegood, T. D. Sharkey, and
S. von Caemmerer), Kluwer.
Sequence CWU 1
1
16 1 15 PRT Artificial Sequence Description of Artificial Sequence
Synthetic peptide 1 Glu Val Met His Glu Arg Asn Ala His Asn Phe Pro
Leu Asp Cys 1 5 10 15 2 17 PRT Artificial Sequence Description of
Artificial Sequence Synthetic peptide 2 Cys Leu Arg Gly Gly Leu Asp
Phe Thr Lys Asp Asp Glu Asn Ile 1 5 10 15 Asn Ser 3 11 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
peptide 3 Cys Thr Asn Ser Tyr Lys Arg Leu Val Pro Gly 1 5 10 4 12
PRT Artificial Sequence Description of Artificial Sequence
Synthetic peptide 4 Val Glu Ser Gly Gly Pro Glu Pro Gly Val Gly Cys
1 5 10 5 12 PRT Artificial Sequence Description of Artificial
Sequence Synthetic peptide 5 Cys His Phe Ser Trp Lys Met Gln Ser
Asp Val Trp 1 5 10 6 9 PRT Artificial Sequence Description of
Artificial Sequence Synthetic peptide 6 His Trp Thr Gly Cys Pro Asn
Ser Cys 1 5 7 8 PRT Artificial Sequence Description of Artificial
Sequence Synthetic peptide 7 Lys Pro Lys Leu Gly Leu Ser Cys 1 5 8
14 PRT Artificial Sequence Description of Artificial Sequence
Synthetic peptide 8 Lys Pro Lys Leu Gly Leu Ser Gly Lys Asn Tyr Gly
Arg Cys 1 5 10 9 8 PRT Artificial Sequence Description of
Artificial Sequence Synthetic peptide 9 Asp Leu Phe Glu Glu Gly Ser
Cys 1 5 10 11 PRT Artificial Sequence Description of Artificial
Sequence Synthetic peptide 10 Ile Phe Ala Glu Val Gly Arg Arg Leu
Gly Phe 1 5 10 11 12 PRT Artificial Sequence Description of
Artificial Sequence Synthetic peptide 11 Val Ser Gln Ser Leu Gly
His His Ile Ala Asn Asp 1 5 10 12 13 PRT Artificial Sequence
Description of Artificial Sequence Synthetic peptide 12 Cys Thr Thr
Cys Met Ala Glu Val Ile Gly Asp Asp Leu 1 5 10 13 10 PRT Artificial
Sequence Description of Artificial Sequence Synthetic peptide 13
Cys Met Ala Glu Val Ile Gly Asp Asp Leu 1 5 10 14 7 PRT Artificial
Sequence Description of Artificial Sequence Synthetic peptide 14
Gly Arg Gln Trp Glu Leu Cys 1 5 15 7 PRT Artificial Sequence
Description of Artificial Sequence Synthetic peptide 15 Gly Arg Glu
Trp Glu Leu Cys 1 5 16 12 PRT Artificial Sequence Description of
Artificial Sequence Synthetic peptide 16 Phe Pro Cys Asp Gly Pro
Gly Arg Gly Gly Thr Cys 1 5 10
* * * * *
References