U.S. patent application number 11/870217 was filed with the patent office on 2009-10-15 for membrane scaffold proteins.
This patent application is currently assigned to THE BOARD OF TRUSTEES OF THE UNIVERSITY OF ILLINOIS. Invention is credited to Timothy H. Bayburt, Natanya R. Civjan, Ilia G. Denisov, Stephen James Grimme, Yelena V. Grinkova, Mary A. Schuler, Stephen G. Sligar.
Application Number | 20090257950 11/870217 |
Document ID | / |
Family ID | 34914740 |
Filed Date | 2009-10-15 |
United States Patent
Application |
20090257950 |
Kind Code |
A1 |
Sligar; Stephen G. ; et
al. |
October 15, 2009 |
Membrane Scaffold Proteins
Abstract
The membrane scaffold proteins (MSP) of the present invention
assemble with hydrophobic or partially hydrophobic proteins to form
soluble nanoscale particles which preserve native structure and
function; they are improved over liposomes and detergent micelles,
both in terms of stability and preservation of biological activity
and native conformation. In the presence of phospholipid, MSPs form
nanoscopic phospholipid bilayer disks, with the MSP stabilizing the
particle at the perimeter of the bilayer domain. The particle
bilayer structure allows manipulation of incorporated proteins in
solution or on solid supports, including for use with such
surface-sensitive techniques as scanning probe microscopy or
surface plasmon resonance. The nanoscale particles, which are
robust in terms of integrity and maintenance of biological activity
of incorporated proteins, facilitate pharmaceutical and biological
research, structure/function correlations, structure
determinations, bioseparations, and drug discovery.
Inventors: |
Sligar; Stephen G.; (Urbana,
IL) ; Bayburt; Timothy H.; (Urbana, IL) ;
Schuler; Mary A.; (Urbana, IL) ; Civjan; Natanya
R.; (Urbana, IL) ; Grinkova; Yelena V.;
(Urbana, IL) ; Denisov; Ilia G.; (Urbana, IL)
; Grimme; Stephen James; (Urbana, IL) |
Correspondence
Address: |
GREENLEE WINNER AND SULLIVAN P C
4875 PEARL EAST CIRCLE, SUITE 200
BOULDER
CO
80301
US
|
Assignee: |
THE BOARD OF TRUSTEES OF THE
UNIVERSITY OF ILLINOIS
Urbana
IL
|
Family ID: |
34914740 |
Appl. No.: |
11/870217 |
Filed: |
October 10, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11033489 |
Jan 11, 2005 |
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11870217 |
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10465789 |
Jun 18, 2003 |
7083958 |
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11033489 |
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09990087 |
Nov 20, 2001 |
7048949 |
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10465789 |
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60536281 |
Jan 13, 2004 |
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60252233 |
Nov 20, 2000 |
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Current U.S.
Class: |
424/1.69 ;
424/178.1; 424/499; 424/9.1; 514/1.1; 514/23; 514/254.05; 514/31;
514/44R; 514/773 |
Current CPC
Class: |
A61P 35/00 20180101;
A61P 31/12 20180101; A61P 43/00 20180101; C07K 14/47 20130101; C07K
14/775 20130101 |
Class at
Publication: |
424/1.69 ;
424/499; 514/773; 514/12; 514/2; 514/44.R; 514/23; 514/31;
514/254.05; 424/178.1; 424/9.1 |
International
Class: |
A61K 51/08 20060101
A61K051/08; A61K 9/14 20060101 A61K009/14; A61K 47/42 20060101
A61K047/42; A61K 38/16 20060101 A61K038/16; A61K 38/02 20060101
A61K038/02; A61K 31/7052 20060101 A61K031/7052; A61K 31/70 20060101
A61K031/70; A61K 31/7048 20060101 A61K031/7048; A61K 31/497
20060101 A61K031/497; A61K 39/395 20060101 A61K039/395; A61K 49/00
20060101 A61K049/00; A61P 43/00 20060101 A61P043/00 |
Goverment Interests
ACKNOWLEDGMENT OF FEDERAL RESEARCH SUPPORT
[0002] This invention was made, at least in part, with funding from
the National Institutes of Health (Grant Nos. R21 GM63574, R01
GM50007, R01 GM31756, R01 GM33775, and 5F32GM19024) and the
National Science Foundation (Grant No. MCB 01-15068). Accordingly,
the United States Government has certain rights in this invention.
Claims
1. A nanoscale particle comprising at least one phospholipid, a
membrane scaffold protein and an additional hydrophobic or
partially hydrophobic molecule.
2. The nanoscale particle of claim 1, wherein the additional
hydrophobic or partially hydrophobic molecule is a therapeutic
molecule, and wherein the membrane scaffold protein is an
artificial membrane scaffold protein.
3. The nanoscale particle of claim 2, wherein the at least one
phospholipid is phosphatidyl choline, phosphatidyl ethanolamine,
phosphatidyl inositol, dipalmitoyl-phosphatidylcholine, dimyristoyl
phosphatidyl choline, 1-palmitoyl-2-oleoyl-phosphatidyl choline,
1-palmitoyl-2-oleoyl-phosphatidyl serine,
1-palmitoyl-2-oleoyl-phosphatidyl ethanolamine, dihexanoyl
phosphatidyl choline, dipalmitoyl phosphatidyl ethanolamine,
dipalmitoyl phosphatidyl inositol, dimyristoyl phosphatidyl
ethanolamine, dimyristoyl phosphatidyl inositol, dihexanoyl
phosphatidyl ethanolamine, dihexanoyl phosphatidyl inositol,
1-palmitoyl-2-oleoyl-phosphatidyl ethanolamine and
1-palmitoyl-2-oleoyl-phosphatidyl inositol.
4. The nanoscale particle of claim 2, wherein the therapeutic
molecule is an antimicrobial agent, an antineoplastic agent, an
angiogenic factor, a thrombolytic agent, a calcium channel blocker,
an antiatherogenic agent, an antihypertensive agent, an inotropic
agent, an anti-inflammatory agent, an antiarrythmia agent, an
antiviral agent, an antifungal agent, an anticoagulant, an
anti-restenosis agent, a therapeutic protein, a photodynamic agent,
a therapeutic peptide, a therapeutic nucleic acid molecule, a
therapeutic carbohydrate or at least one vitamin.
5. The nanoscale particle of claim 4, wherein said antimicrobial
agent is Amphotericin B or ketoconazole.
6. The nanoscale particle of claim 2, wherein said membrane
scaffold protein comprises an amino acid sequence as set forth in
SEQ ID NO: 6, SEQ ID NO:9, SEQ ID NO:17, amino acids 13-414 of SEQ
ID NO:17, SEQ ID NO:19, amino acids 13-422 of SEQ ID NO:19, SEQ ID
NO:23, amino acids 13-168 of SEQ ID NO:23, SEQ ID NO:29, amino
acids 13-168 of SEQ ID NO:29, SEQ ID NO:43, amino acids 13-201 of
SEQ ID NO:43, SEQ ID NO:44, amino acids 13-201 of SEQ ID NO:44, SEQ
ID NO:45, amino acids 13-392 of SEQ ID NO:45, SEQ ID NO:73, amino
acids 13-234 of SEQ ID NO:73, SEQ ID NO:74, amino acids 13-256 of
SEQ ID NO:74, SEQ ID NO:75, amino acids 13-278 of SEQ ID NO:75, SEQ
ID NO:76, amino acids 24-223 of SEQ ID NO:76, SEQ ID NO:77, SEQ ID
NO:78, amino acids 24-212 of SEQ ID NO:78, SEQ ID NO:79, SEQ ID
NO:80, amino acids 24-201 of SEQ ID NO:80, SEQ ID NO:81, amino
acids 13-168 of SEQ ID NO:81, SEQ ID NO:82, amino acids 13-168 of
SEQ ID NO:82, SEQ ID NO:83, amino acids 13-190 of SEQ ID NO:83, SEQ
ID NO:84, amino acids 13-201 of SEQ ID NO:84, SEQ ID NO:85, amino
acids 13-190 of SEQ ID NO:85, SEQ ID NO:86, amino acids 24-381 of
SEQ ID NO:86, SEQ ID NO:91, amino acids 24-201 of SEQ ID NO:91, SEQ
ID NO:92, amino acids 24-190 of SEQ ID NO:92, SEQ ID NO:93, amino
acids 24-179 of SEQ ID NO:93, SEQ ID NO:94, amino acids 24-289 of
SEQ ID NO:94, SEQ ID NO:95, amino acids 24-289 of SEQ ID NO:94, SEQ
ID NO:95, amino acids 24-278 of SEQ ID NO:95, SEQ ID NO:96, amino
acids 24-423 of SEQ ID NO:96, SEQ ID NO:97, amino acids 24-199 of
SEQ ID NO:97, SEQ ID NO:98, amino acids 24-401 of SEQ ID NO:98, SEQ
ID NO:99, amino acids 24-392 of SEQ ID NO:99, SEQ ID NO:111, amino
acids 24-397 of SEQ ID NO:111, SEQ ID NO:113, amino acids 24-383 of
SEQ ID NO:113, SEQ ID NO:115, amino acids 24-379 of SEQ ID NO:115,
SEQ ID NO:117, amino acids 24-381 of SEQ ID NO:117, SEQ ID NO:119,
amino acids 13-1094 of SEQ ID NO:119, SEQ ID NO:129, amino acids
25-214 of SEQ ID NO:129, SEQ ID NO:131, amino acids 25-212 of SEQ
ID NO:131, SEQ ID NO:133, amino acids 25-212 of SEQ ID NO:133, SEQ
ID NO:135 and amino acids 13-212 of SEQ ID 135.
7. The nanoscale particle of claim 2, further comprising a
targeting agent which specifically binds to a surface of a cell to
which the therapeutic agent is to be delivered.
8. The nanoscale particle of claim 7, wherein said targeting agent
is a lectin, single chain antibody or an antigen-binding fragment
of an antibody.
9. The nanoscale particle of claim 8, wherein the targeting agent
is covalently linked to said membrane scaffold protein.
10. The nanoscale particle of claim 8, wherein a targeting agent is
noncovalently bound to said particle.
11. The nanoscale particle of claim 1, wherein the additional
hydrophobic or partially hydrophobic molecule is a protein.
12. The nanoscale particle of claim 11, wherein the protein is a
membrane receptor protein.
13. The nanoscale particle of claim 12, wherein the membrane
receptor protein is a G protein coupled receptor.
14. The nanoscale particle of claim 13, wherein the G protein
coupled receptor is a 5-hydroxytryptamine receptor, a beta
adrenergic receptor, a chemokine receptor, a lysophosphatidic acid
receptor or a dopamine receptor.
15. The nanoscale particle of claim 14, wherein the chemokine
receptor is a CXCR4 receptor or CCR5 receptor.
16. The nanoscale particle of claim 11, wherein the protein is a
transhydrogenase or an aspartate receptor protein.
17. The nanoscale particle of claim 11, wherein the protein is a
cytochrome P450 protein.
18. The nanoscale particle of claim 11, further comprising an
electron transfer system, cytochrome P450 reductase or cytochrome
b5.
19. The nanoscale particle of claim 2, further comprising
cholesterol or a cholesterol ester.
20. The nanoscale particle of claim 11, wherein said membrane
scaffold protein comprises an amino acid sequence selected from the
group consisting of SEQ ID NO: 6, SEQ ID NO:9, SEQ ID NO:17, amino
acids 13-414 of SEQ ID NO:17, SEQ ID NO:19, amino acids 13-422 of
SEQ ID NO:19, SEQ ID NO:23, amino acids 13-168 of SEQ ID NO:23, SEQ
ID NO:29, amino acids 13-168 of SEQ ID NO:29, SEQ ID NO:43, amino
acids 13-201 of SEQ ID NO:43, SEQ ID NO:44, amino acids 13-201 of
SEQ ID NO:44, SEQ ID NO:45, amino acids 13-392 of SEQ ID NO:45, SEQ
ID NO:73, amino acids 13-234 of SEQ ID NO:73, SEQ ID NO:74, amino
acids 13-256 of SEQ ID NO:74, SEQ ID NO:75, amino acids 13-278 of
SEQ ID NO:75, SEQ ID NO:76, amino acids 24-223 of SEQ ID NO:76, SEQ
ID NO:77, SEQ ID NO:78, amino acids 24-212 of SEQ ID NO:78, SEQ ID
NO:79, SEQ ID NO:80, amino acids 24-201 of SEQ ID NO:80, SEQ ID
NO:81, amino acids 13-168 of SEQ ID NO:81, SEQ ID NO:82, amino
acids 13-168 of SEQ ID NO:82, SEQ ID NO:83, amino acids 13-190 of
SEQ ID NO:83, SEQ ID NO:84, amino acids 13-201 of SEQ ID NO:84, SEQ
ID NO:85, amino acids 13-190 of SEQ ID NO:85, SEQ ID NO:86, amino
acids 24-381 of SEQ ID NO:86, SEQ ID NO:91, amino acids 24-201 of
SEQ ID NO:91, SEQ ID NO:92, amino acids 24-190 of SEQ ID NO:92, SEQ
ID NO:93, amino acids 24-179 of SEQ ID NO:93, SEQ ID NO:94, amino
acids 24-289 of SEQ ID NO:94, SEQ ID NO:95, amino acids 24-289 of
SEQ ID NO:94, SEQ ID NO:95, amino acids 24-278 of SEQ ID NO:95, SEQ
ID NO:96, amino acids 24-423 of SEQ ID NO:96, SEQ ID NO:97, amino
acids 24-199 of SEQ ID NO:97, SEQ ID NO:98, amino acids 24-401 of
SEQ ID NO:98, SEQ ID NO:99, amino acids 24-392 of SEQ ID NO:99, SEQ
ID NO:111, amino acids 24-397 of SEQ ID NO:11, SEQ ID NO:113, amino
acids 24-383 of SEQ ID NO:113, SEQ ID NO:115, amino acids 24-379 of
SEQ ID NO:115, SEQ ID NO:117, amino acids 24-381 of SEQ ID NO:117,
SEQ ID NO:119, amino acids 13-1094 of SEQ ID NO:119, SEQ ID NO:129,
amino acids 25-214 of SEQ ID NO:129, SEQ ID NO:131, amino acids
25-212 of SEQ ID NO:131, SEQ ID NO:133, amino acids 25-212 of SEQ
ID NO:133, SEQ ID NO:135 and amino acids 13-212 of SEQ ID
NO:135.
21. The nanoscale particle of claim 11, said membrane scaffold
protein comprising an amino acid sequence of SEQ ID NO: 6, SEQ ID
NO:9, SEQ ID NO:17, amino acids 13-414 of SEQ ID NO:17, SEQ ID
NO:19, amino acids 13-422 of SEQ ID NO:19, SEQ ID NO:23, amino
acids 13-168 of SEQ ID NO:23, SEQ ID NO:29, amino acids 13-168 of
SEQ ID NO:29, SEQ ID NO:43, amino acids 13-201 of SEQ ID NO:43, SEQ
ID NO:44, amino acids 13-201 of SEQ ID NO:44, SEQ ID NO:45, amino
acids 13-392 of SEQ ID NO:45, SEQ ID NO:73, amino acids 13-234 of
SEQ ID NO:73, SEQ ID NO:74, amino acids 13-256 of SEQ ID NO:74, SEQ
ID NO:75, amino acids 13-278 of SEQ ID NO:75, SEQ ID NO:76, amino
acids 24-223 of SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, amino
acids 24-212 of SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, amino
acids 24-201 of SEQ ID NO:80, SEQ ID NO:81, amino acids 13-168 of
SEQ ID NO:81, SEQ ID NO:82, amino acids 13-168 of SEQ ID NO:82, SEQ
ID NO:83, amino acids 13-190 of SEQ ID NO:83, SEQ ID NO:84, amino
acids 13-201 of SEQ ID NO:84, SEQ ID NO:85, amino acids 13-190 of
SEQ ID NO:85, SEQ ID NO:86, amino acids 24-381 of SEQ ID NO:86, SEQ
ID NO:91, amino acids 24-201 of SEQ ID NO:91, SEQ ID NO:92, amino
acids 24-190 of SEQ ID NO:92, SEQ ID NO:93, amino acids 24-179 of
SEQ ID NO:93, SEQ ID NO:94, amino acids 24-289 of SEQ ID NO:94, SEQ
ID NO:95, amino acids 24-289 of SEQ ID NO:94, SEQ ID NO:95, amino
acids 24-278 of SEQ ID NO:95, SEQ ID NO:96, amino acids 24-423 of
SEQ ID NO:96, SEQ ID NO:97, amino acids 24-199 of SEQ ID NO:97, SEQ
ID NO:98, amino acids 24-401 of SEQ ID NO:98, SEQ ID NO:99, amino
acids 24-392 of SEQ ID NO:99, SEQ ID NO:111, amino acids 24-397 of
SEQ ID NO:111, SEQ ID NO:113, amino acids 24-383 of SEQ ID NO:113,
SEQ ID NO:115, amino acids 24-379 of SEQ ID NO:115, SEQ ID NO:117,
amino acids 24-381 of SEQ ID NO:117, SEQ ID NO:119, amino acids
13-1094 of SEQ ID NO:119, SEQ ID NO:129, amino acids 25-214 of SEQ
ID NO:129, SEQ ID NO:131, amino acids 25-212 of SEQ ID NO:131, SEQ
ID NO:133, amino acids 25-212 of SEQ ID NO:133, SEQ ID NO:135 and
amino acids 13-212 of SEQ ID NO:135, wherein said sequence is
modified by from one to five conservative amino acid, an insertion
of from one to 5 amino acids or a deletion of from one to five
amino acids.
22. The nanoscale particle of claim 1, wherein the hydrophobic or
partially hydrophobic molecule is a lipophilic dye or a fluorescent
dye.
23. The nanoscale particle of claim 1, wherein the hydrophobic or
partially hydrophobic molecule is an imaging agent.
24. The nanoscale particle of claim 23, wherein the imaging agent
comprises gadolinium, technetium, tellurium, iridium or iodine.
25. The method of claim 1, wherein the molar ratio of
MSP:solubilizing agent:membrane lipid is from 1:25:50 to
1:2000:1000.
26. The method of claim 25, wherein the molar ratio of
MSP:solubilizing agent:membrane lipid is 1:75:150.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Division of U.S. patent application
Ser. No. 11/033,489, filed Jan. 11, 2005, which is a
Continuation-in-Part of U.S. patent application Ser. No.
10/465,789, filed Jun. 18, 2003, which is a Continuation-in-Part of
U.S. patent application Ser. No. 09/990,087, filed Nov. 20, 2001,
which claims benefit of U.S. Provisional Application No.
60/252,233, filed Nov. 20, 2000, and the present application claims
benefit of U.S. Provisional Application 60/536,281, filed Jan. 13,
2004. All prior applications are incorporated by reference in their
entireties to the extent there is no inconsistency with the present
disclosure.
BACKGROUND OF THE INVENTION
[0003] The field of the present invention encompasses molecular
biology and membrane technology. Specifically, the present
invention relates to membrane scaffold proteins (MSPs), especially
artificial MSPs, and methods of using membrane scaffold proteins to
stabilize, disperse and solubilize fully or partially hydrophobic
proteins including but not limited to tethered, embedded or
integral membrane proteins while maintaining the biological
activities of those proteins or to stabilize, disperse and
solubilize proteins which are purified and chemically solubilized,
or directly from solubilized membrane fragments or membranes into a
mimic of the native membrane environment. The hydrophobic proteins
associate with the membrane scaffold proteins to form nanoscale
disc-like structures termed Nanodiscs herein.
[0004] Several years ago we pursued structural and functional
studies of lipids complexed with apolipoproteins (prepared from
human plasma) and characterized these molecular assemblies by
scanning probe microscopy, for example, using the adsorption of
synthetic high density lipoprotein disks (rHDL, apo A-I) onto mica
in an oriented manner (Carlson et al., 1997; Bayburt et al., 1998;
Bayburt et al., 2000; Carlson et al., 2000). The diameters of the
discoidal structures observed are approximately 10 nm with a height
of 5.5 nanometers. The 5.5 nm high topology observed is most
compatible with a single membrane bilayer epitaxially oriented on
the atomically flat mica surface (Carlson et al., 1997).
[0005] We subsequently discovered that purified membrane proteins
can be reconstituted into the phospholipid bilayer domain of
certain such discoidal structures and studied in solution or
subsequently adsorbed on a suitable surface for examination by
structural or spectroscopic techniques that take advantage of a
surface of oriented protein-bilayer assemblies. In the latter case,
the underlying discoidal structures containing the membrane protein
are easily recognizable and provide a point of reference for
judging the quality of the sample and images.
[0006] High-density lipoproteins (HDL) are spherical assemblies of
a protein component, termed apo A-I, and various phospholipids. HDL
particles play an important role in mammalian cholesterol
homeostasis by acting as the primary conduit for reverse
cholesterol transport (Fielding and Fielding, 1991). The function
of HDL as a cholesterol transporter relies upon the activity of the
HDL-associated enzyme lecithin-cholesterol acyl transferase, or
LCAT (Glomset, 1968; Jonas, 1991), which mediates the insertion of
cholesterol esters into HDL lipoprotein particles. Certain portions
of the apo A-I protein are required for the activity of this enzyme
(Holvoet et al., 1995). In addition, a part of the apo A-I protein
is thought to be in a globular domain at the N-terminus, and to be
responsible for interactions with cell surface receptors. One
nascent form of HDL particles has been assumed to be that of a
discoid based on electron microscopy of stained preparations (Forte
et al., 1971). Our laboratory has confirmed this using atomic force
microscopy (AFM) studies of synthetic forms of rHDL under aqueous
conditions. This form, however, is not the predominant form in
circulation in vivo. Rather, the apo A-I sequence appears to have
evolved to stabilize the more prevalent spherical structural
form.
[0007] Two general models for the nascent structure of HDL disks
have been proposed. One model has the apo A-I protein surrounding a
circular bilayer section as a horizontal band or "belt" composed of
a curving segmented alpha helical rod (Wlodawer et al., 1979). The
other picket fence model has the protein traversing the edges of
the bilayer vertically in a series of helical segments (Boguski et
al., 1986). Both models are based primarily on indirect
experimental evidence, and no three dimensional structure of the
entire particle is available to distinguish between them.
[0008] The currently accepted model is the belt model, which is
consistent with some electron microscopy and neutron scattering
data (Wlodawer et al., 1979), where the helices are arranged
longitudinally around the edge of the bilayer disks like a "belt"
(Segrest et al. 1999). More recent infrared spectroscopy studies
using a new method of sample orientation for dichroism measurements
are more consistent with the belt model, in contrast to earlier
studies (Wald et al., 1990; Koppaka et al., 1999). So far, there is
no complete and direct evidence as to which model is correct, even
though a low resolution x-ray crystal structure for apo A-I
crystallized without lipid (Borhani et al., 1997) has been
obtained. The low resolution crystal structure of an N-terminally
truncated apo A-I shows a unit cell containing a tetrameric species
composed of 4 helical rods which bend into a horseshoe shape and
which combine to give a circular aggregate about
125.times.80.times.40 .ANG.. It was suggested that a dimeric
species in this belt conformation is capable of forming discoidal
particles.
[0009] The information collected to date concerning the reverse
cholesterol transport cycle and the maturation of HDL particles
suggests that the apo A-I protein has unique properties that allow
it to interact spontaneously with membranes resulting in the
formation of lipoprotein particles. Initial apo A-I lipid binding
events have been proposed (Rogers et al., 1998), but the mechanism
for conversion of bilayer-associated forms to discoidal particles
remains unclear. The available evidence suggests that the energy of
stabilization of lipid-free apo A-I is fairly low and that there is
an equilibrium between two conformers (Atkinson and Small, 1986;
Rogers et al., 1998). One conformer may be a long helical rod, and
the other may be a helical "hairpin" structure about half as long.
It has been suggested that the low stabilization energy and
conformational plasticity allow apo A-I to structurally reorganize
when it encounters a lipid membrane, thus facilitating the
structural changes that would have to take place in both the
membrane and the protein to produce discrete lipoprotein particles
(Rogers et al., 1998). Once these particles are formed, apo A-I may
still undergo specific conformational changes that contribute to
the dynamic functionality of the lipoprotein particles and
interaction with enzymes and receptors. All of these interactions
and changes take place at the protein-lipid interface and in
specific topologies providing surface accessibility of critical
residues. Thus, there is little reason to believe that apo A-I
itself would be ideal for generating a stable, nanometer size
phospholipid bilayer of controlled dimension.
[0010] Different types of lipid aggregates are used for
reconstitution and study of purified membrane proteins; these
include membrane dispersions, detergent micelles and liposomes
(FIG. 1). Purified systems for biochemical and physical study
require stability, which is not always inherent in or is limiting
in these systems. Liposomes are closed spherical bilayer shells
containing an aqueous interior. Reconstitution into liposomes by
detergent dialysis or other methods typically results in random
orientation of the protein with respect to outer and lumenal
spaces. Since ligands or protein targets are usually hydrophilic or
charged, they cannot pass through the liposomal bilayer as depicted
in FIG. 1, although this may be advantageous in some instances.
Since both sides of the liposomal bilayer are not accessible to
bulk solvent, coupling effects between domains on opposite sides of
the bilayer are difficult to study. Liposomes are also prone to
aggregation and fusion and are usually unstable for long periods or
under certain physical manipulations, such as stopped flow or
vigorous mixing. The size of liposomes obtained by extruding
through defined cylindrical pore sizes polydisperse in size
distribution rather than exhibiting a uniform diameter.
[0011] Liposomes also may present difficulties due to light
scattering, and aggregation of membrane proteins present in the
bilayer and thermodynamic instability (Angrand et al., 1997;
Savelli et al., 2000). The surface area of a liposome is relatively
large (10.sup.5 to 10.sup.8 .ANG..sup.2). To obtain liposomes with
single membrane proteins incorporated requires a large lipid to
protein molar ratio.
[0012] Detergent micelles allow solubilization of membrane proteins
by interaction with the membrane-embedded portion of the protein
and are easy to use. Detergent micelles are dynamic and undergo
structural fluctuations that promote subunit dissociation and often
present difficulty in the ability to handle proteins in dilute
solutions. An excess of detergent micelles, however, is necessary
to maintain the protein in a non-aggregated and soluble state.
Detergents can also be denaturing and often do not maintain the
properties found in a phospholipid bilayer system. Specific
phospholipid species are often necessary to support and modulate
protein structure and function (Tocanne et al., 1994). Thus, the
structure, function, and stability of detergent solubilized
membrane proteins may be called into question. Since an excess of
detergent micelles is needed, protein complexes can dissociate
depending on protein concentration and the detergent protein ratio.
By contrast, the MSP nanostructures of the present invention are
more robust structurally, having a phospholipid bilayer mimetic
domain of discrete size and composition and greater stability and
smaller surface area than unilamellar liposomes. The disk
structures allow access to both sides of the bilayer like
detergents, and also provide a bilayer structure like that of
liposomes.
[0013] There is a long felt need in the art for stable, defined
compositions for the dispersion of membrane proteins and other
hydrophobic or partially hydrophobic proteins, such that the native
activities and properties of those proteins are preserved.
Compounds other than proteins can also be dispersed in the
nanoscale particles of the present invention.
SUMMARY OF THE INVENTION
[0014] Membrane Scaffold Proteins (MSPs) as used herein are
artificial amphiphilic proteins which self-assemble with
phospholipids and phospholipid mixtures into nanometer size
membrane bilayers. A subset of these nanometer size assemblies are
discoidal in shape, and are referred to as Nanodisc structures.
These nanoscale particles can be from about 5 to about 500 nm,
about 5 to about 100 nm, or about 5 to about 20 nm in diameter.
These structures comprising phospholipid and MSP preserve the
overall bilayer structure of normal membranes but provide a system
which is both soluble in solution and which can be assembled or
affixed to a variety of surfaces.
[0015] The amino acid sequences of specifically exemplified MSPs
are given in SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:19,
SEQ ID NO:23, SEQ ID NO:29, SEQ ID NO:43, SEQ ID NO:44, SEQ ID
NO:45 and SEQ ID NOs:73-86, SEQ ID NO:91-99, SEQ ID NO:11, SEQ ID
NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:129,
SEQ ID NO:131, SEQ ID NO:133 AND SEQ ID NO:135, and the
corresponding sequences lacking the N-terminal 12 amino acid His
tag or 23 amino acid HisTEV tag portion. Also within the scope of
the present invention are artificial variant MSPs having
conservative acid substitutions, insertions or deletions of up to
five amino acids, or artificial variants having from 70 to 100%
amino acid sequence identity with a specifically exemplified MSP
sequence.
[0016] Within the scope of the present invention are those MSPs
which contain a His-tag and/or a His-tag TEV sequence, as well as
those MSPs which are the result of proteolytic or other cleavage to
remove the "tag" portion of the protein, and which contain at the
N-terminus one or more amino acids derived from the protease
recognition sequence, which may be as specifically exemplified in
the tables herein or with a functional modification (such as the
TEV recognition sequence with either Ser or Gly in the P1'
position, as discussed herein). It is also understood that certain
amino acid substitutions at the P2' position are permitted in the
His TEV-MSPs or the proteolytic cleavage products thereof; for
example, the P2' amino acid can be Ser, Gly, Thr, Ala, Asn, Lys or
Met. MSPs with such substitutions are within the scope of the
present invention. In certain embodiments, naturally occurring
membrane scaffold proteins, such as apolipoprotein A-1, A-II, C-I,
C-II, C-III or E, apolipophorin III, among others, can be used in
place of the artificial MSPs of the present invention (i.e., where
the combination has not been reported in the prior art).
[0017] Methods for recombinantly producing the artificial MSPs are
also within the scope of the present invention. Besides the
specifically exemplified artificial MSPs, there can be additional
helical domains included within the primary structure of an
artificial MSP, for example, those derived from Apo A-II, apo C-I,
apo C-II, apo C-III, apo E, apolipophorin III, myoglobin or
hemoglobin. The numbers and orders of helical building blocks (See
Table 19 for particular examples) can be varied, provided that the
self assembly function of Nanodisc formation is preserved.
[0018] The present invention further provides the use of the
nanometer scale phospholipid bilayer structures or Nanodiscs formed
using MSPs for the incorporation of additional hydrophobic or
partially hydrophobic molecules, including hydrophobic or partially
hydrophobic proteins. Those additional proteins can be solubilized,
for example, with the use of detergent, and the solubilized
proteins can be added to a solution of MSP, with or without
phospholipid(s), and the nanoscale particles self-assemble so that
the MSPs and the additional "target" proteins are incorporated into
a stable and soluble particle. Subsequently, any detergent can be
removed by dialysis or treatment with such agents as ion exchange
resins or macroporous polymeric adsorbent beads, e.g., Biobeads
made of styrene divinylbenzene.
[0019] Detergents (or other solubilizing agents) useful in the
dispersion of MSPs, membrane fragments, membranes or preparations
of purified or partially purified hydrophobic or partially
hydrophobic proteins include, without limitation, cholic acid,
neutralized cholic acid, deoxycholic acid, sodium deoxycholate,
including n-dodecyl-.beta.-D-maltoside,
t-octylphenoxypolyethoxyethanol (Triton X-100, Union Carbide
Chemicals and Plastics Co., Inc.), n-octyl-beta-D-glucopyranoside
(octylglucoside), octaethylene glycol monododecyl ether (C12E8),
nonaethylene glycol monododecyl ether (C12E9), Emulgen 913,
myristoyl sulfobetaine, dihexanoyl phosphatidylcholine, digitonin
and JB3-14. Peptidetergents can be used as well. High hydrodynamic
pressures (from about 200 to about 200,000 atmospheres) can also be
used to solubilize (solvate) hydrophobic or partially hydrophobic
proteins or other molecules.
[0020] Phospholipids which can be used in the Nanodisc assembly
methods of the present invention include, without limitation, PC,
phosphatidyl choline; PE, phosphatidyl ethanolamine, PI,
phosphatidyl inositol; DPPC, dipalmitoyl-phosphatidylcholine; DMPC,
dimyristoyl phosphatidyl choline; POPC,
1-palmitoyl-2-oleoyl-phosphatidyl choline; DHPC, dihexanoyl
phosphatidyl choline, dipalmitoyl phosphatidyl ethanolamine,
dipalmitoyl phosphatidyl inositol; dimyristoyl phosphatidyl
ethanolamine; dimyristoyl phosphatidyl inositol; dihexanoyl
phosphatidyl ethanolamine; dihexanoyl phosphatidyl inositol;
1-palmitoyl-2-oleoyl-phosphatidyl ethanolamine;
1-palmitoyl-2-oleoyl-phosphatidyl inositol; among others. The
phospholipids can contain glycerol backbones or they can include
sphingolipids. Generally, the phospholipid has two saturated fatty
acids of from 6 to 20 carbon atoms with a commonly used head group
exemplified by, but not limited to, phosphatidyl choline,
phosphatidyl ethanolamine and phosphatidyl serine. The head group
can be uncharged, positively charged, negatively charged or
zwitterionic. The phospholipids can be natural (those which occur
in nature) or synthetic (those which do not occur in nature), or
mixtures of natural and synthetic. Desirably the molar ratio of
MSPs to total membrane protein is that which produces about 100 to
about 200 phospholipid molecules in each discoidal structure of
about 10 nm in diameter. Those proteins, found in nature or
associated with the various membrane structures of a living
organism, are solubilized in the MSP supported nanobilayer or
Nanodisc through the process of self-assembly, and the native
structure and activity of the target protein are preserved in these
MSP-supported structures.
[0021] Besides purified or solubilized hydrophobic or partially
hydrophobic proteins, hydrophobic or partially hydrophobic proteins
bound to or within membranes or membrane fragments or disrupted
membranes can be assembled with the MSPs of the present invention,
without the need for pre-purification of the target protein. It is
understood by the skilled artisan that the properties of a
particular phospholipid determine its suitability for a particular
application of a Nanodisc; for example, DPPC, which is gel-like in
consistency, is not an appropriate choice for use in certain
applications. Where membrane proteins are incorporated into
Nanodiscs directly from intact or solubilized membranes or membrane
fragments, the use of MSP1 is preferred over MSP2.
[0022] The MSP supported bilayers or Nanodiscs can be used in
solutions or applied to a number of surfaces, such that the native
structure and ligand binding, antigenic determinants and/or
enzymatic activities of the protein incorporated in the MSP
supported structure are maintained. As specifically exemplified,
the MSP supported structures are affixed to a gold surface, e.g.,
for use in surface plasmon resonance technologies, to a multiwell
plate or to solid surfaces including but not limited to, beads,
magnetic particles, chromatography matrix materials and others.
Solid materials to which the MSPs can be affixed include, but are
not limited to, gold, silicon, polystyrene, quartz, silica, silicon
oxides, silicon nitride, and other simple or complex materials.
Where a polyhistidine sequence (His tag) is retained as part of the
MSPs, the Nanodiscs can be bound to a nickel-NTA-coated surface,
for example. Other oligopeptide tags which mediate binding to a
surface or facilitate purification and which can be fused to a
protein of interest, generally at the N- or C-terminus, by such
techniques include, without limitation, strep-tag (Sigma-Genosys,
The Woodlands, Tex.) which directs binding to streptavidin or its
derivative streptactin (Sigma-Genosys); a glutathione-5-transferase
gene fusion system which directs binding to glutathione coupled to
a solid support (Amersham Pharmacia Biotech, Uppsala, Sweden); a
calmodulin-binding peptide fusion system which allows purification
using a calmodulin resin (Stratagene, La Jolla, Calif.); and a
maltose binding protein fusion system allowing binding to an
amylose resin (New England Biolabs, Beverly, Mass.).
[0023] It is noted that a His or other tag does not interfere with
formation of helical domains and the ability to mediate assembly of
a Nanodisc particle, nor is it required for helix formation and
particle assembly. With appropriate modification of the MSP primary
sequence, the polyhistidine or other tag) portion can be removed by
specific proteolytic cleavage, for example using the Tobacco Etch
Virus protease, where there is cognate recognition sequence between
the tag and the first helical domain of the MSP.
[0024] The present invention further relates to methods for the
incorporation of membrane-associated or other hydrophobic or
partially hydrophobic proteins (or other hydrophobic or partially
hydrophobic molecules) into nanoscale lipid bilayers or Nanodiscs
comprising at least one MSP of the present invention. Membrane
proteins (tethered, embedded or integral) can be used in the
methods of the present invention. These proteins can be
incorporated into nanoscale particles with MSPs from solubilized
intact membrane preparations, intact cells (native or recombinant)
or from disrupted membranes or membrane fragments, without
prepurification or prefractionation of the membrane proteins, or
the proteins can be purified prior to incorporation (with
solubilization if needed).
[0025] Tethered membrane proteins, which are associated with the
membrane bilayer via a relatively small portion of the protein, can
be exemplified by cytochrome P450 reductases and cytochrome b5
proteins from various sources.
[0026] Embedded membrane proteins have a more extensive association
with the bilayer, but typically the bulk of the protein is in
contact with the extracellular environment or the cytoplasm.
Examples of embedded membrane proteins include, without limitation,
the general class of membrane associated cytochromes P450, for
example, cytochrome P450 2B4 from rabbit liver microsomes,
cytochrome P450 3A4 from human liver microsomes and cytochrome P450
6B1 from insect microsomes.
[0027] The integral membrane proteins are exemplified by the
general class of proteins which include helical segments in the
membrane bilayer, such as the 7-helix transmembrane proteins,
including, but not limited to, bacteriorhodopsin (bR) from
Halobacterium halobium, the human .beta.-adrenergic receptor, the
5-hydroxy tryptamine 1A G-protein coupled receptor from Homo
sapiens and other G-protein coupled protein receptors from human,
plant, animal or other sources. In general an integral membrane
protein has at least one portion which extends through the membrane
bilayer. Other examples include, without limitation,
channel-forming proteins, transporter proteins, signaling proteins,
cytokine receptors (e.g., tumor necrosis factor receptors),
interleukin receptors, Fas receptor, CD27, CD40, CD30, insulin and
insulin family receptors, dopamine receptors, the lysophosphatidic
acid receptors, and the chemokine receptors, such as CXCR4 and
CCR5, dopamine receptors, and growth factor receptors (e.g.,
epidermal growth factor and/or HGF receptors). There can be from
one to more than twenty domains of the protein passing through the
membrane bilayer. An example of a one-pass protein which has been
successfully incorporated into the nanoscale particles of the
present invention is the aspartate receptor (Tar) from Escherichia
coli; and an example of a twenty six-pass protein incorporated into
Nanodiscs is an E. coli transhydrogenase. Members of each type of
membrane protein have been successfully incorporated into the
nanoscale structures using the MSPs and methods of the present
invention. In particular, cell surface receptors, and especially
G-protein coupled receptors, including but not limited to,
beta-adrenergic, chemokine and other receptor proteins, can be
incorporated into nanobilayer bilayer structures formed with the
membrane scaffold proteins (MSPs) of the present invention. Where
it is desired that a dimer or higher oligomer of a 7-helix
transmembrane protein is incorporated into a Nanodisc, a Nanodisc
of greater than 9 nm in diameter is preferred, which can be
accomplished by the use of a relatively longer MSP sequence such as
MSP1E1, MSP1E2 or MSP1E3.
[0028] The present invention further provides materials and methods
using artificial or naturally occurring MSPs which increase the
stability and monodispersity of the self-assembled nanoparticles.
G-protein coupled receptors (GPCRs) are an important and diverse
class of pharmaceutical targets in mammalian cellular membranes
where they function as signal transducing elements, bind several
classes of bioactive ligands and transmit information to the
intracellular machinery. The artificial MSPs of the present
invention stabilize and solubilize the membrane-associated form of
GPCRs to allow purification and manipulation in solution or on a
solid support for use in flow cytometry, high throughput screening
applications, on surfaces for surface-plasmon biosensor and
scanning-probe techniques, as well as other analytical
applications. The methods for Nanodisc production of the present
invention can be used to facilitate purification of naturally
produced or recombinant membrane proteins in stable, biologically
active and soluble form.
[0029] Also within the scope of the present invention are methods
for adsorbing or binding a molecule or ion of interest to a protein
(or other molecule) within a Nanodisc, where that protein (or other
molecule) binds the compound or ion of interest with sufficient
affinity so as to promote removal of the compound or ion of
interest from a solution containing it. This application of
Nanodisc technology can be used to remove contaminating materials
or it can be use in separation or purification schemes. Similarly,
Nanodiscs containing MSP and phospholipid can be used to separate
hydrophobic materials from a solution by partitioning of the
hydrophobic material into the phospholipid portion of the Nanodisc
in a relatively nonspecific fashion. By way of nonlimiting example,
lipophilic dyes have been shown to incorporate within Nanodiscs
either during the self assembly process or by partitioning into the
bilayer of the Nanodiscs from a solution.
[0030] The present invention further provides Nanodisc particles
wherein proteins or carbohydrates of interest are attached
(covalently or noncovalently) to the MSPs on the exterior of the
Nanodisc. Alternatively, a carbohydrate or protein can be
covalently bound to an alkane or phospholipid, which is then
incorporated within the Nanodisc such that the carbohydrate is
accessible to the outside, aqueous environment. Carbohydrates can
also be in the form of glycoproteins which are incorporated within
a Nanodisc. Such carbohydrate-carrying Nanodiscs can be used to
positively or negatively modulate cellular responses, either in
vivo or in vitro. This can be used also to direct the Nanodisc to a
cell or surface displaying a lectin or the ligand of a lectin, or a
receptor of the ligand of the receptor, depending on the choice of
the carbohydrate or other molecule carried by the Nanodisc.
Proteins which could be covalently or noncovalently linked to the
Nanodisc include, without limitation, antibodies or antigen-binding
fragments thereof, adhesins or other proteins or glycoproteins
capable of binding to target molecules or cells of interest.
[0031] Yet another aspect of the present invention is the
incorporation of a hydrophobic therapeutic or cosmetic molecule
within the hydrophobic core of the Nanodisc. This strategy can
prolong the circulating lifetime of the compound and it can also
provide the benefits of slow release of a relatively insoluble
and/or toxic molecule. Such a hydrophobic therapeutic can include,
without limitation, photodynamic therapeutic agents such psoralens,
porphyrins and phthalacyanin-related molecules, tamoxifen,
paclitaxel, anticancer agents such as adriamycin, daunorubicin or
doxorubicin, cholesterol-lowering drugs, antibacterial agents such
as vancomycin, fat soluble vitamins such as D or E, and antifungal
agents such as the azoles (e.g., ketoconazole) or polyenes (e.g.,
Amphotericin B). The Nanodiscs into which these compounds have been
incorporated are also within the scope of the present
invention.
[0032] Other molecules which can be incorporated within Nanodiscs
or attached to Nanodiscs (such as by covalent attachment to the
MSP) include antibodies, monoclonal antibodies, antibody fragments
capable of binding to a cognate antigen, lectins, hormones,
chemokines, lymphokines, peptides, lipids, albumin, amino sugars
and lectins, nucleic acids, among others. Nanodiscs of the present
invention can also be used to stabilize and deliver lipophilic
agents which improve the appearance or quality of skin, including,
but not limited to, vitamins A and/or E or retinol. Methods for
improving the skin or for treating disease by administering or
applying an effective amount of a therapeutic or cosmetically
active composition comprising Nanodisc particles into which the
therapeutic or cosmetically active ingredient has been incorporated
are within the scope of the present invention. Such hydrophobic
agents are packaged within Nanodiscs either directly or through
self assembly with the hydrophobic (lipophilic) small molecule.
[0033] Drugs (therapeutic agents) discussed herein are exemplary,
and are not meant to be limiting in any way. Hydrophobic
anti-inflammatory agents include, but are not limited to, any known
hydrophobic non-steroidal antiinflammatory agent, and any known
hydrophobic steroidal antiinflammatory agent, any known
non-steroidal antiinflammatory agent such as salicylic acid
derivatives (aspirin), para-aminophenol derivatives (e.g.,
acetaminophen), indole and indene acetic acids (indomethacin),
heteroaryl acetic acids (ketorolac), arylpropionic acids
(ibuprofen), anthranilic acids (mefenamic acid), enolic acids
(oxicams) and alkanones (nabumetone) and any known steroidal
antiinflammatory agent which can include corticosteriods and
biologically active synthetic analogs with respect to their
relative glucocorticoid (metabolic) and mineralocorticoid
(electrolyte-regulating) activities. Additionally, other drugs used
in the therapy of inflammation or anti-inflammatory agents to be
incorporated into Nanodiscs can include, but are not limited to,
the autocoid antagonists such as all histamine and bradykinin
receptor antagonists, leukotriene and prostaglandin receptor
antagonists, and platelet activating factor receptor
antagonists.
[0034] Antimicrobial agents include, without limitation,
antibacterial agents, antiviral agents, antifungal agents, and
anti-protozoan agents. Non-limiting examples of antimicrobial
agents (antibiotics) are sulfonamides,
trimethoprim-sulfamethoxazole, quinolones, penicillins, and
cephalosporins. Antifungal agents include, without limitation
azoles, and especially Amphotericin B and nystatin. Therapeutic
compounds effective against protozoans can be similarly
incorporated within Nanodiscs. Solubilization, reduction of
potential toxicity and controlled release are advantages.
[0035] Antineoplastic agents include, but are not limited to, those
which are suitable for treating tumors that may be present on or
within an organ (such as carcinoma, sarcoma, hematopoietic cancers,
e.g., myxoma, lipoma, papillary fibroelastoma, rhabdomyoma,
fibroma, hemangioma, teratoma, mesothelioma of the AV node,
lymphoma, and tumors that metastasize to the target organ, among
others) including cancer chemotherapeutic agents, a variety of
which are well known in the art, such as adriamycin, daunorubicin,
doxorubicin, tamoxifen and paclitaxel. Antineoplastic agents can
also include antibodies specific for the neoplastic cell and
antibodies to which a therapeutic radionuclide or other therapeutic
agent has been bound.
[0036] Angiogenic factors (e.g., to promote organ repair or for
development of a biobypass to avoid a thrombosis) include, but are
not limited to, basic fibroblast growth factor, acidic fibroblast
growth factor, vascular endothelial growth factor, angiogenin,
transforming growth factors, tumor necrosis factor, angiopoietin,
platelet-derived growth factor, placental growth factor, hepatocyte
growth factor, and proliferin.
[0037] Thrombolytic (clot dissolving) agents include, but are not
limited to, urokinase, plasminogen activator, urokinase,
streptokinase, inhibitors of .alpha.2-plasmin inhibitor, and
inhibitors of plasminogen activator inhibitor-1, angiotensin
converting enzyme (ACE) inhibitors, spironolactone, tissue
plasminogen activator (tPA), an inhibitor of interleukin
1.beta.-converting enzyme, anti-thrombin III, and the like.
[0038] Where the target organ is the heart, exemplary drugs for
delivery include, but are not necessarily limited to drugs which
are poorly soluble in water, growth factors, angiogenic agents,
calcium channel blockers, antihypertensive agents, inotropic
agents, antiatherogenic agents, anti-coagulants, beta-blockers,
anti-arrhythmia agents, cardiac glycosides, antiinflammatory
agents, antibiotics, antiviral agents and the like.
[0039] Calcium channel blockers include, but are not limited to,
dihydropyridines such as nifedipine, nicardipine, nimodipine, and
the like; benzothiazepines such as dilitazem; phenylalkylamines
such as verapamil; diarylaminopropylamine ethers such as bepridil;
and benzimidole-substituted tetralines such as mibefradil.
Antihypertensive agents include, but are not limited to, diuretics,
including thiazides such as hydroclorothiazide, furosemide,
spironolactone, triamterene, and amiloride; antiadrenergic agents,
including clonidine, guanabenz, guanfacine, methyldopa,
trimethaphan, reserpine, guanethidine, guanadrel, phentolamine,
phenoxybenzamine, prazosin, terazosin, doxazosin, propanolol,
methoprolol, nadolol, atenolol, timolol, betaxolol, carteolol,
pindolol, acebutolol, labetalol; vasodilators, including
hydralizine, minoxidil, diazoxide, nitroprusside; and angiotensin
converting enzyme inhibitors, including captopril, benazepril,
enalapril, enalaprilat, fosinopril, lisinopril, quinapril,
ramipril; angiotensin receptor antagonists, such as losartan; and
calcium channel antagonists, including nifedine, amlodipine,
felodipine XL, isadipine, nicardipine, benzothiazepines (e.g.,
diltiazem), and phenylalkylamines (e.g. verapamil). Anticoagulants
include, but are not limited to, heparin, warfarin, hirudin, tick
anti-coagulant peptide, low molecular weight heparins (such as
enoxaparin, dalteparin, and ardeparin), ticlopidine, danaparoid,
argatroban, abciximab and tirofiban.
[0040] Antiarrhythmic agents include, but are not limited to,
sodium channel blockers (e.g., lidocaine, procainamide, encainide,
flecanide, and the like), beta adrenergic blockers (e.g.,
propranolol), prolongers of the action potential duration (e.g.,
amiodarone), and calcium channel blockers (e.g., verpamil,
diltiazem, nickel chloride, and the like). Delivery of cardiac
depressants (e.g., lidocaine), cardiac stimulants (e.g.,
isoproterenol, dopamine, norepinephrine, etc.) and combinations of
multiple cardiac agents (e.g., digoxin/quinidine to treat atrial
fibrillation) is possible using the Nanodiscs of the present
invention.
[0041] Agents to treat congestive heart failure, include, but are
not limited to, a cardiac glycoside, inotropic agents, a loop
diuretic, a thiazide diuretic, a potassium ion sparing diuretic, an
angiotensin converting enzyme inhibitor, an angiotensin receptor
antagonist, a nitrovasodilator, a phosphodiesterase inhibitor, a
direct vasodilator, an adrenergic receptor antagonist, a calcium
channel blocker, and a sympathomimetic agent. Agents suitable for
treating cardiomyopathies include, but are not limited to,
dopamine, epinephrine, norepinephrine, and phenylephrine.
[0042] Also suitable are agents that prevent or reduce the
incidence of restenosis including, but not limited to, taxol
(paclataxane) and related compounds; and antimitotic agents. Other
compounds that can be incorporated include vitamins A, D and E and
cholesterol-controlling drugs such as the statins.
[0043] Small molecule therapeutic agents can be incorporated into
the nanoscale discoid particles of the present invention. An
advantageous plasma lifetime of Nanodiscs, the rendering of
partially hydrophobic compounds soluble via the amphipathic
membrane scaffold protein encircled Nanodisc, and the ability for
potential targeting through modification of the MSP or phospholipid
components of the Nanodisc, are important advantages provided.
Examples 15-18 provide specific exemplification using Amphotericin
B, ketoconazole and photodynamic agents, but other therapeutic
molecules can be incorporated using the same or similar ratios and
protocols. Similarly, the MSP specifically exemplified is MSP1T2,
but others having the properties taught herein can substitute
therefor.
[0044] Therapeutic Nanodisc compositions are desirably maintained
as stable soluble solutions; solutions of Nanodiscs can also be
lyophilized and stored as dry powders. Administration to a patient
in need of the particular therapeutic compound contained in the
Nanodiscs is preferably by a parenteral route, which can include
intravenous, intraarterial, intramuscular, intradermal,
subcutaneous, or there can be contact with a mucosal surface, for
example by aerosolization (especially dry powder drug-Nanodisc
preparations) and inhalation either intranasally or via the lower
respiratory system, in a dosage sufficient for the intended patient
response.
[0045] Nanodiscs of the present invention can also be used to
stabilize and deliver lipophilic agents which improve the
appearance and/or quality of skin, including but not limited to
vitamin E or retinol. Methods for improving the appearance of skin
or for treating disease are also within the scope of the present
invention. Such hydrophobic agents are packaged within Nanodiscs
either directly or through self-assembly of the lipid and
phospholipid component. Administration is desirably by topical of
an amount of composition comprising Nanodiscs into which the
cosmetically active ingredient(s) has been incorporated application
to an area of skin in need of improvement, in an amount (and at a
frequency) effective for improving the appearance of the skin in
need of improvement.
[0046] The scope of the present invention includes the use of
Nanodiscs which carry a hydrophobic or partially hydrophobic
antigen, which can be a protein, lipopolysaccharide,
lipooligosaccharide or a lipoprotein. Such Nanodiscs can be used in
immunogenic compositions, for example, as vaccine components. Viral
proteins of interest include, without limitation, gp120 of Human
Immunodeficiency Virus, envelope glycoproteins of Herpes simplex
virus or measles virus, the "spike" protein of the SARS virus,
hemagglutinin ligand of influenza virus or parainfluenza virus.
Exemplary bacterial antigens include, but are not limited to, cell
surface proteins such as the M6 protein or M proteins of
Streptococcus pyogenes, fimbrillin of Porphryomonas gingivalis,
InIB or ActA of Listeria monocytogenes, YadA of Yersinia
enterocolitica, IcsA of Shigella flexneri, invasin of Yersinia
pseudotuberculosis, products of the acf gene of Vibrio cholerae,
capsular material comprising the poly-D-glutamate polypeptide of
Bacillus anthracis, fibrinogen/fibrin binding protein of
Staphylococcus aureus, V and/or W antigens of Yersinia pestis
(especially from a vaccine strain such as EV76) or from Yersinia
enterocolytica or Yersinia pseudotuberculosis, and flagellin or
porin of Campylobacter jejeuni. Similarly, O antigens of Salmonella
typhi, Salmonella choleraesuis, Salmonella enteritidis can be
incorporated into nanodiscs, using the proteins and methods
described herein.
[0047] The present invention further provides immunogenic
compositions comprising Nanodiscs into which has been incorporated
at least one hydrophobic or partially hydrophobic antigen, together
with a pharmaceutically acceptable carrier. Optionally an adjuvant
and/or an immune stimulant, such as a chemokine, can be
incorporated into the composition. The Nanodiscs allow the
stabilization and solubilization of a hydrophobic antigen, with the
maintenance of the native conformation of the antigen, and with the
presentation of hydrophilic regions of the antigen exposed to the
aqueous environment, leading to an improved immune response in the
human or animal to which the immunogenic composition has been
administered.
[0048] An additional application of the present Nanodisc technology
is in diagnostic and/or imaging procedures used in medical or
veterinary settings. In this application, a targeting agent is
embedded within or bound to the Nanodisc such that a binding site
is accessible to the aqueous environment and an imaging compound,
such as a dye, a radionuclide or a fluorescent or luminescent
molecule, is incorporated within the Nanodisc. Imaging techniques
useful with the Nanodiscs carrying the appropriate imaging agent,
as well known to the art, can be used in magnetic resonance
imaging, electron paramagnetic resonance imaging, optical imaging
and ultrasound imaging. The binding site of the targeting agent is
specific for a bacterial surface antigen, a tumor antigen, or other
cell surface or tissue-specific marker. The discs are allowed to
assembly from an aqueous mixture comprising imaging agent, MSP and
target-specific protein. As for introduction of therapeutics, the
MSP is desirably antigenically neutral, i.e., it should not trigger
an immunological response within a human or animal to which it is
administered.
[0049] Additional applications of antigen-containing Nanodiscs
include assay kits and methods for the detection of an antibody
specific for the particular antigen in a biological sample.
Detection of the antibody bound to the Nanodisc-bound antigen can
be by any means known to the art. Detection of the antibody in the
biological sample indicates prior exposure of the human or animal
to the antigen of interest, often this approach is used to
recognize exposure to a pathogen. The biological sample can be
blood, serum, plasma or tissue, especially from the lymph
system.
[0050] We have developed Nanodiscs for use in structural,
biochemical and pharmaceutical strategies by engineering the
scaffold protein (MSP) for greater stability, size homogeneity
through various size classes and useful functionalities in the
resultant nanoscale lipoprotein particle. These particles can
include tags for purification, binding to surfaces and physical
manipulation of disks such as in hydrogels or on a gold biosensor
surface, and they can serve as robust entities for rapid and
reproducible assays and solution-based NMR screening and in solid
state NMR structural studies. In NMR applications, the Nanodiscs
provide a stable monodisperse environment for proteins and other
hydrophobic molecules of interest, especially receptor proteins for
which ligand binding is studied. For example, compounds that bind
to the Nanodisc-supported receptor can exhibit broadened signals
and hence, a difference spectrum between +/-target ligand can
reveal the identity of bound ligands. The nanoparticles and
membrane protein scaffolds are useful in biotechnology, the
pharmaceutical industry as well as in basic research. In addition,
the structural and functional principles uncovered through our
discovery and the related techniques facilitate understanding the
interactions of proteins with lipid bilayers at the molecular
level.
[0051] The Nanodiscs can contain a single type of functional
protein, or, where the MSP and the resulting Nanodiscs are large,
they can contain macromolecular assemblies, for example cellular
motility motors (flagella or cilia), multicomponent bioreactors
such as multienzyme complexes, energy transduction complexes, or
photosynthetic complexes. Where the incorporation of macromolecular
assemblies, including a combination of a cytochrome and a reductase
protein, a relatively large MSP is used to prepare the Nanodiscs,
using for example, MSP1 E1, MSP1 E2 or MSP1 E3. Where an MSP larger
than MSP1 is used, the results are improved by the incorporation of
a higher molar ratio of lipid to MSP (from 70:1 to 140:1, or from
90:1 to 115:1).
[0052] Also within the scope of the present invention are complexes
in which more than one Nanodisc particles are associated with one
another. The complexes can have longer time in circulation in a
human or animal than single Nanodisc particles. These associations
of particles can beheld together by electrostatic interactions
(where different portions of the hydrophilic face of the helices
have different charge) or they can be covalently bonded by
disulfide bonds, for example, where the hydrophilic faces of at
least some of the helices of the MSPs contain cysteine residues, or
they can be constructed through genetic engineering of MSP fusion
constructs. The particles in the complex can include therapeutic
compounds, antibodies or imaging compounds. For example, gadolinium
can be incorporated into Nanodiscs for use in ischemia imaging in
humans.
[0053] The present invention further encompasses compositions and
methods useful in detoxification and/or remediation of certain
chemicals, where the appropriate binding protein or enzyme is
incorporated within a Nanodisc in its bioactive conformation. An
example is a cytochrome P450 coding sequence which encodes a
protein capable of oxidizing (and/or dehalogenating) at least one
halogenated hydrocarbon or light hydrocarbon. Examples include, but
are not limited to, trichloroethylene, ethylene dibromide,
chloroform, carbon tetrachloride, styrene, benzene,
1,2-dichloropropane, vinyl chloride, dichloromethane, methyl
chloride, methyl chloroform, 1,2-dichloroethane,
1,2-dichloropropane, perchloroethylene, dichloroethylene, vinyl
bromide, acrylonitrile, vinyl carbonate, ethyl carbamate,
acetaminophen and methyl tertyl-butyl ether. The incorporation of
human cytochrome P450s, either with our without membrane bound
redox transfer partners, into Nanodiscs, in which the overall
stoichiometry and homo- and hetero-oligomerization state can be
controlled, is a significant improvement on the crude membrane
preparations now used by the pharmaceutical industry for
quantitation of drug metabolism, pharmacokinetics, and metabolite
toxicity studies of lead compounds and drug candidates. In such
examples, the Nanodiscs can be used in solution or they can be
covalently or noncovalently bound to a solid support.
BRIEF DESCRIPTION OF THE DRAWINGS
[0054] FIG. 1 schematically illustrates different types of lipid
aggregates incorporating a membrane protein. Small circles and
triangles represent ligand for intracellular and extracellular
domains of the receptor proteins, respectively.
[0055] FIG. 2 shows the wheel structure of an alpha helix, with the
placement of hydrophobic and hydrophilic amino acid side chains
that give the helix its amphipathic character.
[0056] FIG. 3 is a schematic of a belt model of an MSP supported
bilayer. The rectangles represent single helices with a diameter of
about 1.5 nm and a helix length of about 3.9 nm.
[0057] FIGS. 4A-4G illustrate various engineered MSP structures,
shown with picket fence topology and helical assignments based on
sequence analysis. FIG. 4A: MSP1 showing positions of half-repeats.
Half-repeat 1 is disordered based on molecular dynamics simulation
(Phillips, 1997). FIG. 4B: Hinge domain movement. FIG. 4C: Removal
of half-repeats. FIG. 4D: Hinge domain replacement with helices 3
and 4. FIG. 4E: MSP2, with a tandem duplication of the sequence of
MSP1. FIG. 4F: Removal of half-repeat 1 to make MSP1D1. FIG. 4G:
Tandem repeat of MSP1D1 to form MSP2D1.
[0058] FIGS. 5A-5B diagrammatically illustrate the PCR strategy
used to amplify artificial MSPs.
[0059] FIGS. 6A-6B show diagrams of the tandem repeat MSP2 with a
long linker (FIG. 6A) and with a short linker sequence (FIG.
6B).
[0060] FIGS. 7A-7B show the membrane proteins incorporated into
disks and attached to solid supports. FIG. 7A: Disk-associated
receptor and ligand-induced assembly of receptor-target complex on
gold. FIG. 7B: Disk-associated receptor in a gel matrix.
[0061] FIG. 8 is a chromatogram of cytochrome P450 3A4 incorporated
into 10 nm bilayer disks composed of 100% DPPC as phospholipid.
[0062] FIG. 9 illustrates the results of PAGE with sample 1
(Nanodiscs prepared with microsomal membranes from cells
coexpressing cytochrome P450 6B1 and NADPH P450 reductase). Sample
2 was prepared from microsome lacking expression of CYP6B1.
[0063] FIG. 10 provides a characteristic optical spectrum of active
cytochrome P450 6B1 incorporated within Nanodiscs; the
characteristic peak is at 450 nm. Such spectra indicate a correct
thiolate heme ligation and no evidence for the presence of an
inactive P420 form of the cytochrome in the solubilized membrane
bilayer system.
[0064] FIG. 11 depicts a chromatogram of sample separated by a
Superdex sizing column. Retention times indicated rHDL particles 10
nm in size.
[0065] FIG. 12 illustrates co-incorporation of cytochrome P450
reductase and cytochrome P450 6B1 in MSP Nanodiscs. The ratio of
absorbances at 456 nm (predominantly reductase) to that at 420 nm
(predominantly P450) is plotted as a function of retention time.
The peak at about 26 min indicates a Nanodisc population containing
both reductase and cytochrome.
[0066] FIG. 13 illustrates the binding of DPPC Nanodiscs containing
carboxyl terminated thiols to a gold surface, as monitored by
surface plasmon resonance.
[0067] FIG. 14 provides a schematic describing the formation of
nanoscale supported lipid bilayers (Nanodiscs) through
self-assembly. A cell membrane preparation containing the target
membrane protein is solubilized with detergent in the presence of
membrane scaffold protein (MSP) (see herein below). Upon removal of
the detergent, by dialysis or Biobeads J, a soluble MSP-supported
Nanodisc, is formed with the target incorporated into the resulting
phospholipid bilayer.
[0068] FIG. 15 shows the results of one dimensional SDS-PAGE of
Nanodisc mixture. Lanes 1, low molecular weight markers. Lane 2
(left panel), Sf9 insect cell membranes from insect cells
genetically modified for the overexpression of CYP6B1. The band at
55 kDa represents the overexpressed target membrane-bound protein.
Lane 2 (right panel) illustrates the Nanodisc mixture assembled
from Sf9 insect cell membranes overexpressing CYP6B1. MSP1 and
CYP6B1 run at molecular weights of 25 kDa and 55 kDa,
respectively.
[0069] FIGS. 16A-16B show the results of size exclusion
chromatography of Nanodiscs made using MSP1 and containing a
heterologously expressed cytochrome P450, CYP6B1. The target
protein is incorporated into the Nanodisc through the simple
self-assembly process described in the text. FIG. 16A: Chromatogram
showing the size separation of the reconstituted particles
(Superdex J 200). Dotted line shows size separation of a membrane
sample in the absence of MSP showing the presence of high molecular
weight non-specific and non-functional aggregates. FIG. 16B:
Re-chromatogram of the CYP6B1 containing fraction demonstrating the
homogeneity of the self-assembled CYP6B1-bilayer structure.
[0070] FIG. 17 shows the preservation of phospholipid content of
starting membrane preparation in the resulting soluble Nanodisc
bilayers. Vertical bars represent phospholipid type determined from
three replicate samples of starting membranes or self-assembled
Nanodiscs. PC: phosphatidylcholine, PE: phosphatidylethanolamine,
PI: phosphatidylinositol.
[0071] FIG. 18 shows ligand binding to CYP6B1 incorporated into
Nanodisc membrane bilayers with MSP1. The characteristic Type I
binding spectra (decrease in substrate low spin cytochrome with
absorbance at about 417 nm and concomitant increase in the high
spin fraction absorbing at about 390 nm) is obtained in microtiter
plates using high-throughput plate reader following incremental
addition of the environmental furanocoumarin xanthotoxin. A
dissociation constant of roughly 30 .mu.M was calculated.
[0072] FIG. 19 shows the stoichiometry of phospholipid:MSP for
various MSPs. The H1 helix domain does not play a significant role
in the formation of the protein "belt" surrounding the Nanodiscs.
We have found that the sizes of Nanodiscs constructed with MSP1 and
those Nanodiscs constructed with MSPs missing either half or all of
H1 are the same and have the same number of phospholipid molecules
incorporated per Nanodisc.
[0073] FIG. 20 shows size exclusion chromatography elution profiles
for the Nanodiscs self-assembled with: A-MSP1-DPPC; B-MSP1E1-DPPC;
C-MSP1E2-DPPC; D-MSP1E3-DPPC. Curve E shows the elution profile of
the set of calibration proteins: 1-Bovine serum albumin, 2-Bovine
liver catalase, Stokes diameter 10.4 nm; 3-Ferritin, diameter 12.2
nm; 4-Thyroglobulin.
[0074] FIG. 21A shows the inhibition of Candida albicans by
Nanodiscs loaded with ketoconazole and by a solution of
ketoconazole in 1% DMS. There was no inhibition by "empty"
Nanodiscs. FIG. 21B shows that there is no growth inhibition by the
Nanodisc buffer, 1% DMSO or by the lower amount of
ketoconazole.
DETAILED DESCRIPTION OF THE INVENTION
[0075] Abbreviations used in this application include A, Ala,
Alanine; M, Met, Methionine; C, Cys, Cysteine; N, Asn, Asparagine;
D, Asp, Aspartic Acid; P, Pro, Proline; E, Glu, Glutamic Acid; Q,
Gln, Glutamine; F, Phe, Phenylalanine; R, Arg, Arginine; G, Gly,
Glycine; S, Ser, Serine; H, His, Histidine; T, Thr, Threonine; I,
Ile, Isoleucine; V, Val, Valine; K, Lys, Lysine; W, Try,
Tryptophan; L, Leu, Leucine; Y, Tyr, Tyrosine; MSP, membrane
scaffold protein; DPPC, dipalmitoyl phosphatidylcholine; PC,
phosphatidylcholine; PS, phosphatidyl serine; BR,
bacteriorhodopsin; apo A-I, apolipoprotein A-I; GABA, gamma
aminobutyric acid; PACAP, pituitary adenylate cyclase-activating
polypeptide.
[0076] The simplest single-celled organisms are composed of central
regions filled with an aqueous material and a variety of soluble
small molecules and macromolecules. Enclosing this region is a
membrane which is composed of phospholipids arranged in a bilayer
structure. In more complex living cells, there are internal
compartments and structures that are also enclosed by membranes.
There are numerous protein molecules embedded or associated within
these membrane structures, and these so-called membrane proteins
are often the most important for determining cell functions
including communication and processing of information and energy.
The largest problem in studying membrane proteins is that the
inside of the phospholipid bilayer is hydrophobic and the embedded
or anchored part of the membrane protein is itself also
hydrophobic. In isolating these membrane proteins from their native
membrane environments, it is very difficult to prevent them from
forming aggregates, which may be inactive or insoluble in the
aqueous environments commonly used for biochemical investigations.
The present invention provides ways to generate a soluble
nanoparticle that provides a native-like phospholipid bilayer into
which hydrophobic proteins of interest (target proteins) can be
incorporated to maintain the target protein or smaller hydrophobic
molecule as a soluble and monodisperse entity. This is accomplished
by incorporating hydrophobic proteins such as membrane proteins
into nanometer scale structures using the MSPs as described
herein.
Solubilizing Agents
[0077] In the context of the present application, a solubilizing
agent is one which disrupts hydrophobic interactions which lead to
assembly or aggregation of hydrophobic and/or amphiphilic molecules
into three dimension structures. For example, a solubilizing agent
such as a detergent is used to put into solution hydrophobic
proteins within membranes or membrane fragments. Detergents useful
in the present context include, but are not limited to, cholate,
deoxycholate, 1-palmitoyl-2-oleoyl-sn-glycerophosphocholate,
1-palmitoyl-2-oleoyl-sn-glycerophosphoserine,
1-palmitoyl-2-oleoyl-sn-glycerophosphoethanolamine, CHAPS,
n-dodecyl-.beta.-D-maltoside, octyl-glucopyranoside, Triton X-100,
myristoyl sulfobetaine, dihexanoyl-phosphotidylcholine, digitonin,
emulgen 913 o4r JB3-14. Peptidetergents can also be used; see, for
example, Schafineister et al. (1993).
Membrane Scaffold Proteins
[0078] Membrane Scaffold Proteins (MSPs) as used herein may be
artificial (non-naturally occurring, those which do not occur in
nature, i.e., those which differ in amino acid sequence from any
naturally occurring proteins) amphiphilic proteins which
self-assemble with phospholipids and phospholipid mixtures into
nanometer size membrane bilayers. A subset of these nanometer size
assemblies are discoidal in shape, and are referred to as Nanodiscs
or Nanodisc structures. Desirably the MSPs comprise several helical
domains, where the pairs of helical domains are separated by a
punctuation region, made up of one to five amino acids which do not
favor helix formation or which tend to stop helix formation of
adjacent amino acids. Exemplary helical regions are provided in
Table 19. These building blocks can be combined in orders and
numbers other than those specifically exemplified, provided that
the function of self assembly into stable, soluble nanoscale
disc-like particles is maintained. Similarly, these specifically
exemplified building blocks can be combined with other helical
building blocks from other proteins such as other apolipoproteins,
apolipophorins and the like. These assembled structures of MSP and
phospholipid preserve the overall bilayer structure of normal
membranes but provide a system which is both soluble and can be
assembled or affixed to a variety of surfaces. A naturally
occurring example of an MSP is human apo-A1. In addition, MSPs can
be designed using helical segments of proteins other than human
apoprotein A-1, for example, apo A-1 of other species, or apo C,
apo E, myoglobin or hemoglobin proteins of various species. Helical
segments from more than one protein can be combined, with the
appropriate punctuation sequences, to form a MSP having the useful
properties described herein. Additionally, functional MSPs can be
generated by de novo protein design wherein the desired traits of
amphipathic helical protein structures are generated. It is also
understood that conservative amino acid substitutions can be made
in the sequences specifically exemplified, with the proviso that
the self-association function is maintained. Such substitution
variants can be termed homologs of the specifically exemplified
sequences. Various proteins of interest are described in
Bolanos-Garcia et al. (2003) Progress in Biophys. Molec. Biol.
83:47-68.
[0079] Hydrophobic or partially hydrophobic proteins, e.g.,
membrane proteins, or membrane fragments can associate with these
particles such that the hydrophobic proteins or membrane fragments
are effectively solubilized in a stable structure which maintains
the functionality of the protein with respect to enzymatic activity
or ligand binding. Similarly, other hydrophobic or partially
hydrophobic molecules of interest can also be incorporated within
the nanoscale discoid particles of the present invention.
[0080] The Nanodisc particles are stable in solution or they can be
fixed to a surface, advantageously in a uniform orientation with
respect to the surface. As used herein, a nanoparticle comprising
MSPs (with or without another hydrophobic or a partially
hydrophobic protein) can be from about 5 to about 500 nm, desirably
about 5 to about 100 nm, or about 5 to about 20 nm in diameter.
Nanoparticles (disks) of about 5 to about 15 nm in diameter are
especially useful.
[0081] It is also readily within the grasp of the skilled artisan
to design MSPs for packaging hydrophobic passenger compounds,
proteins or complexes where the MSP assumes an amphiphilic
conformation based on beta sheets, where the amino acid sequence of
the protein is punctuated so that there are regions of beta sheet
forming portions separated by a flexible (hinge) region of amino
acids. The region of beta sheet-forming sequence is desirably from
about 10 to about 30 amino acids, and the punctuation region can
include from 3 to 10 amino acids, where there are antiparallel beta
sheets in the MSP or from about 10 to about 30 amino acids where
the beta sheets are parallel.
[0082] Functional MSPs may or may not have punctuation between
domains of secondary structure. The punctuation region disrupts
regions of secondary structure within a protein. Proline and/or
glycine residues are preferred punctuation regions in a protein
having helical domains. Besides disrupting a domain with a
particular characteristic secondary structure, the punctuation
regions can provide flexibility to a protein=s structure, serving
to create a hinge region, especially in the case of two to three
amino acids, desirably including proline, glycine and alanine
residues. A punctuation region (or punctuation sequence, hinge
region or hinge sequence) can include from 1 to 30 amino acids,
desirably 1 to 2 amino acids when the domains of secondary
structure are alpha helices, and, where there are antiparallel beta
sheets in the MSP, 5 to 30, and especially 3 to 10 amino acids.
[0083] The necessary properties of the linker (punctuation, hinge)
sequence between fused MSPs are flexibility and solubility so that
the fused proteins assemble into particles in a manner similar to
two separate MSP molecules. Linker sequences consisting of repeats
of Gly-Gly-Gly-Ser/Thr- (SEQ ID NO:46) have these properties. It is
also desirable, in at least some MSPs, to minimize the length of
the linker. We constructed a fusion with the minimal linker
sequence-GT-, which corresponds to the consensus DNA restriction
site for Kpn I, as described herein below. The Kpn I site provides
an easy way of inserting any desired linker sequence by restriction
with Kpn I and insertion of double-stranded synthetic DNA encoding
any desired linker (Robinson et al. 1998). We have also made a
fusion construct with the longer linker sequence -GTGGGSGGGT-(SEQ
ID NO:15). The MSP2 with the minimal linker, however, assembles
into particles very similar to particles containing two MSP1
proteins, but which are more stable than those comprised of two
MSP1 proteins. It is understood that the best choice of the
particular MSP depends on the particular protein with which is to
be assembled. In general, the assembly with larger proteins or
protein complexes requires the use of larger MSPs.
[0084] One important goal in utilizing a membrane scaffold protein
(MSP) to provide membrane proteins in general, and G-protein
Coupled Receptors (GPCRs) in particular, with a suitable
environment for homogeneous biochemical assay or crystallization is
to have homogeneous preparations of particles. The engineered
membrane scaffold proteins we have described, including, but not
limited to, truncated human apo A-I (MSP1) where the amino terminal
soluble domain has been removed, deletion or insertion mutants
where one or more protein segments are removed or inserted, tandem
repeats of MSP1 or deletion mutants, respectively, and any of the
above materials where a histidine tag is incorporated, primarily
form 8-10 nm (in diameter) particles when self-assembled with
phospholipids in solution. Desirably the MSP does not include the
helix H1. However, upon assembly with non-optimal stoichiometry of
MSP and phospholipid, particles of other sizes may be present.
While standard size separation chromatography can be used to purify
a single size class of particle, it is preferable to minimize the
size distribution of the initial reconstitution mixture of target
protein, MSP and phospholipid. Engineered Nanodiscs of various
sizes can be formed by appropriate choice of the length of the
membrane scaffold protein. The particle 8-10 nm in diameter
nominally comprises two MSP proteins.
Apolipoprotein Sequences
[0085] Sequences of several apolipoproteins, hemoglobins and
myoglobins are available on the internet at the site of The
National Center for Biotechnology Information (NCIB), National
Institutes of Health. The coding sequences can be found on the
internet and used in the construction of artificial MSP coding
sequences or the sequences can be tailored to optimize expression
in the recombinant host cell of choice. There is a large body of
information about codon choice and nontranslated sequences in the
art. Apolipoprotein C sequences include, without limitation,
bovine, XP 77416; mouse, AAH 28816; human NP 000032; and monkey,
Q28995. Myoglobin sequences include, for example, those of mouse,
NP 038621; bovine, NP 776306; rat, NP 067599; and human, NP 005359.
Hemoglobin alpha chain sequences include human, AAH 32122 or NP
000549; beta chain sequences include human, NP 000509 or P02023;
rat, NP 150237; mouse NP032246; bovine, NP 776342, all of which are
incorporated by reference herein. Others may be found at the NCBI
website and in the scientific literature as well.
[0086] As used herein, amphiphilic and amphipathic are used
synonymously in reference to membrane scaffold proteins. An
amphiphilic protein or an amphiphilic helical region of a protein
is one which has both hydrophobic and hydrophilic regions.
MSP Design
[0087] The MSPs of the present invention must be amphipathic, with
one part of its structure more or less hydrophilic and facing the
aqueous solvent and another part more or less hydrophobic and
facing the center of the hydrophobic bilayer that is to be
stabilized. The elements of secondary structure of the protein
generate the hydrophilic and hydrophobic regions in three
dimensional space. Examination of the basic biochemical literature
reveals two candidate protein structures that can have this
required amphipathic character: the helix and the pleated sheet. We
designed the MSPs described herein to have a helix as the
fundamental amphipathic building block. Each MSP has an amino acid
sequence which forms amphipathic helices with more hydrophobic
residues (such as A, C, F, I, L, M, V, W or Y) predominantly on one
face of the helix and more polar or charged residues (such as D, E,
N, Q, S, T, H, K or R and sometimes C) on the other face of the
helix. See FIG. 2 for a schematic representation. In addition, each
helical building block can be, but is not necessarily, punctuated
with residues such as proline (P) or glycine (G) periodically,
which can introduce flexibility into the overall structure by
interrupting the general topology of the helix. In one embodiment,
these punctuations occur about every 20-25 amino acids to form
kinks or to initiate turns to facilitate the wrapping of the MSP
around the edge of a discoidal phospholipid bilayer. The
punctuation region (or sequence) can include from one to 10 amino
acids, especially 3 to 10 where there are antiparallel beta sheets
in the MSP. See FIG. 2, which depicts a generalized linear amino
acid sequence and a helical wheel diagram showing the placement of
predominantly hydrophobic amino acids on one face of the helix.
[0088] We created an additional artificial variant MSP (MSP2) by
designing a tandem repeat of MSP1 connected by a short linker
sequence to create a new molecule. This type of artificial MSP is
termed a tandem repeat MSP. See FIG. 4G and SEQ ID NO:17.
Relatively large quantities (tens of milligrams/liter cell culture)
of the artificial MSPs of the present invention are produced in a
bacterial expression system. Our constructs reduce the number of
size classes that can be formed (those corresponding to three MSP1
molecules). As used herein a tandem repeat membrane scaffold
protein is one in which at least four helices of a membrane
scaffold are repeated in linear order in a new membrane scaffold
protein (e.g.,
H1-H2-H3-H4-H5-H6-H7-H8-spacer-H1-H2-H3-H4-H5-H6-H7-H8). Examples
of tandem repeat MSPs are also given in FIGS. 4E and 4G. See also
SEQ ID NO:17 and SEQ ID NO:19, among others.
[0089] Nanodiscs made with tandem repeats (two) of MSP1 sequences
were larger, but less stable, than those using certain other MSP
structures, at least in some instances. Designing MSPs lacking at
least one copy of H1I allowed the preparation of stable Nanodiscs
which are also larger in size. In particular, the absence of the
first helix in the second half of dimeric structure plays an
important role in the improved results.
[0090] The complete amino acid and nucleic acid sequences for the
MSP2 tandem repeat scaffold protein is shown in Tables 7 and 8; see
also SEQ ID NO:16 and SEQ ID NO:17. The MSP2 fusion protein was
expressed in E. coli and purified to homogeneity using basically
the same procedure as described for the single MSPs. The MSP2
protein serves as an effective scaffold protein, self-assembling
with phospholipid upon removal of solubilizing detergent. At a
lipid/dimer ratio of 200 corresponding to nominally 10 nm
particles, there is the much greater monodispersivity afforded by
the MSP2 protein. Importantly, the overall stability of the disks,
as monitored by chemically induced unfolding and exposure of
tryptophan residues to solvent, is not altered by the fusion of the
monomeric membrane scaffold proteins.
[0091] We have generated two new membrane scaffold protein dimers
described below and self-assembled these with phospholipids. The
resultant Nanodiscs have an overall Stokes diameter determined by
small angle x-ray scattering of approximately 15.5 nm which
corresponds to a calculated overall physical diameter of a discoid
of 17 nm. These are the largest Nanodiscs constructed to date. The
modular sequences (see also Table 19) of these new tandem repeat
MSPs are as follows:
TABLE-US-00001 MSP2N2:
HisTev-H1/2-H2-H3-H4-H5-H6-H7-H8-H9-H10-GT-H2-H3-
H4-H5-H6-H7-H8-H9-H10 MSP2N3:
HisTev-H1/2-H2-H3-H4-H5-H6-H7-H8-H9-H10-GTREQLG-
H2-H3-H4-H5-H6-H7-H8-H9-H10
[0092] Other MSPs have also been made and characterized. We have
optimized the conditions of self-assembly to obtain the
monodisperse nanoparticles with MSP1 E1, MSP1 E2 and MSP1 E3, shown
that the length of the protein is the determinant of the particle
diameter, measured the stoichiometry of lipid/protein ratio in
these particles and demonstrated the structural difference between
the particles formed with lipids above or below melting point (270
K for POPC, 314 K for DPPC). We also prepared the series of
deletion mutant membrane scaffold proteins, in which one quarter,
one half, or the whole first helix (residues 44-65) was deleted.
Experiments with Nanodiscs formed with the truncated proteins
indicated that the first helix is of not required for the
self-assembly of these Nanodiscs. This observation is believed to
explain the earlier disagreement about the size of discoidal
particles formed with apo A-1 and their heterogeneity. SAXS data
for these Nanodiscs formed with scaffold proteins of different
sizes are consistent with the structural model shown in FIG. 19.
FIG. 19 also shows that H1 does not play a significant role in the
formation of the "belt" of MSP around the outside of the
Nanodisc.
[0093] In order to generate smaller belts around the bilayer
structure, the overall length of the helical building blocks can be
reduced, and the punctuations may be introduced more frequently.
The exact amino acid sequence can vary in the positioning and
number of the hydrophobic amino acids within the designed linear
sequence. Simple models in which either the helical axis is
parallel or perpendicular to the normal of the Nanodisc bilayer can
be generated. To generate a disk with a diameter of roughly 10 nm,
an MSP comprises about 12 to about 20 or more repeating units
having this generalized amphipathic sequence. Preferably, this
protein would be composed of amphipathic alpha helices each with a
length of between 14 and 25 amino acids, punctuated in the linear
sequence by a residue unfavorable for helix formation, such as
proline or glycine or a sequence from about 1 to 5 amino acids
which does not favor helix formation, which form small helical
building blocks that stabilize the hydrophobic core of the
phospholipid bilayer. A helix of about 20-25 amino acids (a helical
building block, in the context of the present application) has a
height comparable to the thickness of a membrane bilayer. These
small helical segments are linked together (punctuated) with from 0
to about 5 amino acid residues, especially G or P. To cover the
edge of a 10 nm discoidal particle in either the belt model
presented, one would need between 10-20 such helices, with 16 being
a useful number based on the simple graphic analysis of FIG. 3.
Desirably, the helix contains from about 3 to about 18 amino acids
per turn, and the type of helix can be an alpha, pi or 3,10 helix,
among others. Helices with three to sixteen, three to eight,
desirably three to four, amino acids per turn of the helix. An MSP
of the present invention can comprise from 50 to 400 turns.
Secondary structure predictions can be determined using programs
readily accessible to the art; see, for example, on the internet at
the ExPASy proteomics server of the Swiss Institute of
Bioinformatics. Guidance in predicting secondary structure is also
given in publications such as Chou et al. (1974) Biochemistry
13:211, 222; Chou et al. (1978) Ann. Rev Biochem. 47:251-278;
Fasman (1987) Biopolymers 26(supp.):S59-S79. Where there is a dimer
or higher oligomer of a protein such as a 7-TM membrane protein or
where more than one protein is to be incorporated within a single
Nanodisc, for example a reductase and a cytochrome, the MSP used
must be capable of forming a Nanodisc particle with a diameter
greater than 9-10 nm. The larger Nanodiscs are prepared using
longer MSP sequences, such as MSP1E1, MSP1E2 or MSP1E3.
[0094] In an alternative embodiment, the engineered amphiphilic MSP
contain regions of secondary structure in three dimensional space,
such as parallel or antiparallel beta sheets, with spacer regions
of appropriate length to allow association of hydrophobic regions
with a target hydrophobic target molecule which is protected from
the aqueous milieu, and thus stabilized and solubilized.
[0095] Certain critical systems controlling cellular function are
located in membrane compartments. Many of these membrane protein
assemblies represent important pharmaceutical targets that are
typically difficult to isolate in soluble and active form because
particular phospholipid environments are often essential for
maintaining optimal enzymatic turnover or ligand binding activity.
Several pharmacologically significant examples indicate specific
phospholipid requirements for individual enzymes and receptors,
which are perturbed by detergents typically used to solubilize
membrane proteins. Examples include the human .beta.-adrenergic
receptor that requires neutral lipids for efficient receptor
hormone response (Kirolovsky et al., 1985) and the human cytochrome
P450 monooxygenase (P450) superfamily that requires several
phospholipid types for efficient drug metabolism (Imaoka et al.,
1992). An inability to faithfully reconstitute the lipid
requirements of detergent solubilized protein in purified systems
can, and often does, affect the measured activity of these
enzymes.
[0096] One of the most widely used alternatives for
characterization of these native proteins involves the
sub-fractionation of natural cellular membranes and incorporation
into micron-sized liposomes. However, liposomes are compromised by
thermodynamic instability, size heterogeneity and sequestration of
target membrane proteins on the solvent-inaccessible side of the
bilayer (Angrand et al., 1997; Savelli et al., 2000). Other
convenient methods for obtaining large quantities of soluble
functional membrane proteins assembled in phospholipid bilayers
have not been available and, as a consequence, our understanding of
the numerous protein complexes functioning within cell membranes
has been hindered. In this application, we report a rapid method
for compartmentalizing heterologously-expressed or native membrane
proteins into stable, soluble nanometer-scale bilayer structures
which are characterized by sufficient target stability, biological
activity and sufficient robustness to survive operation in
high-throughput analyses.
[0097] The MSPs of the present invention can be used to solubilize
tethered, embedded or integral membrane proteins in nanoscale
structures. A tethered membrane protein is composed mostly of a
single relatively soluble globular domain external to the bilayer
and a relatively simple (e.g., a single membrane-spanning or
membrane-inserting domain) which anchors this simple globular
domain to the membrane bilayer. The globular domain, in nature, can
be extracellular or cytoplasmic in orientation.
[0098] Tethered membrane proteins are exemplified by
NADPH-cytochrome P450 reductases (e.g., from rat liver endoplasmic
reticulum or from insect) and cytochrome b5. NADPH-Cytochrome P450
reductase is a membrane protein found in the endoplasmic reticulum.
It catalyzes pyridine nucleotide dehydration and electron transfer
to membrane bound cytochrome P450s. Isozymes of similar structure
are found in humans, plants, other mammals, insects etc
[0099] Cytochrome b5 is a membrane-anchored (tethered) heme protein
having a single membrane anchor domain that penetrates the membrane
bilayer. Cytochrome b5 solubilized from its native membrane exists
as large aggregates in the absence of detergent and appears as a
smear rather than a discrete band on native polyacrylamide gel
electrophoresis (PAGE). Formation of Nanodiscs through the
self-assembly process using MSPs taught in our invention, wherein
cytochrome b5 is added to the preparation of MSP and phospholipid
results in incorporation of cytochrome b5 into disk structures. The
disk complexes containing cytochrome b5 can be chromatographically
separated and purified from undesired aggregated material. The
optical absorption properties of the heme chromophore of the
purified material show that the heme active site is in a native
conformation. Tethered membrane proteins can be incorporated into
Nanodiscs either during disc formation, or they can associate with
preformed Nanodiscs.
[0100] Embedded membrane proteins, as defined herein, are those
which include a membrane anchoring segment of the polypeptide, but
which also have groupings of hydrophobic amino acids on the surface
of the protein, which hydrophobic domains are embedded within the
membrane bilayer. Examples of embedded membrane proteins include,
without limitation, the interferon receptor superfamily, the nerve
growth factor/tumor and the necrosis factor receptor superfamily as
well as the cytochrome P450 proteins.
[0101] Tissue factor (TF), or thromboplastin, is a 30,000 Da type-I
membrane protein critical to initiation of the blood coagulation
cascade. This membrane-bound protein acts as an activation cofactor
for factor VII, the soluble serine protease which carries out the
first enzymatic step in blood coagulation. Expression of tissue
factor is limited to cells that are not in direct contact with
blood plasma, which cells form a hemostatic envelope." The TF:VII
complex must be assembled on a membrane surface to exhibit high
activity, and optimal activity is seen only when the membrane
contains phospholipids with negatively charged headgroups.
[0102] Another integral membrane protein which has been
incorporated into Nanodiscs is a bacterial aspartate receptor. In
E. Coli and Salmonella the chemoreceptors Tsr and Tar mediate taxis
towards serine and aspartate, respectively, mediated through
stereospecific binding of those amino acids. In E. coli the Tar
receptor protein mediates taxis towards maltose via recognition of
a ligand-occupied soluble maltose-binding protein. Membranes from
E. coli containing over-expressed Tar protein (provided by Gerald
Hazelbauer, University of Missouri, Columbia, Mo.) were solubilized
with CHAPS detergent and mixed with the scaffold protein MSP1T2.
Detergent was removed by adsorption (using Biobead treatment as
described herein below). The Tar receptor was incorporated into
Nanodiscs, which were then purified by Ni-affinity column
chromatography and analyzed by HPLC size-exclusion chromatography.
Incorporation of the target was verified by SDS PAGE.
[0103] Examples of embedded cytochrome P450 membrane proteins
include, without limitation, cytochrome P450 2B4 from rabbit liver
microsomes, cytochrome P450 3A4 from human liver microsomes,
cytochrome P450 6B1 from insect fat bodies, and cytochrome P450
86A1, 73A5 and 86A8 from plants. The cytochromes P450 are a
superfamily of enzymes that are found in all forms of life. One
role of many P450s is to detoxify xenobiotics; for instance, human
liver P450s detoxify most endogenous and exogenous compounds, and
these enzymes determine the mean plasma lifetime of all drugs
ingested. One of the most widely studied human liver cytochrome
P450s is cytochrome P450 3A4 (CYP 3A4). This membrane bound P450 is
the most highly expressed P450 in human liver and is responsible
for metabolizing almost 50% of all pharmaceuticals (Guengerich, F.
P., Cytochrome P450. Cytochrome P450, ed. P. R. Ortiz de
Montellano, 1995, New York: Plenum Press. 473-535). In order to
demonstrate the utility of Nanodisc technology for the study of the
cytochrome P450, we incorporated CYP 3A4 into MSP supported
nanobilayer discs. Further evidence from size separation
chromatography and PAGE analysis supports the conclusion of
incorporation of CYP 3A4 into Nanodiscs.
[0104] Cytochrome P450 6B1 (CYP 6B1) is a member of the large
cytochrome P450 monooxygenase protein superfamily, and it is
another example of an embedded membrane protein. CYP 6B1 has been
isolated from Papilio polyxenes, the black swallow tail, which
feeds exclusively on plants producing furanocoumarins, plant
psoralen derivatives that are phototoxic to most organisms. CYP 6B1
catalyzes the detoxification of furanocoumarins by what is believed
to be an epoxidation reaction (Ma et al. (1994)).
[0105] Integral membrane proteins have predominant and critical
regions of structure located within the membrane bilayer.
Alternatively, there can be relatively large soluble domains on
both sides of the bilayer which are linked by one or more passes of
the primary sequence through the hydrophobic bilayer core,
especially cytokine-type molecules and receptors, which have simple
one-pass connectivity but with soluble domains on both sides of the
bilayer. As used herein, integral membrane proteins are exemplified
by the general class of proteins in which there one or more helical
segment in the membrane bilayer, including but not limited to the
well known 7 helix transmembrane proteins (e.g., GPCRs).
[0106] We have shown that MSP1, MSP2, MSP1E1, MSP1E2, MSP1E3 and
MSP2 assemble with bacteriorhodopsin. From the initial
reconstitution mixture, two bacteriorhodopsin-containing species
are observed when particles are formed with MSP1 or MSP2 in the
absence of added phospholipid. MSP is absolutely required for the
solubilization of bacteriorhodopsin to form these species because
omission of an MSP from the formation mixture results in large
non-specific bacteriorhodopsin aggregates that elute in the void
volume of the gel filtration column. The majority of
bacteriorhodopsin appeared solubilized in the presence of MSPs.
[0107] An especially valuable advantage of the MSP-containing
nanoparticles of the present invention as a means to solubilize
hydrophobic or partially hydrophobic target proteins is that the
protein incorporated into the nanoparticle has a naturalistic
presentation. Native target protein conformation is maintained, the
native target protein-membrane interaction and topology are
preserved, the target protein is maintained in a native-like
environment, thereby increasing the stability of the target protein
to inactivation and denaturation, and the topology of the target
protein is maintained relative to the membrane. The maintenance of
target protein topology relative to the membrane is especially
important for screening targets for cell-cell or cell-virus
interaction, elicitation of ligand or antibody binding to
extra-membrane regions of the target protein or delivery of the
target protein through specific trafficking pathways.
Incorporation from Membranes and Membrane Fragments
[0108] We have demonstrated that membranes or membrane fragments
containing their natural repertoire of membrane proteins and lipids
can be incorporated into Nanodiscs comprising MSPs. This can be
effected directly without pre-purification or solubilization of the
membrane protein populations. A particularly important embodiment
is the use of this technology in a variety of commonly used
heterologous expression systems for membrane proteins. These
include, but are not limited to, insect cells, yeast cells,
mammalian cells such as HEK cells, Vero cells and CHO cells, and
bacterial cells. Virus envelope proteins or cell membranes of
pathogens (e.g., bacteria), either of which can contain multiple
copies of antigenic proteins or other molecules, can also be used.
A specifically exemplified embodiment is the use of the common
insect cell-baculovirus expression system. We used a commercially
available Sf9 insect cell line co-infected such that a microsomal
preparation containing over-expressed insect CYP6B1 and an
over-expressed insect NADPH cytochrome P450 reductase was produced.
Hence, we not only demonstrated that MSP Nanodiscs can be used to
incorporate another cytochrome P450 system into soluble
monodisperse particles, but also that the source of this P450 could
be the whole membranes from the Sf9 cell line that was infected
with a baculovirus carrying a cloned CYP6B1 gene.
[0109] The Nanodiscs generated by the procedure described herein
contain the fatty acids and phospholipids from the original native
membrane starting material and therefore provide a reliable in
vitro environment in which to assay any membrane-bound enzyme or
receptor of interest. Thus, MSP-supported Nanodiscs can be used in
high-throughput screening ventures such as the identification of
ligands for membrane-associated proteins, for example, using
combinatorial libraries of peptides, proteins or chemical
compounds, and for the identification of new pharmaceuticals.
Additionally, the simple procedure of incorporation into Nanodiscs
can be used to generate samples for structure determination using
x-ray crystallography or NMR spectroscopy. A particular advantage
of the Nanodisc system over alternative methods for membrane
protein solubilization is the increase in sensitivity of optical
measurements due to a significant decrease in light scattering of
the particles. The methods of the present invention can be extended
to any other source of membrane fragments containing target
proteins of interest, such as any yeast, insect, bacterial or
mammalian cell culture system or expression system.
High Throughput Screening
[0110] An important utility of the Nanodisc technology of the
present invention is in high throughput screening for enzymatic or
ligand binding activity. In many such systems, it is advantageous
to have more than one target membrane protein incorporated into the
Nanodiscs, for example, the electron transfer partner needed for
P450 monooxygenase catalysis or the corresponding G-protein
incorporated with a G-protein coupled receptor.
[0111] In order to demonstrate the utility of the MSP Nanodisc
technology in these situations, we successfully incorporated the
NADPH cytochrome P450 reductase and a cytochrome P450 6B1 into
Nanodiscs. As demonstrated herein, each target membrane protein can
be individually incorporated into Nanodiscs using MSPs or they can
be incorporated in combinations. The endogenous relative amount of
cytochrome P450 to reductase is about 10-20 P450 molecules per
reductase molecule (Feyereisen, R. (1999) Ann. Rev. Entomol. 44,
501-533). To obtain activity of CYP6B1 after reconstitution into
disks, an excess amount of reductase can be added to the
reconstitution mixture.
[0112] BR, an integral membrane protein, has been incorporated into
the MSP Nanodiscs as described herein, and we have also used a
commercially available insect cell expression system that provides
a membrane fraction hosting the G-protein coupled receptor human
for 5-HT-1A (serotonin). The ligand binding activity documented for
5-HT-1A incorporation into Nanodiscs proves that the protein is in
the active conformation in the Nanodiscs of the present invention.
Subsequent experiments show that the beta-2 adrenergic receptor,
the dopamine D2 and D1 receptors and the cytokine receptors CXCR4
and CCR5, all of which belong to the 7-transmembrane protein family
and G-protein coupled receptor type, are easily incorporated into
Nanodiscs by the methods of the present invention.
[0113] Other examples of membrane proteins and membrane protein
complexes which have successfully been incorporated into Nanodiscs
include cytochrome P450 reductases from rat, insects and plants, a
bacteriorhodopsin trimer, a photosynthetic reaction center complex,
a twenty-six transmembrane domain Escherichia coli transhydrogenase
and integrin.
[0114] Stoichiometry of protein and lipid is an important factor in
the formation of monodisperse discs with all MSPs. The set of
extended MSPs shows that there is a well-defined optimum of
lipid/protein ratio, which is crucial for quantitative assembly of
monodisperse discoidal particles. In addition, the stoichiometric
ratio is determined by the MSP length, because of the well-defined
topology of discoidal structure with a cylindrical lipid bilayer
surrounded by the scaffold protein (FIG. 19), which determines the
lipid/protein ratio. The lipid/protein stoichiometry ratios for
discs of different sizes were calculated as shown at FIG. 19 and
tested experimentally. Concentrations of lipids were measured using
tritiated lipids of the same chemical structure and scintillation
counting of the column fractions, as described (Bayburt et al.
(2002) supra). Concentrations of scaffold proteins were determined
spectrophotometrically (Jones et al. (1990) J. Biol. Chem.
274:22123-22129), using the molar absorption coefficients
calculated for the known amino acid sequences according to the
modified method of Gill-von Hippel, as described in Pace et al.
(1995) Protein Science 4:2411-2423. All measurements were done on
the narrow fractions after separation of the assembled
lipid-protein particles by HPLC (Millenium System, Waters, Milford,
Mass.) on the calibrated Superdex 200 (size exclusion
chromatography) column.
[0115] Small angle X-ray scattering (SAXS) was measured at an
ambient temperature of 295 K at the vacuum chamber with 1500 mm
distance from the sample to the 2D detector at the photon energy 15
keV (wavelength 0.826 A). The solutions of the Nanodiscs were
sealed in glass capillaries with a diameter of 1.5 mm and placed on
the holder in the sample chamber, together with calibrant (Ag
behenate, spacing 58.38 .ANG.,) and reference buffer solvents. The
raw data were processed using the program FIT2D (Hammersley, A. P.
(1998) ESRF Internal Report, ESRF98HA01T, FITD2D V9.129 Reference
Manual V3.1; Hammersley et al. (1996) High Pressure Research
14:235-248) to give the scattering curves in the form 1
g(I/I.sub.0) vs. Q=4.pi. sin(q)/2. Analysis of SAXS data was with
the program CRYSOL (Svergun et al. (1995) J. Appl. Cryst.
28:768-773) and home written modeling and fitting subroutines using
MATLAB (MathWorks, Natick, Mass.). The fitting program is based on
the Debye equation and modeling of the nanoparticle by close packed
spherical beads with different contrast, as it is done in other
popular programs CRYSOL, DUMMIN, SAXS3D and DALAI_GA, reviewed in
Takahasi et al. (2003) J. Appl. Cryst. 36:549-552; Koch et al.
(2003) Quart. Rev. Biophys. 36:147-227. The models for fitting were
constructed using the information on size and composition of
Nanodiscs obtained by other methods described in this paper. The
initial estimate for the scattering contrast, i.e. the difference
between the electron density for water, 0.334 e/.ANG..sup.-3, and
the average electron density for the methylene groups in the
central part of bilayer, lipid acyl chains, lipid polar head groups
(Wiener et al (1989) Biophys. J. 55:315-325), and scaffold protein
at the circumference of the particle (Svergun et al. (1998) Proc.
Natl. Acad. Sci. USA 95:2267-2272), was assigned to each bead
representing a correspondent phase. Experimental curves for each
type of Nanodisc were fitted using five parameters, four electron
densities and the radius of the disc. No attempt to made to
introduce size or shape heterogeneity into the fitting. Modeling of
such heterogeneity within 5% (as suggested by other experimental
data) did not give a large difference in the calculated scattering
curves, more significant variance in size and shape resulted in the
loss of observed features on the scattering curves and gave
considerably worse fits.
[0116] MSPs have been engineered to minimize the variability in the
structure of the discoidal phospholipid bilayer entities, provide
greater structural stability and increased size homogeneity of the
disk structures, and incorporate useful functionalities such as
peptide tags for purification and physical manipulation of disks.
Such oligopeptide tags which can be fused to a protein of interest
(by molecular biological or chemical methods) include, without
limitation, strep-tag (Sigma-Genosys, The Woodlands, Tex.) which
directs binding to streptavidin or its derivative streptactin
(Sigma-Genosys); a glutathione-S-transferase gene fusion system
which directs binding to glutathione coupled to a solid support
(Amersham Pharmacia Biotech, Uppsala, Sweden); a calmodulin-binding
peptide fusion system which allows purification using a calmodulin
resin (Stratagene, La Jolla, Calif.); a maltose binding protein
fusion system allowing binding to an amylose resin (New England
Biolabs, Beverly, Mass.); and the oligo-histidine fusion peptide
system which allows purification using a Ni.sup.2+-NTA column
(Qiagen, Valencia, Calif.).
[0117] Disk homogeneity is necessary for efficient incorporation of
single membrane proteins or single membrane protein complexes into
a single size class of disk. The parent molecule, apo A-I, has
functions beyond disk structure stabilization (Forte et al., 1971;
Holvoet et al., 1995; Fidge, 1999). These functional regions are
unnecessary and often deleterious in the artificial bilayer systems
of the present invention.
[0118] Secondary structure prediction allows assessment of
structural features of the scaffold protein. The apo A-I structure
consists of mostly helix, sometimes punctuated by proline or
glycine residues in the repeat sequences. Eight to nine helices are
believed to associate with lipid in the form of disks. The
N-terminal GLOB region (SEQ ID NO:89) of apo A-I is predicted to be
more globular in character. This portion of the molecule has been
removed to produce the engineered MSP1. An MSP that produces disk
assemblies with high monodispersity is desirable. To ascertain the
roles of half repeats and to further characterize and optimize the
MSP structure and function, mutagenesis was used to generate
variants as described herein below. See Tables 2-21 below.
[0119] Hydrophobic or partially hydrophobic receptors incorporated
into MSP disks are useful in structural, biochemical and
pharmaceutical research. Membrane protein study was previously
limited to insoluble membrane dispersions, detergent micelles, and
liposomes. Purified systems for biochemical and physical study
require stability, which may or may not be obtainable with
detergents. Detergent micelles are dynamic and undergo structural
fluctuations that promote subunit dissociation and present
difficulties in the handling of proteins in dilute solution. MSP
nanobilayers (Nanodiscs) are more robust structurally, having a
phospholipid bilayer mimetic domain of discrete size and
composition, and greater stability and smaller surface area than
unilamellar liposomes. The particles of the present invention are
stable in size, conformation and biological activity for at least a
month at 4 C.
Surface Technology
[0120] The MSPs of the present invention, when formulated into
Nanodiscs, can be used in analyses in surface technology such as
biosensor chips for high throughput screening or solid phase assay
techniques, including but not limited to multiwell plates made, for
example, of polystyrene. Where the MSP comprises a His tag, the
Nanodiscs can be bound to an immobilized metal, for example
divalent nickel cation. Our work on disk scaffolds has also
involved surface-associated assemblies.
[0121] For instance, the surface plasmon resonance (SPR) biosensor
utilizes an approximately 50 nm gold film on an optical component
to couple surface plasmons to a dielectric component (sample) at
the surface of the gold film. MSP-stabilized bilayers can be
attached to the surface for use as a biomimetic layer containing
proteins or other targets of interest by engineering cysteines into
the MSP (FIG. 7A). The use of thiols is well known for attaching
molecules to gold surfaces. Based on the belt model, cysteine
residues can be placed along the polar side of the amphipathic
helix axis, provided that a cysteine residue is not positioned at
the helix-helix interface. In cases wherein the MSP is so
engineered, multiple cysteine residues can form disulfide-linked
dimers (Segrest et al., 1999). An alternative is to introduce
cysteines within a flexible NB or C-terminal linker. Such a
construct is, in theory, capable of associating the belt (or the
picket fence) model of disk to a gold surface. Alternatively, thiol
lipids can be incorporated within the bilayer domain. Methodologies
which utilize differences in optical refractive index with layered
structures, such as total internal reflection spectroscopy,
resonant mirrors, optical diffraction grating engineered on optical
surfaces, and the like, can be likewise utilized by direct
extrapolation. In addition to SPR, surface-associated disks on gold
can be used in STM and electrochemical studies, for example, such
as with membrane associated redox proteins, e.g. cytochrome P450
and its flavoprotein, as well as ion channels.
[0122] SPR data can also be obtained from measurements made using a
thin film of dielectric such as silicon dioxide applied over the
metal film normally used as the substrate in SPR. This variation of
the technique has been termed coupled plasmon waveguide resonance
(CPWR) (Salamon et al., 1997a). Because silica can be used as the
active surface in these plasmon resonance experiments, the process
of producing a self-assembled bilayer can be adapted according to
the procedures used to produce surfaces on mica or other silicon
oxide surfaces. This has the added advantage of making the
conditions used for the SPR experiments directly comparable to
those used for AFM experiments. The CPWR technique can easily be
performed on an SPR instrument by simply adding the silica coating
to the metal film slides that are presently used for SPR
spectroscopy.
[0123] MSPs with available cysteine groups also enable specific
labeling with chemically reactive groups or affinity tags for
immobilization in gel matrices. Hydrogels with reactive coupling
groups are useful for immobilizing proteins for SPR measurements.
In a hydrogel configuration, the disk serves as a carrier for
bilayer-embedded membrane proteins in a monodisperse form with both
intra- and extracellular domains available for ligand binding. We
have already demonstrated that disks containing a His tag bind to a
metal chelate matrix, which can be used to immobilize Nanodiscs
containing a His-tagged MSP. His tag vectors are commercially
available (e.g., from Qiagen, Valencia, Calif.) and are described
in U.S. Pat. Nos. 5,284,933 and 5,130,663. Other tag peptide
sequences known to the art, including but not limited to, Flag tag
(flagellar antigen) or Step tag (streptavidin binding), can be
engineered into the MSP by molecular biological methods. Besides
mediating attachment to a support of choice, the tag sequences can
facilitate purification of the MSPs or of Nanodiscs containing
them. Nanodiscs can also be used in preparing affinity matrices for
bioseparation processes and measurements of ligand affinities. The
particles produced by the methods of the present invention are
useful for drug discovery, structure/function correlation, and
structure determination of membrane proteins.
Membrane Protein Structure and Function Analysis
[0124] Structure determination of membrane proteins has been
limited by the abilities to produce large amounts of membrane
proteins and to crystallize these proteins. Nanodiscs and MSPs are
useful as carriers for membrane protein stabilization and
expression. MSP can serve to solubilize membrane proteins for
crystallization in lieu of detergents. Indeed, where the lipid
bound form of MSP is structurally stable and rigid, crystallization
can be enhanced by introduction of crystal contacts through the
MSP. We have demonstrated that MSP1, the extended forms of MSP1,
and MSP2 or other tandem repeat MSPs can be used to solubilize BR
from purple membranes in the presence and absence of exogenous
lipid.
[0125] Fusion constructs with a membrane (or other) protein and an
MSP region can be expressed in Escherichia coli using any of a
number of art-known vectors to produce a stable and soluble form of
the membrane protein that contains a membrane anchor in large
quantity. The exciting discovery that MSP solubilizes BR in the
absence of added phospholipid allows the use of the artificial MSP
to stabilize membrane proteins in the absence of detergents or
lipid additives. The (artificial) MSPs disclosed herein can be used
in solubilization of other membrane proteins including, but not
limited to, cytochrome P450, cytochrome P450 reductase, and the
5-HT-1A receptor, as well as other membrane-associated receptor
proteins and enzymes.
[0126] Signal transducing elements occur across membranes, while
vesicles render one side of membrane inaccessible to hydrophilic
reagents and effector proteins. A specific embodiment of the
present invention uses disks to solubilize and stabilize
pharmaceutical targets such as GPCRs, ion channels, receptor
kinases, and phosphatases in a naturalistic presentation. We have
incorporated proteins with multiple membrane spanning domains into
the disks of the present invention, with a focus on GPCRs. We had
successfully incorporated the model serpentine membrane protein,
bacteriorhodopsin, into Nanodiscs. Bacteriorhodopsin is a model for
GPCRs, which are current targets for drug discovery. Currently,
over 1000 probable G-protein receptors from various organisms have
been cloned and many of the so-called orphan receptors await
identification of natural (or synthetic) ligands. Ligand classes
include peptide hormones, neurotransmitters, eicosanoids, lipids,
calcium, nucleotides, and biogenic amines. GPCRs are believed be
targets for more than half of currently marketed pharmaceuticals.
This structural class of membrane proteins can readily be
incorporated into Nanodiscs when contacted with MSPs as
pre-solubilized proteins or as membrane-associated proteins.
G-protein coupled receptors inserted into Nanodiscs are completely
functional in this trans-membrane signaling process. Structural
characterization of the reconstituted receptors is performed using
chemical analysis, spectroscopy and atomic force microscopy.
[0127] Cytochrome proteins and reductases can be derived from
plant, insect, mammalian, avian or other sources. Specific examples
include, insect cytochrome P450 reductase and cytochrome P450
CYP6B1 and plant cytochrome P450 CYP7B12, CYP7B13, CYP73A5,
CYP86A1, CYP86A2, CYP86A4, CYP86A7 or CYP86A8. "Derived from" can
mean that the target protein is present in a natural (native)
membrane when contacted with MSP to produce Nanodiscs, or the
target protein can be isolated, purified or presolublized, or the
target protein can be associated with the membranes of cells in
which it is recombinantly produced.
[0128] GPCRs which can be solubilized in nanoscale phospholipid
bilayers include the Class A (Rhodopsin-like) GPCRs which bind
amines, peptides, hormone proteins, rhodopsin, olfactory
prostanoid, nucleotide-like compounds, cannabinoids, platelet
activating factor, gonadotropin-releasing hormone,
thyrotropin-releasing hormone and secretagogue, melatonin and
lysosphingolipid and lysophosphatidic acid (LPA), among other
compounds. GPCRs with amine ligands include, without limitation,
acetylcholine or muscarinic, adrenoceptors, dopamine, histamine,
serotonin or octopamine receptors; peptide ligands include, but are
not limited to, angiotensin, bombesin, bradykinin, anaphylatoxin,
Fmet-leu-phe, interleukin-8, chemokine, cholecystokinin,
endothelin, melanocortin, neuropeptide Y, neurotensin, opioid,
somatostatin, tachykinin, thrombin vasopressin-like, galanin,
proteinase activated, orexin and neuropeptide FF, adrenomedullin
(G10D), GPR37/endothelin B-like, chemokine receptor-like and
neuromedin U.
[0129] Other exemplary proteins include mammalian, especially
human, CCR5 and CXCR4 chemokine receptors. These were incorporated
into Nanodiscs by contacting membranes containing native or
recombinant protein. The native protein conformation is maintained,
as evidenced by the reaction of the CCR5-containing and
CXCR4-containing Nanodiscs with CCR5- and CXCR4-specific
antibodies. Nanodiscs containing the human bet-2 adrenergic
receptor have also been made.
[0130] Ligands of other specific GPCRs include hormone proteins,
rhodopsin, olfactory compounds, prostanoid, nucleotide-like
(adenosine, purinoceptors), cannabinoid, platelet activating
factor, gonadotropin-releasing hormone, thyrotropin-releasing
hormone and secretagogue, melatonin and lysosphingolipid and LPA,
among others. Class B secretin-like GPCRs include, without
limitation, those which bind calcitonin, corticotropin releasing
factor, gastric inhibitory peptide, glucagon, growth
hormone-releasing hormone, parathyroid hormone, pituitary adenylate
cyclase activating polypeptide (PACAP), secretin, vasoactive
intestinal polypeptide, diuretic hormone, EMR1 and latrophilin.
Class C metabotropic glutamate receptors include those which bind
metabotropic glutamate, extracellular calcium-sensing receptors or
GABA-B receptors, among others. "Orphan" receptors whose ligands
are not yet known are also potential targets of assays of the
present invention.
[0131] In the assays of the present invention which demonstrate
binding of a particular ligand or which are used to identify
inhibitors or competitors of ligand binding to an MSP-supported
GPCR, a variety of detectable moieties (labels) can be incorporated
within the ligand molecule (such as radioactive isotope, e.g.,
.sup.3H, .sup.14C, .sup.35S, .sup.32P, .sup.125I, .sup.131I,
fluorescent compounds, luminescent compounds, etc.) can be attached
to the ligand molecule provided that binding to the cognate
receptor is not significantly reduced due to the label.
Scanning Probe Microscopy
[0132] An important technique used in the characterization of disk
structures and associated proteins is scanning probe microscopy
(SPM). SPM is an umbrella term for any microscope that utilizes the
scanning principles first pioneered in the scanning tunneling
microscope (STM), but these microscopes can vary so greatly they
are best discussed in terms of their guiding central principle. The
technology has been used in the analysis of biological membranes
and their associated proteins, bilayer structures and incorporated
membrane proteins surfaces. SPM combines independent mobility in
all three spatial directions (scanning) with a detection system
capable of detecting some characteristic of the surface (probing).
The various surface characteristics that can be probed
(conductivity, surface forces, compressibility, capacitance,
magnetic, fluorescence emission) demonstrate the wealth of
information that can be obtained. The excellent z-axis sensitivity
of atomic force microscopy makes the presence of proteins binding
to an rHDL monolayer or in Nanodiscs easily detectable (Bayburt et
al., 1998). Precise height measurements are possible with AFM, and
membrane protein height measurements obtained by modulating the
force of the AFM probe on various Nanodisc assemblies (Bayburt et
al., 2000). The surface association of disks formed from MSPs
allows direct investigation of the biophysical properties of single
membrane proteins incorporated into phospholipid bilayers on
surfaces by SPM. The ability to attach disks to atomically flat
conductive surfaces (such as gold or silica) is necessary for
scanning tunneling microscopy (STM). Without wishing to be bound by
theory, it is believed that tunneling through a redox-active system
can be used to probe the functional state of an enzyme (Friis et
al., 1999; Mukhopadhyay et al., 2000). These two techniques provide
complementary data and can be used in concert to study events
occurring at the bilayer/solution interface. The ability to place
disks on a gold surface also allows the use of surface plasmon
resonance (SPR). Insertion of membrane proteins into such
artificial lipid bilayers, or their interaction with
surface-associated proteins can be detected and quantified by
SPR.
[0133] Other useful solid surfaces onto which Nanodiscs can be
bound include, without limitation, quartz, silica, silicon, silicon
oxide, silicon nitride, polystyrene, plastic and resins.
Disc Stability and Size Dispersion
[0134] Measurements of disk stabilities and determination of size
dispersion among classes are necessary to evaluate the constructs
and Nanodiscs. Gel filtration and native gel electrophoresis are
used to separate and quantitate sizes of particles. Spectroscopy is
used to quantitate secondary structure (CD) and lipid association
(fluorescence) characteristics of the engineered MSPs, including
stabilities based on thermal and chemical denaturation.
Compositions and stoichiometries of components in disks can be
quantitated by traditional methods, using radioactive or
fluorescent labels, mass spectrometry, etc. of protein and lipid
components.
[0135] Advances in the incorporation of fluorophores into the lipid
bilayer of Nanodiscs have been accomplished. Such experiments
provide important information for the incorporation of small
molecules into Nanodiscs for therapeutic use and in the generation
of labeled structures for tissue localization and ADME/toxicology
studies. Fluorescence is one of the most widely used techniques to
track proteins and to analyze protein binding events. Nanodiscs can
be prepared to contain lipophilic fluorescent dyes and to label
proteins. Several different fluorophores have been incorporated
into the lipid bilayer of Nanodiscs during or after self-assembly.
Due to the small size of the lipid bilayer of Nanodiscs (.about.8
nm in diameter) the dye is held within a few nanometers of a
protein incorporated into the bilayer. In addition, the
protein-to-dye stoichiometry can be strictly controlled. This
methodology allows a desired number of dyes to label a protein
without directly attaching the dyes through mutations or other
invasive or potentially destructive techniques.
[0136] In related experiments, numerous fluorescein-labeled lipids
were used in the formation of Nanodiscs. Results have suggested
that as many as 30 to 40 small molecule organic molecules can be
incorporated into a single Nanodiscs without perturbing the
discoidal bilayer structure, as monitored hydromatically. These
highly fluorescent Nanodiscs are useful in optical sensing and
sorting applications, including use in microfluidic arrays and
on-chip analytical systems for diagnostics.
[0137] As an example of this technology, Nanodiscs were assembled
using DPPC doped with DHPE-fluorescein lipids and MSP1. Lipid
mixtures containing 10 and 20% DHPE-fluorescein yielded Nanodiscs
as shown by size exclusion chromatography. These percentages
correspond to 16 and 32 fluorescently labeled lipids per Nanodisc
which have been shown to contain 160 DPPCs when assembled with
MSP1. A variety of lipophilic fluorophores have been incorporated
into Nanodiscs. These include a lipophilic derivative of
fluorescein, the lipid phase state marker laurdan and a derivative
of hydroxycoumarin, a pH sensitive probe. These fluorophores have
been incorporated into Nanodiscs both during and after the assembly
process. Laurdan has been incorporated into Nanodiscs containing
DPPC and DMPC. All of the fluorophores have been incorporated into
Nanodiscs containing DMPC as well as into Nanodiscs which have been
preassembled to incorporate an integral membrane protein
target.
Incorporation of Hydrophobic or Amphipathic Compounds
[0138] Hydrophobic or amphipathic organic compounds, for example
fluorescent and/or lipophilic dyes such as those used to probe
membrane structure, can be readily incorporated into Nanodiscs in
one of two ways. Most commonly, such a compound can be added to the
detergent solubilized mixture. The compound of interest is then
assembled naturally into the final structure during the Nanodisc
assembly which is initiated by detergent removal. Alternately,
these compounds can be incorporated into pre-formed Nanodiscs by
simple incubation. In this case, there is an expected more facile
incorporation into a fluid phospholipid state which is determined
by the incubation temperature relative to the phase transition
temperature of the phospholipid mixture. However, strong
partitioning of such compounds into the hydrophobic bilayer
structure allows successful incorporation even at room temperature
(about 25.degree. C.) with DPPC (phase transition temperature about
42.degree. C.). Lipophilic dyes which partition into Nanodiscs can
include, without limitation, diphenylhexatriene,
octyldecylindocarbocyanine (Dil), C1-BODIPY 500/510,
dihexadecanoylglycerophosphoethanolamine fluorescein.
[0139] Hydrophobic or partially hydrophobic imaging agents,
therapeutic and/or cosmetically active molecules and the like can
also be incorporated using the same or similar protocols.
Atomic Force Microscopy
[0140] AFM is used to provide molecular resolution data on the
structural organization of the lipid and protein components of the
Nanodiscs of the present invention. This technique can be used in
air, vacuum, and under aqueous and non-aqueous fluids. The latter
capability has made it the most important scanning probe technique
in the biological sciences. The AFM is a very versatile instrument
as it is capable of acquiring images and other forms of force data
in contact, tapping, phase, and lateral force modes (Sarid, 1994).
These scanning modes are available on the Digital Instruments
Multimode Scanning Probe Microscope (Digital Instruments,
Plainview, N.Y.), and they have been successfully used to image
rHDL and proteins associated with Nanodiscs both with and without
incorporated proteins. This instrument can also be used in STM and
electrochemical modes to study characteristics of gold-associated
Nanodiscs and incorporated redox proteins.
[0141] Modifications of MSP primary structure can generate
alternative and more effective and stable membrane scaffold
proteins. For instance, we have deleted and/or duplicated helical
regions of MSP1 to produce novel artificial membrane scaffold
proteins. See Table 21 herein below for examples of such membrane
scaffold protein constructs.
[0142] Careful attention to the concentrations of MSP in the
reconstitution mixture is necessary to insure homogeneity with
respect to the sizes of Nanodiscs produced. The optimal
phospholipid to MSP ratio depends on the overall size Nanodisc
generated, which is in turn determined by the overall length of the
encircling membrane scaffold protein. For example, the MSP1
scaffold protein self assembles to form a nominally 9.7 nm diameter
disc with 163 DPPC phospholipid (PL) molecules incorporated per
Nanodisc (81.6 per MSP1). For Nanodiscs which are engineered to be
larger by adding additional helical segments within the MSP, more
phospholipids (PL) are enclosed. MSPE1 with an additional 22-mer
helix generates particles of diameter 10.4 nm and 105.7 PL per
MSP1E1. With two 22-mer helices inserted into the MSP, a Nanodisc
of diameter 11.1 nm is generated with 138.2 PL molecules per
MSP1E2. With three 22-mer helices added, a 12 nm particle is
produced with 176.6 DPPC molecules per resulting Nanodisc.
[0143] We have studied the lipid composition of Nanodiscs formed
with natural cell membranes. The successful application of MSP
technology to the assembly of nanobilayers from natural biological
membranes provides a unique opportunity for the direct isolation of
membrane proteins from cells and their solubilization and dispersal
into a system that closely mimics the native cell environment. To
further clarify the extent to which the phospholipid content of the
isolated Nanodiscs mimics that of the original Sf9 microsomal
membranes, nickel affinity-purified nanostructures assembled with
Sf9 microsomal membranes were analyzed by thin-layer
chromatography. Comparisons of these Nanodisc phospholipid
populations with the major phospholipid types found in insect cell
membranes, which are phosphatidylcholine, phosphatidylinositol, and
phosphatidylethanolamine (Marheineke et al., 1998) (FIG. 17),
clearly indicate that the phospholipid composition of endogenous
Sf9 microsomal membranes is preserved in assembled Nanodiscs.
Functional Proteomics
[0144] To adapt MSP technology to a format compatible with a
functional proteomic analysis of heterologously-expressed membrane
proteins, membranes from Sf9 cells overexpressing CYP6B1 were
completely solubilized with detergent in the presence of the
engineered membrane scaffold protein MSP1. Removal of the detergent
(using Biobeads) initiated self-assembly, allowing for the
incorporation of the membrane protein population into MSP-supported
phospholipid nanobilayers, as outlined in FIG. 14. The
MSP1-containing particles were subsequently isolated using a
nickel-chelating resin to bind the His6-tag on the N-terminus of
the scaffold protein. Analysis of the affinity-purified soluble
nanobilayers by denaturing polyacrylamide gel electrophoresis
confirmed the presence of the CYP6B1 target protein as well as an
array of endogenous proteins present in the original Sf9 cell
membranes (FIG. 15). The nickel affinity-purified sample was
fractionated by size exclusion chromatography (FIG. 16A) and
analyzed by absorbance at 417 nm to identify a 10 nm fraction
containing over 90% of the solubilized heme-containing target
protein.
[0145] Size exclusion chromatography of CYP6B1-expressing Sf9 cell
membranes treated and fractionated in the absence of the membrane
scaffold protein shows that the target elutes as large,
non-specific aggregates (FIG. 16A, dotted line). The homogeneity of
the MSP1-supported Nanodiscs generated is dependent on the identity
of lipid and its ratio of lipid to the amount of MSP used in the
reconstitution procedure (Bayburt et al., 2002) supra). Our
analysis of MSP disks assembled with the natural lipid pool from
Sf9 insect cell membranes indicates other size populations in the
initial nickel affinity-purified Nanodiscs (FIG. 16A). These
variations are due to the difficulty in determining a priori the
precise concentration of MSP protein ideally matched to the lipid
composition in membrane preparations expressing variable amounts of
the heterologous P450 protein and to the significant size
distribution of the endogenous membrane proteins that are also
assembled into nanostructures in this process. These other size
classes represent non-specific aggregates that are easily separated
from the about 10 nm diameter nanobilayer assemblies.
Size-fractionated populations of Nanodiscs containing the P450
target protein are uniform and stable through re-fractionation on a
sizing column, such as Superdex.TM. 200. The final
CYP6B1-containing population displays a stoichiometry of
approximately one CYP6B1 protein per 10 Nanodiscs (FIG. 16B).
[0146] We have examined the integrity of the membrane protein
assembled into Nanodiscs. CYP6B1-containing nanostructures were
assayed by reduction of the iron and binding of carbon monoxide
(CO), which monitors via an absorbance maximum at 450 nm the
quantity of protein that is intact and correctly configured for
P450-mediated catalysis (Omura and Sato (1964) (See FIG. 18). This
spectral assay indicates a clear absence of absorbance at 420 nm
and documents the fact that normally labile proteins, such as
P450s, are incorporated in their native form into Nanodiscs
suitable for subsequent fractionation and biochemical analysis. To
further demonstrate that the solubilized membrane protein is
accessible for binding substrate and suitable for use in
high-throughput optical analysis, binding of xanthotoxin, one of
several furanocoumarin substrates metabolized by this P450, to
MSP1- and CYP6B1-containing Nanodiscs was analyzed in 96-well
microtiter plates using a sample volume of only 200 .mu.l Nanodiscs
(10 picomoles enzyme) and varying concentrations of substrate. The
Type-I binding spectra (Estabrook and Werringloer, 1978) obtained
at varying concentrations of xanthotoxin show an absorbance shift
from 420 nm to 390 nm that is characteristic of substrates
effectively displacing water as the sixth ligand to the heme iron
in the P450 catalytic site and converting the iron from low spin to
high spin. The data presented in FIG. 18 clearly illustrate that
the ability of CYP6B1 to bind substrate is maintained throughout
the Nanodisc assembly and subsequent fractionation process.
[0147] In summary, the present invention provides an important tool
for the study of membrane protein targets as well as the
complicated multi-component assemblies present in cellular
bilayers. When coupled with our ability to express individual
cloned P450s or other membrane proteins in the frequently used
baculovirus, yeast and mammalian expression systems, these
technologies present the opportunity to display single membrane
proteins supported in native membrane bilayers in the development
of biochemical methodologies previously restricted to soluble
proteins. The lipid composition of the particles derived from MSP
and membranes or membrane fragments mimics that of the starting
membranes or fragments, especially where solubilized membrane or
membrane fragment preparations are used as the source of the
phospholipid(s) and hydrophobic protein or other hydrophobic
molecule of interest. This contributes to maintaining the native
conformation and activity of the membrane (or other hydrophobic)
protein which becomes incorporated into the particles with MSP.
[0148] The ability to bind substrates, inhibitors and other
interacting molecules with these solubilized membrane proteins
using sensitive optical difference spectra in microtiter plates
enables the development of high throughput screening methods for
many different types of membrane proteins. For instance, cytochrome
P450 and its reductase stabilized in a functional state through
incorporation into Nanodiscs offer an attractive means for
measurements of drug metabolism and pharmokinetics, with
applications in the pharmaceutical industry. The fact that the
Nanodisc solubilization procedures can be applied nonspecifically
to all membrane proteins means that this technology can be used to
solubilize and fractionate many pharmacological target proteins
directly out of cellular membranes. Coupled with the histidine (or
other) tag on the MSP molecule, this technology enables the
immobilization of target proteins on surfaces suitable for high
throughput screening. All the MSPs described herein can be used in
preparing Nanodiscs with purified and solubilized hydrophobic or
partially hydrophobic proteins or with hydrophobic or partially
hydrophobic membrane proteins solubilized from membrane or membrane
fragment preparations.
Immunogenic Compositions
[0149] Antigens which are hydrophobic or partially hydrophobic can
be formulated into immunogenic compositions for administration to a
human or animal in which an immune response, either cellular or
humoral, is desired. The incorporation of the antigen into a
Nanodisc with a MSP of the present invention allows the preparation
of stable aqueous preparations which do not have a tendency to
aggregate. At least one antigenic determinant of the antigen is
presented to the aqueous phase, with the more hydrophobic portions
of the antigen being buried within the hydrophobic central region
of the Nanodisc. The antigen incorporated within the Nanodisc can
be a protein, such as a cell membrane protein or a viral envelope
protein, or it can be a lipopolysaccharide or a
lipooligosaccharide.
[0150] The antigen can be derived from a virus, especially an
enveloped virus, a bacterium including, but not limited to, a
bacterium, fungus, protozoan, parasite, or it can be derived from a
particular type of tumor or cancer. The antigen-containing Nanodisc
preparation can be administered in prophylactic or therapeutic
treatment regimens to generate an immune response, and
administration of these Nanodiscs can be carried out in combination
with other vaccine preparations for priming and/or boosting.
[0151] Cancers (neoplastic conditions) from which cells can be
obtained for use as an antigen source in the methods of the present
invention include carcinomas, sarcomas, leukemias and cancers
derived from cells of the nervous system. These include, but are
not limited to bone cancers (osteosarcoma), brain cancers,
pancreatic cancers, lung cancers such as small and large cell
adenocarcinomas, rhabdosarcoma, mesiothelioma, squamous cell
carcinoma, basal cell carcinoma, malignant melanoma, other skin
cancers, bronchoalveolar carcinoma, colon cancers, other
gastrointestinal cancers, renal cancers, liver cancers, breast
cancers, cancers of the uterus, ovaries or cervix, prostate
cancers, lymphomas, myelomas, bladder cancers, cancers of the
reticuloendothelial system (RES) such as B or T cell lymphomas,
melanoma, and soft tissue cancers.
[0152] The terms "neoplastic cell", "tumor cell", or "cancer cell",
used either in the singular or plural form, refer to cells that
have undergone a malignant transformation that makes them harmful
to the host organism. Primary cancer cells (that is, cells obtained
from near the site of malignant transformation) can be readily
distinguished from non-cancerous cells by well-established
techniques, particularly histological examination. The definition
of a cancer cell, as used herein, includes not only a primary
cancer cell, but also any cell derived from a cancer cell ancestor.
This includes metastasized cancer cells, and in vitro cultures and
cell lines derived from cancer cells. When referring to a type of
cancer that normally manifests as a solid tumor, a "clinically
detectable" tumor is one that is detectable on the basis of tumor
mass; e.g., by such procedures as CAT scan, magnetic resonance
imaging (MRI), X-ray, ultrasound, or palpation. Biochemical or
immunologic findings alone may be insufficient to meet this
definition.
[0153] Pathogens to which multiple antigen immunological responses
are advantageous include viral, bacterial, fungal and protozoan
pathogens. Viruses to which immunity is desirable include, but are
not limited to, hemorrhagic fever viruses (such as Ebola virus),
immune deficiency viruses (such as feline or human immunodeficiency
viruses), herpesviruses, coronaviruses, adenoviruses, poxviruses,
picornaviruses, orthomyxoviruses, paramyxoviruses, rubella,
togaviruses, flaviviruses, bunyaviruses, reoviruses, oncogenic
viruses such as retroviruses, pathogenic alphaviruses (such as
Semliki forest virus or Sindbis virus), rhinoviruses, hepatitis
viruses (Group B, C, etc), influenza viruses, among others.
Bacterial pathogens to which immune responses are helpful include,
without limitation, staphylococci, streptococci, pneumococci,
salmonellae, escherichiae, yersiniae, enterococci, clostridia,
corynebacteria, hemophilus, neisseriae, bacteroides, francisella,
legionella, pasteurellae, brucellae, mycobacteriae, bordetella,
spirochetes, actinomycetes, chlamydiae, mycoplasmas, rickettsias,
and others. Pathogenic fungi of interest include but are not
limited to Candida, cryptococci, blastomyces, histoplasma,
coccidioides, phycomycetes, trichodermas, aspergilli, pneumocystis,
and others. Protozoans to which immunity is useful include, without
limitation, toxoplasma, plasmodia, schistosomes, amoebae, giardia,
babesia, leishmania, and others. Other parasites include the
roundworms, hookworms and tapeworms, filiaria and others.
[0154] A further object of the present invention is the
administration of the antigen-containing immunogenic Nanodisc
compositions of the present invention to a human or animal (e.g.
horse, pig, cow, goat, rabbit, mouse, hamster) to generate immune
responses, such as production of antibody specific to the antigen
or a cellular response such that cells or tissues sharing the
antigen are the subject of a cellular or cytotoxic immune response.
Sera or cells collected from such humans or animals are useful in
providing polyclonal sera or cells for the production of hybridomas
that generate monoclonal sera, such antibody preparations being
useful in research, diagnostic, and therapeutic applications.
[0155] While the generation of an immune response includes at least
some level of protective immunity directed to the tumor cell (or
neoplastic condition), pathogen or parasite, the clinical outcome
in the patient suffering from such a neoplastic condition or
infection with a parasite or a pathogen can be improved by also
treating the patient with a suitable chemotherapeutic agent, as
known to the art. Where the pathogen is viral, an anti-viral
compound such as acyclovir can be administered concomitantly with
antigen-containing Nanodisc vaccination in patients with herpes
virus infection, or HAART (highly active anti-retroviral therapy)
in individuals infected with HIV. Where the pathogen is a bacterial
pathogen, an antibiotic to which that bacterium is susceptible is
desirably administered and where the pathogen is a fungus, a
suitable antifungal antibiotic is desirably administered.
[0156] Similarly, chemical agents for the control and/or
eradication of parasitic infections are known and are
advantageously administered to the human or animal patients using
dosages and schedules well known to the art. Where the patient is
suffering from a neoplastic condition, for example, a cancer, the
administration of the immunogenic composition comprising the
Nanodiscs carrying one or more multiplicity of cancer-associated
antigens in the patient to which it has been administered is
desirably accompanied by administration of antineoplastic agent(s),
including, but not limited to, such chemotherapeutic agents as
daunorubicin, taxol, thioureas, cancer-specific antibodies linked
with therapeutic radionuclides, with the proviso that the agent(s)
do not ablate the ability of the patient to generate an immune
response to the administered Nanodiscs and the antigens whose
expression they direct in the patient. Nucleic acids for modulating
gene expression or for directing expression of a functional protein
can be incorporated within Nanodiscs, especially where the nucleic
acid molecules are complexed with a cationic lipids, many of which
are commercially available.
[0157] Pharmaceutical formulations, such as vaccines or other
immunogenic compositions, of the present invention comprise an
immunogenic amount of the antigen-bearing Nanodiscs in combination
with a pharmaceutically acceptable carrier. An immunogenic amount"
is an amount of the antigen-bearing Nanodiscs which is sufficient
to evoke an immune response in the subject to which the
pharmaceutical formulation is administered. An amount of from about
10.sup.3 to about 10.sup.11 particles per dose, preferably 10.sup.5
to 10.sup.9, is believed suitable, depending upon the age and
species of the subject being treated. Depending on the setting for
administration (i.e., disease treatment or prevention), the dose
(and repetition of administration) can be chosen to be
therapeutically effective or prophylactically effective.
[0158] Exemplary pharmaceutically acceptable carriers include, but
are not limited to, sterile pyrogen-free water and sterile
pyrogen-free physiological saline solution. Subjects which may be
administered immunogenic amounts of the antigen-carrying Nanodiscs
of the present invention include, but are not limited to, human and
animal (e.g., dog, cat, horse, pig, cow, goat, rabbit, donkey,
mouse, hamster, monkey) subjects. Immunologically active compounds
such as cytokines and/or BCG can also be added to increase the
immune response to the administered immunogenic preparation.
[0159] Immunogenic compositions comprising the Nanodiscs which
incorporate antigens of interest produced using the methods of the
present invention may be formulated by any of the means known in
the art. Such compositions, especially vaccines, are typically
prepared as injectables, either as liquid solutions or suspensions.
Solid forms suitable for solution in, or suspension in, liquid
prior to injection may also be prepared.
[0160] The active immunogenic ingredients (the Nanodiscs) are
advantageously mixed with excipients or carriers that are
pharmaceutically acceptable and compatible with the active
ingredient. Suitable excipients include but are not limited to
sterile water, saline, dextrose, glycerol, ethanol, or the like and
combinations thereof.
[0161] In addition, if desired, the immunogenic compositions,
including vaccines, may contain minor amounts of auxiliary
substances such as wetting or emulsifying agents, pH buffering
agents, and/or adjuvants which enhance the effectiveness of the
vaccine. Examples of adjuvants which may be effective include but
are not limited to aluminum hydroxide;
N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP);
N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred
to as nor-MDP);
N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dip-
almitoyl-sn-glycero-3hydroxyphosphoryloxy)-ethylamine (CGP 19835A,
referred to as MTP-PE); and RIBI, which contains three components
extracted from bacteria, monophosphoryl lipid A, trehalose
dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2%
squalene/Tween 80 emulsion. The effectiveness of an adjuvant may be
determined by measuring the amount of antibodies directed against
the immunogenic component of the nanoscale particles after
administration. Such additional formulations and modes of
administration as known in the art may also be used.
[0162] The immunogenic (or otherwise biologically active)
antigen-containing Nanodisc compositions are administered in a
manner compatible with the dosage formulation, and in such amount
as will be prophylactically and/or therapeutically effective. The
quantity to be administered, which is generally in the range of
about 10.sup.3 to about 10.sup.10 particles, preferably 10.sup.5 to
10.sup.8, in a dose, depends on the subject to be treated, the
capacity of the individual's immune system to synthesize
antibodies, and the degree of protection desired. Precise amounts
of the active ingredient required to be administered may depend on
the judgment of the physician, veterinarian or other health
practitioner and may be peculiar to each individual, but such a
determination is within the skill of such a practitioner.
[0163] The vaccine or other immunogenic composition may be given in
a single dose or multiple dose schedule. A multiple dose schedule
is one in which a primary course of vaccination may include 1 to 10
or more separate doses, followed by other doses administered at
subsequent time intervals as required to maintain and or reinforce
the immune response, e.g., at weekly, monthly or 1 to 4 months for
a second dose, and if needed, a subsequent dose(s) after several
months or years. Hydrophobic or partially hydrophobic antigens can
be incorporated into Nanodiscs as described for other molecules
(such as membrane proteins or small molecules). Where the antigen
is in nature associated with or is within a membrane, either a
solubilized pure or partially pure preparation or a solubilized
membrane or membrane fragment preparation can be used as the source
of the input antigen in the Nanodisc assembly mixture.
Nuclear Magnetic Resonance
[0164] A current method for the diagnosis of myocardial ischemia
utilizes an NMR relaxation agent containing gadolinium (Gd). The
current market leader is Magnevist (Trademark of Berlex). The Gd
metal is chelated in the form of gadopentetate dimeglumine.
Unfortunately, the half life of this compound is only a few minutes
in humans, due to its small size and rapid clearance. Nanodiscs are
believed to have a half life of several hours in human plasma.
[0165] Various organic and inorganic complexes can be incorporated
into the Nanodisc bilayer by conjugation (covalent attachment) with
fatty-acid like chains that then partition into the Nanodisc
bilayer. We have used this technique to affix fluorescent molecules
to the Nanodisc at various loadings. For a typical 10 nm diameter
Nanodisc containing about 160 DPPC phospholipid molecules, up to
about 40-50 such alkyl chain-anchored species can be incorporated,
replacing the native phospholipids, without comprising the Nanodisc
structure. This same procedure can be used to affix other organics
or inorganics to the Nanodisc, wherein the Nanodisc then becomes a
carrier of the compound and conveys the advantageously controlled
circulation lifetime while providing small and robust size. Such
compounds include sugars, imaging agents, lipophilic dyes,
photoactive (photodynamic) agents, etc. Photodynamic agents
include, but are not limited to, those useful for treating tumors
or atherosclerotic plaques, for example, porphyrins and
phthalacyanin-related molecules.
[0166] Various chelating agents can be so constructed to provide a
Nanodisc with approximately 50 Gd relaxation agents in a 10 nm
diameter package. This should have great benefit in providing a
longer lifetime imaging agent for cardiovascular imaging. We have
completed a first experiment along these lines using a commercially
available chelating agent, but which provides an incomplete
coordination of the Gd molecule. This compound then is prone to
precipitation. It is straightforward chemistry (J. Med. Chem. 42,
2852 (1999)) to affix a long alkyl chain to the Magnevist
structure, for example at the methylene carbon position, to have
the same chelating properties as Magnevist but now in a more
concentrated entity with increased plasma lifetime.
Functional Equivalents
[0167] It is understood that a variant of a specifically
exemplified MSP can be made with an amino acid sequence which is
substantially identical (at least about 80 to 99% identical, and
all integers therebetween) to the amino acid sequence to an MSP of
the present invention and it forms a functionally equivalent,
amphiphilic, three dimensional structure and retains the ability to
form Nanodiscs with phospholipid and/or a passenger molecule such
as a hydrophobic or partially hydrophobic protein, among others. It
is well known in the biological arts that certain amino acid
substitutions can be made in protein sequences without affecting
the function of the protein.
[0168] Generally, conservative amino acid substitutions or
substitutions of similar amino acids are tolerated without
affecting protein function. Similar amino acids can be those that
are similar in size and/or charge properties, for example,
aspartate and glutamate and isoleucine and valine are both pairs of
similar amino acids. Nonpolar amino acids include alanine, valine,
leucine, phenylalanine, tryptophan, methionine, isoleucine,
cysteine and glycine. Uncharged polar amino acids include serine,
threonine, asparagine, glutamine and tyrosine. Charged polar basic
amino acids include lysine, arginine and histidine. Substitutions
of one for another are permitted when helix formation is not
disrupted except as intended. Similarity between amino acid pairs
has been assessed in the art in a number of ways. For example,
Dayhoff et al. (1978) in Atlas of Protein Sequence and Structure,
Volume 5, Supplement 3, Chapter 22, pages 345-352, which is
incorporated by reference herein, provides frequency tables for
amino acid substitutions which can be employed as a measure of
amino acid similarity. Dayhoff et al.'s frequency tables are based
on comparisons of amino acid sequences for proteins having the same
function from a variety of evolutionarily different sources.
[0169] Substitution mutation, insertional, and deletional variants
of the disclosed nucleotide (and amino acid) sequences can be
readily prepared by methods which are well known to the art. These
variants can be used in the same manner as the exemplified MSP
sequences so long as the variants have substantial sequence
identity with a specifically exemplified sequence of the present
invention. As used herein, substantial sequence identity refers to
homology (or identity) which is sufficient to enable the variant
polynucleotide or protein to function in the same capacity as the
polynucleotide or protein from which the variant is derived.
Preferably, this sequence identity is greater than 70% or 80%, more
preferably, this identity is greater than 85%, or this identity is
greater than 90%, and or alternatively, this is greater than 95%,
and all integers between 70 and 100%. It is well within the skill
of a person trained in this art to make substitution mutation,
insertional, and deletional mutations which are equivalent in
function or are designed to improve the function of the sequence or
otherwise provide a methodological advantage. No variants which may
read on any naturally occurring proteins or which read on a prior
art variant are intended to be within the scope of the present
invention as claimed.
[0170] It is well known in the art that the polynucleotide
sequences of the present invention can be truncated and/or mutated
such that certain of the resulting fragments and/or mutants of the
original full-length sequence can retain the desired
characteristics of the full-length sequence. A wide variety of
restriction enzymes which are suitable for generating fragments
from larger nucleic acid molecules are well known. In addition, it
is well known that Bal31 exonuclease can be conveniently used for
time-controlled limited digestion of DNA. See, for example,
Maniatis (1982) Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor Laboratory, New York, pages 135-139, incorporated herein by
reference. See also Wei et al. (1983 J. Biol. Chem.
258:13006-13512. By use of Bal31 exonuclease (commonly referred to
as erase-a-base procedures), the ordinarily skilled artisan can
remove nucleotides from either or both ends of the subject nucleic
acids to generate a wide spectrum of fragments which are
functionally equivalent to the subject nucleotide sequences. One of
ordinary skill in the art can, in this manner, generate hundreds of
fragments of controlled, varying lengths from locations all along
the original MSP-encoding sequence. The ordinarily skilled artisan
can routinely test or screen the generated fragments for their
characteristics and determine the utility of the fragments as
taught herein. It is also well known that the mutant sequences of
the full length sequence, or fragments thereof, can be easily
produced with site directed mutagenesis. See, for example,
Larionov, O. A. and Nikiforov, V. G. (1982) Genetika 18(3):349-59;
Shortle, D, DiMaio, D., and Nathans, D. (1981) Annu. Rev. Genet.
15:265-94; both incorporated herein by reference. The skilled
artisan can routinely produce deletion-, insertion-, or
substitution-type mutations and identify those resulting mutants
which contain the desired characteristics of the full length
wild-type sequence, or fragments thereof, i.e., those which retain
membrane scaffold protein activity, i.e., ability to self assemble
with phospholipid to form nanoscale disc-like particles.
[0171] As used herein percent sequence identity of two nucleic
acids is determined using the algorithm of Altschul et al. (1997)
Nucl. Acids Res. 25: 3389-3402; see also Karlin and Altschul (1990)
Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and
Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an
algorithm is incorporated into the NBLAST and XBLAST programs of
Altschul et al. (1990) J. Mol. Biol. 215:402-410. BLAST nucleotide
searches are performed with the NBLAST program, score=100,
wordlength=12, to obtain nucleotide sequences with the desired
percent sequence identity. To obtain gapped alignments for
comparison purposes, Gapped BLAST is used as described in Altschul
et al. (1997) Nucl. Acids. Res. 25:3389-3402. When utilizing BLAST
and Gapped BLAST programs, the default parameters of the respective
programs (NBLAST and XBLAST) are used. See the National Center for
Biotechnology Information on the internet.
Antibody Technology
[0172] Monoclonal or polyclonal antibodies, preferably monoclonal,
specifically reacting with an MSP of the present invention (or to
another protein of interest) can be made by methods known in the
art. See, e.g., Harlow and Lane (1988) Antibodies: A Laboratory
Manual, Cold Spring Harbor Laboratories; Goding (1986) Monoclonal
Antibodies: Principles and Practice, 2d ed., Academic Press, New
York; and Ausubel et al. (1993) Current Protocols in Molecular
Biology, Wiley Interscience, New York, N.Y.
[0173] Standard techniques for cloning, DNA isolation,
amplification and purification, for enzymatic reactions involving
DNA ligase, DNA polymerase, restriction endonucleases and the like,
and various separation techniques are those known and commonly
employed by those skilled in the art. A number of standard
techniques are described in Sambrook et al. (1989) Molecular
Cloning, Second Edition, Cold Spring Harbor Laboratory, Plainview,
N.Y.; Maniatis et al. (1982) Molecular Cloning, Cold Spring Harbor
Laboratory, Plainview, N.Y.; Wu (ed.) (1993) Meth. Enzymol. 218,
Part I; Wu (ed.) (1979) Meth. Enzymol. 68; Wu et al. (eds.) (1983)
Meth. Enzymol. 100 and 101; Grossman and Moldave (eds.) Meth.
Enzymol. 65; Miller (ed.) (1972) Experiments in Molecular Genetics,
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Old and
Primrose (1981) Principles of Gene Manipulation, University of
California Press, Berkeley; Schleif and Wensink (1982) Practical
Methods in Molecular Biology; Glover (ed.) (1985) DNA Cloning Vol.
I and II, IRL Press, Oxford, UK; Hames and Higgins (eds.) (1985)
Nucleic Acid Hybridization, IRL Press, Oxford, UK; Setlow and
Hollaender (1979) Genetic Engineering: Principles and Methods,
Vols. 1-4, Plenum Press, New York; and Ausubel et al. (1992)
Current Protocols in Molecular Biology, Greene/Wiley, New York,
N.Y. Abbreviations and nomenclature, where employed, are deemed
standard in the field and commonly used in professional journals
such as those cited herein.
[0174] All references cited in the present application are
incorporated by reference herein to the extent that there is no
inconsistency with the present disclosure.
[0175] The description provided herein is not intended to limit the
scope of the invention as claimed herein. Any variations in the
exemplified articles and methods which occur to the skilled artisan
are intended to fall within the scope of the present invention.
EXAMPLES
Example 1
Construction of Recombinant DNA Molecules for Expression of
MSPs
[0176] The human proapo A-I coding sequence as given below was
inserted between NcoI and HindIII sites (underlined) in pET-28
(Novagen, Madison, Wis.). Start and stop codons are in bold type.
The restriction endonuclease recognition sites used in cloning are
underlined.
TABLE-US-00002 TABLE 1 ProApo A-I coding sequence (SEQ ID NO: 1)
CCATGGCCCATTTCTGGCAGCAAGATGAACCCCCCCAGAGCCCCTGGGAT
CGAGTGAAGGACCTGGCCACTGTGTACGTGGATGTGCTCAAAGACAGCGG
CAGAGACTATGTGTCCCAGTTTGAAGGCTCCGCCTTGGGAAAACAGCTAA
ACCTAAAGCTCCTTGACAACTGGGACAGCGTGACCTCCACCTTCAGCAAG
CTGCGCGAACAGCTCGGCCCTGTGACCCAGGAGTTCTGGGATAACCTGGA
AAAGGAGACAGAGGGCCTGAGGCAAGAGATGAGCAAGGATCTGGAGGAGG
TGAAGGCCAAGGTGCAGCCCTACCTGGACGACTTCCAGAAGAAGTGGCAG
GAGGAGATGGAGCTCTACCGCCAGAAGGTGGAGCCGCTGCGCGCAGAGCT
CCAAGAGGGCGCGCGCCAGAAGCTGCACGAGCTGCAAGAGAAGCTGAGCC
CACTGGGCGAGGAGATGCGCGACCGCGCGCGCGCCCATGTGGACGCGCTG
CGCACGCATCTGGCCCCCTACAGCGACGAGCTGCGCCAGCGCTTGGCCGC
GCGCCTTGAGGCTCTCAAGGAGAACGGCGGCGCCAGACTGGCCGAGTACC
ACGCCAAGGCCACCGAGCATCTGAGCACGCTCAGCGAGAAGGCCAAGCCC
GCGCTCGAGGACCTCCGCCAAGGCCTGCTGCCCGTGCTGGAGAGCTTCAA
GGTCAGCTTCCTGAGCGCTCTCGAGGAGTACACTAAGAAGCTCAACACCC AGTAATAAGCTT-3'
Restriction sites used in cloning are underlined, and the
translation start and stop signals are shown in bold.
TABLE-US-00003 TABLE 2 ProApo A-I amino acid sequence (SEQ ID NO:
2) MAHFWQQDEPPQSPWDRVKDLATVYVDVLKDSGRDYVSQFEGSALGKQLN
LKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEV
KAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSP
LGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYH
AKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ
[0177] The construction of the MSP1 coding sequence was
accomplished as follows. Primers were designed to produce DNA
encoding MSP1, the truncated protein lacking the N-terminal domain
of proApo A-I, by polymerase chain reaction (PCR) mutagenesis
(Higuchi et al., 1988).
Primer 1 (SEQ ID NO:3) (5'-TATACCATGGGCCATCATCATCATCATCATATAGAAGGAA
GACTAAAGCTCCTTGACAACT-3') introduces an N-terminal 6-histidine tag
for purification and manipulation of MSP1, and a factor Xa cleavage
site for removal of the histidine tag. Factor Xa cleaves after R in
the protein sequence IEGR. Primer 2 (SEQ ID NO:4)
(5'-GCAAGCTTATTACTGGGTGTTGAGCTTCTT-3') was used as a reverse
primer.
TABLE-US-00004 TABLE 3 Histidine-tagged MSP1 coding sequence (SEQ
ID NO: 5). TATACCATGGGCCATCATCATCATCATCATATAGAAGGAAGACTAAAGCT
CCTTGACAACTGGGACAGCGTGACCTCCACCTTCAGCAAGCTGCGCGAAC
AGCTCGGCCCTGTGACCCAGGAGTTCTGGGATAACCTGGAAAAGGAGACA
GAGGGCCTGAGGCAGGAGATGAGCAAGGATCTGGAGGAGGTGAAGGCCAA
GGTGCAGCCCTACCTGGACGACTTCCAGAAGAAGTGGCAGGAGGAGATGG
AGCTCTACCGCCAGAAGGTGGAGCCGCTGCGCGCAGAGCTCCAAGAGGGC
GCGCGCCAGAAGCTGCACGAGCTGCAAGAGAAGTTGAGCCCACTGGGCGA
GGAGATGCGCGACCGCGCGCGCGCCCATGTGGACGCGCTGCGCACGCATC
TGGCCCCCTACAGCGACGAGCTGCGCCAGCGCTTGGCCGCGCGCCTTGAG
GCTCTCAAGGAGAACGGCGGCGCCAGACTGGCCGAGTACCACGCCAAGGC
CACCGAGCATCTGAGCACGCTCAGCGAGAAGGCCAAACCCGCGCTCGAGG
ACCTCCGCCAAGGCCTGCTGCCCGTGCTGGAGAGCTTCAAGGTCAGCTTC
CTGAGCGCTCTCGAGGAGTACACTAAGAAGCTCAACACCCAGTAATAAGC TTGC Restriction
sites used in cloning are underlined, and the translation start and
stop signals are shown in bold.
TABLE-US-00005 TABLE 4 Histidine-tagged MSP1 amino acid sequence
(SEQ ID NO: 6) MGHHHHHHIEGRLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEG
LRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGAR
QKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEAL
KENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLS ALEEYTKKLNTQ
[0178] For production of MSP1 without a N-terminal histidine tag,
primer 1 was replaced with primer 1a:
5'-TACCATGGCAAAGCTCCTTGACAACTG-3' (SEQ ID NO:7) to produce the
sequence provided in SEQ ID NO:8.
TABLE-US-00006 TABLE 5 Non-Histidine-tagged MSP1 DNA sequence (SEQ
ID NO: 8). TACCATGGCAAAGCTCCTTGACAACTGGGACAGCGTGACCTCCACCTTCA
GCAAGCTGCGCGAACAGCTCGGCCCTGTGACCCAGGAGTTCTGGGATAAC
CTGGAAAAGGAGACAGAGGGCCTGAGGCAGGAGATGAGCAAGGATCTGGA
GGAGGTGAAGGCCAAGGTGCAGCCCTACCTGGACGACTTCCAGAAGAAGT
GGCAGGAGGAGATGGAGCTCTACCGCCAGAAGGTGGAGCCGCTGCGCGCA
GAGCTCCAAGAGGGCGCGCGCCAGAAGCTGCACGAGCTGCAAGAGAAGTT
GAGCCCACTGGGCGAGGAGATGCGCGACCGCGCGCGCGCCCATGTGGACG
CGCTGCGCACGCATCTGGCCCCCTACAGCGACGAGCTGCGCCAGCGCTTG
GCCGCGCGCCTTGAGGCTCTCAAGGAGAACGGCGGCGCCAGACTGGCCGA
GTACCACGCCAAGGCCACCGAGCATCTGAGCACGCTCAGCGAGAAGGCCA
AACCCGCGCTCGAGGACCTCCGCCAAGGCCTGCTGCCCGTGCTGGAGAGC
TTCAAGGTCAGCTTCCTGAGCGCTCTCGAGGAGTACACTAAGAAGCTCAA
CACCCAGTAATAAGCTTGC Restriction sites used in cloning are
underlined, and the translation start and stop signals are shown in
bold.
TABLE-US-00007 TABLE 6 Non-Histidine-tagged MSP1 amino acid
sequence (SEQ ID NO: 9).
MAKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEE
VKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLS
PLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEY
HAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNT Q
[0179] The production of an MSP with tandem repeats (MSP2) was
carried at as described below. The following primers were used to
generate MSP2 (see FIGS. 6A-6B):
TABLE-US-00008 Primer 3 (SEQ ID NO: 10):
5'-TACCATGGCAAAGCTCCTTGACAACTG-3' primer3a (SEQ ID NO: 11):
5'-TATACCATGGGCCATCATCATCATCATCATATAGAAGGAAGACTAAA
GCTCCTTGACAACT-3' Primer 4 (SEQ ID NO: 12):
5'-TAAGAAGCTCAACACCCAGGGTACCGGTGGAGGTAGTGGAGGTGGTA CCCTA-3' Primer
5 (SEQ ID NO: 13):
5'-CAGGGTACCGGTGGAGGTAGTGGAGGTGGTACCCTAAAGCTCCTTGA CAA-3' Primer 6
(SEQ ID NO: 14): 5'-GCAAGCTTATTACTGGGTGTTGAGCTTCTT-3'
[0180] In a first PCR, primer 2 (or primer 2a for N-terminal
histidine tag) and primer 4 were used to add a linker sequence
(encoding the amino acid sequence GTGGGSGGGT; SEQ ID NO:15) to the
3' end of the MSP gene to produce MSP-A. In a second PCR, the
linker was added to the 5' end of the MSP gene to produce MSP-B.
Treatment of MSP-A and MSP-B with KpnI and subsequent ligation
produced the following constructs, one with and one without the
linker. The Kpn I site provides an easy way to inserting any
desired linker sequence by restriction with Kpn I and religation
with double-stranded synthetic DNA encoding desired linker. See
FIGS. 6A-6B.
TABLE-US-00009 TABLE 7 MSP2 (with histidine tag, without long
linker) DNA sequence (SEQ ID NO: 16).
TATACCATGGGCCATCATCATCATCATCATATAGAAGGAAGACTAAAGCT
CCTTGACAACTGGGACAGCGTGACCTCCACCTTCAGCAAGCTGCGCGAAC
AGCTCGGCCCTGTGACCCAGGAGTTCTGGGATAACCTGGAAAAGGAGACA
GAGGGCCTGAGGCAGGAGATGAGCAAGGATCTGGAGGAGGTGAAGGCCAA
GGTGCAGCCCTACCTGGACGACTTCCAGAAGAAGTGGCAGGAGGAGATGG
AGCTCTACCGCCAGAAGGTGGAGCCGCTGCGCGCAGAGCTCCAAGAGGGC
GCGCGCCAGAAGCTGCACGAGCTGCAAGAGAAGCTGAGCCCACTGGGCGA
GGAGATGCGCGACCGCGCGCGCGCCCATGTGGACGCGCTGCGCACGCATC
TGGCCCCCTACAGCGACGAGCTGCGCCAGCGCTTGGCCGCGCGCCTTGAG
GCTCTCAAGGAGAACGGCGGCGCCAGACTGGCCGAGTACCACGCCAAGGC
CACCGAGCATCTGAGCACGCTCAGCGAGAAGGCCAAGCCCGCGCTCGAGG
ACCTCCGCCAAGGCCTGCTGCCCGTGCTGGAGAGCTTCAAGGTCAGCTTC
CTGAGCGCTCTCGAGGAGTACACTAAGAAGCTCAACACCCAGGGTACCCT
AAAGCTCCTTGACAACTGGGACAGCGTGACCTCCACCTTCAGCAAGCTGC
GCGAACAGCTCGGCCCTGTGACCCAGGAGTTCTGGGATAACCTGGAAAAG
GAGACAGAGGGCCTGAGGCAGGAGATGAGCAAGGATCTGGAGGAGGTGAA
GGCCAAGGTGCAGCCCTACCTGGACGACTTCCAGAAGAAGTGGCAGGAGG
AGATGGAGCTCTACCGCCAGAAGGTGGAGCCGCTGCGCGCAGAGCTCCAA
GAGGGCGCGCGCCAGAAGCTGCACGAGCTGCAAGAGAAGCTGAGCCCACT
GGGCGAGGAGATGCGCGACCGCGCGCGCGCCCATGTGGACGCGCTGCGCA
CGCATCTGGCCCCCTACAGCGACGAGCTGCGCCAGCGCTTGGCCGCGCGC
CTTGAGGCTCTCAAGGAGAACGGCGGCGCCAGACTGGCCGAGTACCACGC
CAAGGCCACCGAGCATCTGAGCACGCTCAGCGAGAAGGCCAAGCCCGCGC
TCGAGGACCTCCGCCAAGGCCTGCTGCCCGTGCTGGAGAGCTTCAAGGTC
AGCTTCCTGAGCGCTCTCGAGGAGTACACTAAGAAGCTCAACACCCAGTA ATAAGCTTGC The
translation start and stop codons are in bold type, and the
restriction endonuclease recognition sites used in cloning are
underlined.
TABLE-US-00010 TABLE 8 MSP2 (with histidine tag, without long
linker) amino acid sequence (SEQ ID NO: 17)
MGHHHHHHIEGRLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEG
LRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGAR
QKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEAL
KENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLS
ALEEYTKKLNTQGTLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKET
EGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEG
ARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLE
ALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSF
LSALEEYTKKLNTQ
TABLE-US-00011 TABLE 9 MSP2L (with histidine tag, with long linker)
DNA sequence (SEQ ID NO: 18).
TACCATGGGCCATCATCATCATCATCATATAGAAGGAAGACTAAAGCTCC
TTGACAACTGGGACAGCGTGACCTCCACCTTCAGCAAGCTGCGCGAACAG
CTCGGCCCTGTGACCCAGGAGTTCTGGGATAACCTGGAAAAGGAGACAGA
GGGCCTGAGGCAGGAGATGAGCAAGGATCTGGAGGAGGTGAAGGCCAAGG
TGCAGCCCTACCTGGACGACTTCCAGAAGAAGTGGCAGGAGGAGATGGAG
CTCTACCGCCAGAAGGTGGAGCCGCTGCGCGCAGAGCTCCAAGAGGGCGC
GCGCCAGAAGCTGCACGAGCTGCAAGAGAAGCTGAGCCCACTGGGCGAGG
AGATGCGCGACCGCGCGCGCGCCCATGTGGACGCGCTGCGCACGCATCTG
GCCCCCTACAGCGACGAGCTGCGCCAGCGCTTGGCCGCGCGCCTTGAGGC
TCTCAAGGAGAACGGCGGCGCCAGACTGGCCGAGTACCACGCCAAGGCCA
CCGAGCATCTGAGCACGCTCAGCGAGAAGGCCAAGCCCGCGCTCGAGGAC
CTCCGCCAAGGCCTGCTGCCCGTGCTGGAGAGCTTCAAGGTCAGCTTCCT
GAGCGCTCTCGAGGAGTACACTAAGAAGCTCAACACCCAGGGTACCGGTG
GAGGTAGTGGAGGTGGTACCCTAAAGCTCCTTGACAACTGGGACAGCGTG
ACCTCCACCTTCAGCAAGCTGCGCGAACAGCTCGGCCCTGTGACCCAGGA
GTTCTGGGATAACCTGGAAAAGGAGACAGAGGGCCTGAGGCAGGAGATGA
GCAAGGATCTGGAGGAGGTGAAGGCCAAGGTGCAGCCCTACCTGGACGAC
TTCCAGAAGAAGTGGCAGGAGGAGATGGAGCTCTACCGCCAGAAGGTGGA
GCCGCTGCGCGCAGAGCTCCAAGAGGGCGCGCGCCAGAAGCTGCACGAGC
TGCAAGAGAAGCTGAGCCCACTGGGCGAGGAGATGCGCGACCGCGCGCGC
GCCCATGTGGACGCGCTGCGCACGCATCTGGCCCCCTACAGCGACGAGCT
GCGCCAGCGCTTGGCCGCGCGCCTTGAGGCTCTCAAGGAGAACGGCGGCG
CCAGACTGGCCGAGTACCACGCCAAGGCCACCGAGCATCTGAGCACGCTC
AGCGAGAAGGCCAAGCCCGCGCTCGAGGACCTCCGCCAAGGCCTGCTGCC
CGTGCTGGAGAGCTTCAAGGTCAGCTTCCTGAGCGCTCTCGAGGAGTACA
CTAAGAAGCTCAACACCCAGTAATAAGCTTGC Translation start and stop codons
are in bold type; restriction endonuclease sites used in cloning
are underlined.
TABLE-US-00012 TABLE 10 MSP2 (with histidine tag, with long linker,
in bold type) amino acid sequence (SEQ ID NO: 19).
MGHHHHHHIEGRLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEG
LRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGAR
QKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEAL
KENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLS
ALEEYTKKLNTQGTGGGSGGGTLKLLDNWDSVTSTFSKLREQLGPVTQEF
WDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEP
LRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELR
QRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPV
LESFKVSFLSALEEYTKKLNTQ
[0181] To delete hinge regions, deletion of helices 4 and 5 was
carried out by constructing the C-terminal portion of MSP1 using
the following PCR primers and the Sac I and Hind III fragment of
the MSP1 coding sequence as template.
TABLE-US-00013 Primer A (SEQ ID NO: 20):
5'-TGGAGCTCTACCGCCAGAAGGTGGAGCCCTACAGCGACGAGCT-3' Primer B (SEQ ID
NO: 21): 5'-GCAAGCTTATTACTGGGTGTTGAGCTTCTT-3'.
[0182] This amplification product was digested with SacI and
HindIII and ligated into pLitmus 28 for sequencing. The Sac
I+HindIII treated histidine-tagged MSP1 construct in pET 28 vector
was then ligated with the above fragment to produce MSP1 Da.
TABLE-US-00014 TABLE 11 MSP1D5D6 DNA sequence (SEQ ID NO: 22).
Translations start and stop codons are in bold type; restriction
endonuclease recognition sites are underlined.
TATACCATGGGCCATCATCATCATCATCATATAGAAGGAAGACTAAAGCT
CCTTGACAACTGGGACAGCGTGACCTCCACCTTCAGCAAGCTGCGCGAAC
AGCTCGGCCCTGTGACCCAGGAGTTCTGGGATAACCTGGAAAAGGAGACA
GAGGGCCTGAGGCAGGAGATGAGCAAGGATCTGGAGGAGGTGAAGGCCAA
GGTGCAGCCCTACCTGGACGACTTCCAGAAGAAGTGGCAGGAGGAGATGG
AGCTctaccgccagaaggtggagcCCTACAGCGACGAGCTGCGCCAGCGC
TTGGCCGCGCGCCTTGAGGCTCTCAAGGAGAACGGCGGCGCCAGACTGGC
CGAGTACCACGCCAAGGCCACCGAGCATCTGAGCACGCTCAGCGAGAAGG
CCAAACCCGCGCTCGAGGACCTCCGCCAAGGCCTGCTGCCCGTGCTGGAG
AGCTTCAAGGTCAGCTTCCTGAGCGCTCTCGAGGAGTACACTAAGAAGCT
CAACACCCAGTAATAAGCTTGC
TABLE-US-00015 TABLE 12 MSP1D5D6 amino acid sequence (SEQ ID NO:
23). MGHHHHHHIEGRLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEG
LRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPYSDELRQRLA
ARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESF
KVSFLSALEEYTKKLNTQ
[0183] Deletion of helices 5 and 6 was performed in a similar
manner, but two separate PCR steps using the following primers were
employed in a first reaction (Reaction 1, Primer C:
5'-CAGAATTCGCTAGCCGAGTACCACGCCAA-3', SEQ ID NO:24; and Primer D:
5'-GCAAGCTTATTACTGGGTGTTGAGCTTCTT-3', SEQ ID NO:25) and a second
reaction
(Reaction 2, Primer E: 5'-ATACCATGGGCCATCATCATCATCATCATA-3', SEQ ID
NO:26; and Primer F: 5'-CAGAATTCGCTAGCCTGGCGCTCAACTTCTCTT-3', SEQ
ID NO:27.
[0184] The PCR products encode the NB and C-terminal portions of an
MSP both lacking helices 5 and 6 and each contain a NheI
restriction site. After digestion of the PCR products with NheI,
NcoI and HindIII, the fragments was ligated into NcoI+HindIII
treated pET 28 to produce the DNA sequence of MSP1D6D7 See FIGS.
9A-9B.
TABLE-US-00016 TABLE 13 MSP1D6D7 DNA sequence (SEQ ID NO: 28).
Translation start and stop codons are shown in bold type, and
restriction endonuclease recogni- tion sites used in cloning are
underlined. TATACCATGGGCCATCATCATCATCATCATATAGAAGGAAGACTAAAGCT
CCTTGACAACTGGGACAGCGTGACCTCCACCTTCAGCAAGCTGCGCGAAC
AGCTCGGCCCTGTGACCCAGGAGTTCTGGGATAACCTGGAAAAGGAGACA
GAGGGCCTGAGGCAGGAGATGAGCAAGGATCTGGAGGAGGTGAAGGCCAA
GGTGCAGCCCTACCTGGACGACTTCCAGAAGAAGTGGCAGGAGGAGATGG
AGCTCTACCGCCAGAAGGTGGAGCCGCTGCGCGCAGAGCTCCAAGAGGGC
GCGCGCCAGAAGCTGCACGAGCTGCAAGAGAAGTTGAGCGCCAGGCTAGC
CGAGTACCACGCCAAGGCCACCGAGCATCTGAGCACGCTCAGCGAGAAGG
CCAAACCCGCGCTCGAGGACCTCCGCCAAGGCCTGCTGCCCGTGCTGGAG
AGCTTCAAGGTCAGCTTCCTGAGCGCTCTCGAGGAGTACACTAAGAAGCT
CAACACCCAGTAATAAGCTTGC
TABLE-US-00017 TABLE 14 MSP1D6D7 amino acid sequence (SEQ ID NO:
29). MGHHHHHHIEGRLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEG
LRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGAR
QKLHELQEKLSARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESF
KVSFLSALEEYTKKLNTQ
Example 2
Construction of Synthetic MSP Gene
[0185] A synthetic gene for MSP1 is made using the following
overlapping synthetic oligonucleotides which are filled in using
PCR. The codon usage has been optimized for expression in E. coli,
and restriction sites have been introduced for further genetic
manipulations of the gene.
TABLE-US-00018 Synthetic nucleotide taps1a (SEQ ID NO: 30)
TACCATGGGTCATCATCATCATCATCACATTGAGGGACGTCTGAAGCTGT
TGGACAATTGGGACTCTGTTACGTCTA Synthetic nucleotide taps2a (SEQ ID NO:
31) AGGAATTCTGGGACAACCTGGAAAAAGAAACCGAGGGACTGCGTCAGGAA ATGTCCAAAGAT
Synthetic nucleotide taps3a (SEQ ID NO: 32)
TATCTAGATGACTTTCAGAAAAAATGGCAGGAAGAGATGGAATTATATCG TCAA Synthetic
nucleotide taps4a (SEQ ID NO: 33)
ATGAGCTCCAAGAGAAGCTCAGCCCATTAGGCGAAGAAATGCGCGATCGC
GCCCGTGCACATGTTGATGCACT Synthetic nucleotide taps5a (SEQ ID NO: 34)
GTCTCGAGGCGCTGAAAGAAAACGGGGGTGCCCGCTTGGCTGAGTACCAC GCGAAAGCGACAGAA
Synthetic nucleotide taps6a (SEQ ID NO: 35)
GAAGATCTACGCCAGGGCTTATTGCCTGTTCTTGAGAGCTTTAAAGTCAG TTTTCT Synthetic
nucleotide taps1b (SEQ ID NO: 36)
CAGAATTCCTGCGTCACGGGGCCCAGTTGTTCGCGAAGTTTACTGAAGGT AGACGTAACAG
Synthetic nucleotide taps2b (SEQ ID NO: 37)
TCATCTAGATATGGCTGAACCTTGGCCTTCACCTCTTCTAAATCTTTGGA CATTT Synthetic
nucleotide taps3b (SEQ ID NO: 38)
TGGAGCTCATGGAGTTTTTGGCGTGCCCCCTCTTGCAGTTCCGCACGCAG
CGGTTCCACCTTTTGACGATATAATTCCAT Synthetic nucleotide taps4b (SEQ ID
NO: 39) GCCTCGAGACGTGCGGCCAAACGCTGGCGAAGTTCATCCGAATACGGCGC
CAAATGAGTCCGGAGTGCATCAACAT Synthetic nucleotide taps5b (SEQ ID NO:
40) GTAGATCTTCCAGCGCCGGTTTCGCTTTTTCGCTCAAGGTGCTCAGGTGT TCTGTCGCTTT
Synthetic nucleotide taps6b (SEQ ID NO: 41)
CCAAGCTTATTACTGGGTATTCAGCTTTTTAGTATATTCTTCCAGAGCTG
ACAGAAAACTGACTTT
TABLE-US-00019 TABLE 15 Full synthetic gene sequence for MSP1 (SEQ
ID NO: 42). Restriction sites used in cloning are underlined, and
the translation start and stop signals are shown in bold.
ACCATGGGTCATCATCATCATCATCACATTGAGGGACGTCTGAAGCTGTT
GGACAATTGGGACTCTGTTACGTCTACCTTCAGTAAACTTCGCGAACAAC
TGGGCCCCGTGACGCAGGAATTCTGGGACAACCTGGAAAAAGAAACCGAG
GGACTGCGTCAGGAAATGTCCAAAGATTTAGAAGAGGTGAAGGCCAAGGT
TCAGCCATATCTAGATGACTTTCAGAAAAAATGGCAGGAAGAGATGGAAT
TATATCGTCAAAAGGTGGAACCGCTGCGTGCGGAACTGCAAGAGGGGGCA
CGCCAAAAACTCCATGAGCTCCAAGAGAAGCTCAGCCCATTAGGCGAAGA
AATGCGCGATCGCGCCCGTGCACATGTTGATGCACTCCGGACTCATTTGG
CGCCGTATTCGGATGAACTTCGCCAGCGTTTGGCCGCACGTCTCGAGGCG
CTGAAAGAAAACGGGGGTGCCCGCTTGGCTGAGTACCACGCGAAAGCGAC
AGAACACCTGAGCACCTTGAGCGAAAAAGCGAAACCGGCGCTGGAAGATC
TACGCCAGGGCTTATTGCCTGTTCTTGAGAGCTTTAAAGTCAGTTTTCTG
TCAGCTCTGGAAGAATATACTAAAAAGCTGAATACCCAGTAATAAGCTTG G
[0186] The following is the amino acid sequence of a MSP
polypeptide in which half repeats are deleted:
TABLE-US-00020 TABLE 16 MSP1D3 (SEQ ID NO: 43).
MGHHHHHHIEGRLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEG
LRQEMSPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLS
PLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEY
HAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNT Q
TABLE-US-00021 TABLE 17 MSP1D9 (SEQ ID NO: 44).
MGHHHHHHIEGRLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEG
LRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGAR
QKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEAL
KENGGARLAEYHAKATEHLSTLSEKAKPVLESFKVSFLSALEEYTKKLNT Q
TABLE-US-00022 TABLE 18 MSP tandem repeat with first half-repeats
deleted (MSP2delta1) (SEQ ID NO: 45)
MGHHHHHHIEGRLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEG
LRQEMSPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLS
PLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEY
HAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNT
QGTLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSPYL
DDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDR
ARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLS
TLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ
[0187] Plasmids for the expression of extended MSPs were
constructed from plasmid for MSP1 described in Bayburt et al.
(2002) Nanoletters 2:853-856 using a "Seamless" cloning kit
(Stratagene) according to the manufacturer recommendations. An
alternative N-terminus for MSP1 TEV was added by PCR; the primers
were designed to include Nco I and Hind III restriction sites. The
PCR product was cloned into the pET28a plasmid (Novagen). Truncated
mutants of MSP were produced with a Quick-change kit (Stratagene)
using the MSP1TEV plasmid as a template. The presence of the
desired insertions or deletions and absence of PCR-induced
mutations were verified by DNA sequencing.
[0188] Expression and purification of the MSP proteins was
performed as described herein. Protein purity was characterized by
SDS-PAGE and Electrospray Mass Spectrometry; it was found to be
greater than 95%. The TEV protease expression system was purchased
(Science Reagents, Inc., Atlanta, Ga.) and used after some minor
modifications. The sequences of new scaffold proteins were
optimized with respect to salt link scores for the belt model of
the antiparallel dimer as described in Segrest et al. (1999) J.
Biol. Chem. 274:31755-31758. At first, the amino acid sequences of
the extended mutants were generated so that each of the central
helices (from H3 to H7) (see FIG. 19), was inserted sequentially at
every position between other central helices, i.e. after H3, H4,
H5, and H6, and the number of favorable salt links minus number of
unfavorable contacts of the same charges was calculated for all
possible configurations of antiparallel dimers in the resulting
scaffold protein (Segrest (1999) supra). As a result, the insertion
mutants shown at FIG. 20 were selected as optimal for maximum salt
link scores. These extended scaffold proteins, as well as truncated
scaffold proteins, also containing different tag sequences at the
N. terminus, were engineered in E. coli and expressed with a high
yield and purified by standard procedures.
[0189] With reference to the following protein and DNA sequences,
the MSPs we have utilized can be summarized as the following linked
structures. Note H1, H2 refer to the sequences of Helix #1 etc. His
is a (His)6 tag, TEV is the tobacco viral protease, X is the Factor
X (ten) protease site.
TABLE-US-00023 TABLE 19 Amino Acid Sequences of MSP Building Blocks
GLOB DEPPQSPWDRVKDLATVYVDVLKDSGRDYVSQFEGSALGKQLN (SEQ ID NO: 89)
HisX MGHHHHHHIEGR (SEQ ID NO: 47) HisTEV MGHHHHHHHDYDIPTTENLYFQG
(SEQ ID NO: 48) Helix 1 (H1): LKLLDNWDSVTSTFSKLREQLG (SEQ ID NO:
49) Helix 2 (H2): PVTQEFWDNLEKETEGLRQEMS (SEQ ID NO: 50) Helix 3
(H3): KDLEEVKAKVQ (SEQ ID NO: 51) Helix 4 (H4):
PYLDDFQKKWQEEMELYRQKVE (SEQ ID NO: 52) Helix 5 (H5):
PLRAELQEGARQKLHELQEKLS (SEQ ID NO: 53) Helix 6 (H6):
PLGEEMRDRARAHVDALRTHLA (SEQ ID NO: 54) Helix 7 (H7):
PYSDELRQRLAARLEALKENGG (SEQ ID NO: 55) Helix 8 (H8):
ARLAEYHAKATEHLSTLSEKAK (SEQ ID NO: 56) Helix 9 (H9): PALEDLRQGLL
(SEQ ID NO: 57) Helix 10 (H10): PVLESFKVSFLSALEEYTKKLNTQ (SEQ ID
NO: 58) Helix 0.5 (H0.5): STFSKLREQLG (SEQ ID NO: 59) Helix 10.5
(H10.5): SALEEYTKKLNTQ (SEQ ID NO: 87) Helix 2S (H2):
PVTQEFWDNLEKETEGLRQEMS (SEQ ID NO: 136)
TABLE-US-00024 TABLE 20 Sequences encoding the MSP Building Blocks
of Table 19. HisX ATGGGTCATCATCATCATCATCACATTGAGGGACGT (SEQ ID NO:
60) HisTEV ATGGGTCATCATCATCATCATCATCACGATTATGATATTCCTA (SEQ ID NO:
61) CTACTGAGAATTTGTATTTTCAGGGT Helix 1 (H1):
CTGAAGCTGTTGGACAATTGGGACTCTGTTACGTCTACCTTC (SEQ ID NO: 62)
AGTAAACTTCGCGAACAACTGGGC Helix 2 (H2):
CCCGTGACGCAGGAATTCTGGGACAACCTGGAAAAAGAAAC CGAGGGACTGCGTCAGGAAATGTCC
(SEQ ID NO: 63) Helix 3 (H3): AAAGATTTAGAAGAGGTGAAGGCCAAGGTTCAG
(SEQ ID NO: 64) Helix 4 (H4):
CCATATCTCGATGACTTTCAGAAAAAATGGCAGGAAGAGATG (SEQ ID NO: 65)
GAATTATATCGTCAAAAGGTGGAA Helix 5 (H5):
CCGCTGCGTGCGGAACTGCAAGAGGGGGCACGCCAAAAAC (SEQ ID NO: 66)
TCCATGAGCTCCAAGAGAAGCTCAGC Helix 6 (H6):
CCATTAGGCGAAGAAATGCGCGATCGCGCCCGTGCACATGT (SEQ ID NO: 67)
TGATGCACTCCGGACTCATTTGGCG Helix 7 (H7):
CCGTATTCGGATGAACTTCGCCAGCGTTTGGCCGCACGTCT (SEQ ID NO: 68)
CGAGGCGCTGAAAGAAAACGGGGGT Helix 8 (H8):
GCCCGCTTGGCTGAGTACCACGCGAAAGCGACAGAACACCT (SEQ ID NO: 69)
GAGCACCTTGAGCGAAAAAGCGAAA Helix 9 (H9):
CCGGCGCTGGAAGATCTACGCCAGGGCTTATTG (SEQ ID NO: 70) Helix 10 (H10):
CCTGTTCTTGAGAGCTTTAAAGTCAGTTTTCTGTCAGCTCTGG (SEQ ID NO: 71)
AAGAATATACTAAAAAGCTGAATACCCAG Helix 0.5 (H0.5):
TCTACCTTCAGTAAACTTCGCGAACAACTGGGC (SEQ ID NO: 72) Helix 10.5
(H10.5): CAGTTTTCTGTCAGCTCTGGAAGAATATACTAAAAAGCTGAATACCCAG (SEQ ID
NO: 88) Helix 2S (H2S):
TCCGTGACGCAGGAATTCTGGGACAACCTGGAAAAAGAAACCGAGGGACTGCGTCAGG (SEQ ID
NO: 90) AAATGTCC
[0190] Several particular MSP sequences useful in the present
invention are the following combinations of the above sequences, as
given in Table 21 and others.
TABLE-US-00025 TABLE 21 Engineered MSPs Useful in Nanodisc
Preparation. MSP1 HisX-H1-H2-H3-H4-H5-H6-H7-H8-H9-H10 (SEQ ID NO:
6) MSP1E1 HisX-H1-H2-H3-H4-H4-H5-H6-H7-H8-H9-H10 (SEQ ID NO: 73)
MSP1E2 HisX-H1-H2-H3-H4-H5-H4-H5-H6-H7-H8-H9-H10 (SEQ ID NO: 74)
MSP1E3 HisX-H1-H2-H3-H4-H5-H6-H4-H5-H6-H7-H8-H9-H10 (SEQ ID NO: 75)
MSP1TEV HisTev-H1-H2-H3-H4-H5-H6-H7-H8-H9-H10 (SEQ ID NO: 76)
MSP1NH H1-H2-H3-H4-H5-H6-H7-H8-H9-H10 (SEQ ID NO: 77) MSP1T2
HisTev-H0.5-H2-H3-H4-H5-H6-H7-H8-H9-H10 (SEQ ID NO: 78) MSP1T2NH
H0.5-H2-H3-H4-H5-H6-H7-H8-H9-H10 (SEQ ID NO: 79) MSP1T3
HisTev-H2-H3-H4-H5-H6-H7-H8-H9-H10 (SEQ ID NO: 80) MSP1D3
HisX-H1-H2-H4-H5-H6-H7-H8-H9-H10 (SEQ ID NO: 43) MSP1D9
HisX-H1-H2-H3-H4-H5-H6-H7-H8-H10 (SEQ ID NO: 44) MSP1D5D6
HisX-H1-H2-H3-H4-H7-H8-H9-H10 (SEQ ID NO: 23) MSP1D6D7
HisX-H1-H2-H3-H4-H5-H8-H9-H10 (SEQ ID NO: 82) MSP1D3D9
HisX-H1-H2-H4-H5-H6-H7-H8-H10 (SEQ ID NO: 83) MSP1D10.5
HisX-H1-H2-H3-H4-H5-H6-H7-H8-H9-H10.5 (SEQ ID NO: 84) MSP1D3D10.5
HisX-H1-H2-H4-H5-H6-H7-H8-H9-H10.5 (SEQ ID NO: 85) MSP1T4
HisTEV-H2S-H3-H4-H5-H6-H7-H8-H9-H10 (SEQ ID NO: 91) Apo A-I
GLOB-H1-H2-H3-H4-H4-H5-H6-H5-H6-H7-H8-H9-H10 MSP1T5
HisTev-H2.5-H3-H4-H5-H6-H7-H8-H9-H10 (SEQ ID NO: 92) MSP1T6
HisTev-H3-H4-H5-H6-H7-H8-H9-H10 (SEQ ID NO: 93) MSP1E3TEV:
HisTev-H1-H2-H3-H4-H5-H6-H4-H5-H6-H7-H8-H9-H10 (SEQ ID NO: 94)
MSP1E3D1: HisTev-H0.5-H2-H3-H4-H5-H6-H4-H5-H6-H7-H8-H9-H10 (SEQ ID
NO: 95) MSP2TEV:
HisTev-H1-H2-H3-H4-H5-H6-H7-H8-H9-H10-GT-H1-H2-H3-H4-H5- (SEQ ID
NO: 96) H6-H7-H8-H9-H10 MSP1N1: His-TEV-H2S-H3-H4-H4-H5-H6-H7-H8-H9
(SEQ ID NO: 97) MSP2N1:
HisTev-H0.5-H2-H3-H4-H5-H6-H7-H8-H9-H10-GT-H0.5-H2-H3-H4- (SEQ ID
NO: 98) H5-H6-H7-H8-H9-H10 MSP2N2:
HisTev-H0.5-H2-H3-H4-H5-H6-H7-H8-H9-H10-GT-H2-H3-H4- (SEQ ID NO:
99) H5-H6-H7-H8-H9-H10
[0191] In addition to these sequences, there are two fusion protein
(tandem repeat MSP) constructs of reference. These are composed of
two MSP1 constructs linked by a Gly-Thr linker:
TABLE-US-00026 MSP2 (MSP1BGlyBThrBMSP1, SEQ ID NO: 17) and MSP2D1D1
(MSP1T3BGlyBThrB H2-H3-H4-H5-H6-H7-H8-H9-H10, SEQ ID NO: 86).
[0192] Other constructs that can be readily produced include
permutations of the above, i.e., MSP1 or a tandemly repeated MSP
with either a short or long linker sequence with any combination of
the following: hinge deletion, hinge replacement, half-repeat
deletion, histidine tag, different linkers for MSP2 analogs.
[0193] The coding and amino acid sequences of MSP1T4 are given in
Tables 22 and 23, respectively.
TABLE-US-00027 TABLE 22 DNA sequence encoding MSP1T4 (SEQ ID NO:
100)
atgggtcatcatcatcatcatcatcacgattatgatattcctactactgagaatttgtattttcagggttccgt-
gacgcaggaattc
tgggacaacctggaaaaagaaaccgagggactgcgtcaggaaatgtccaaagatttagaagaggtgaaggccaa-
ggt
tcagccatatctcgatgactttcagaaaaaatggcaggaagagatggaattatatcgtcaaaaggtggaaccgc-
tgcgtg
cggaactgcaagagggggcacgccaaaaactccatgagctccaagagaagctcagcccattaggcgaagaaatg-
cg
cgatcgcgcccgtgcacatgttgatgcactccggactcatttggcgccgtattcggatgaacttcgccagcgtt-
tggccgcac
gtctcgaggcgctgaaagaaaacgggggtgcccgcttggctgagtaccacgcgaaagcgacagaacacctgagc-
acc
ttgagcgaaaaagcgaaaccggcgctggaagatctacgccagggcttattgcctgttcttgagagctttaaagt-
cagttttct gtcagctctggaagaatatactaaaaagctgaatacccag
TABLE-US-00028 TABLE 23 Amino acid sequence of MSP1T4 (SEQ ID NO:
91) MGHHHHHHHDYDIPTTENLYFQGSVTQEFWDNLEKETEGLRQEMSKDLEE
VKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLS
PLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEY
HAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNT Q
[0194] In the schematic for MSP1T5, H2.5 indicates the second half
of the H2 helical sequence, i.e. the last 33 nucleotides or 11
amino acids is not included in the MSP sequence. The coding and
amino acid sequence for this protein is given in Tables 24 and 25,
respectively.
TABLE-US-00029 TABLE 24 DNA sequence encoding MSP1T5 (SEQ ID NO:
101)
atgggtcatcatcatcatcatcatcacgattatgatattcctactactgagaatttgtattttcagggtaaaga-
aaccgaggga
ctgcgtcaggaaatgtccaaagatttagaagaggtgaaggccaaggttcagccatatctcgatgactttcagaa-
aaaatg
gcaggaagagatggaattatatcgtcaaaaggtggaaccgctgcgtgcggaactgcaagagggggcacgccaaa-
aa
ctccatgagctccaagagaagctcagcccattaggcgaagaaatgcgcgatcgcgcccgtgcacatgttgatgc-
actcc
ggactcatttggcgccgtattcggatgaacttcgccagcgtttggccgcacgtctcgaggcgctgaaagaaaac-
gggggt
gcccgcttggctgagtaccacgcgaaagcgacagaacacctgagcaccttgagcgaaaaagcgaaaccggcgct-
gg
aagatctacgccagggcttattgcctgttcttgagagctttaaagtcagttttctgtcagctctggaagaatat-
actaaaaagct gaatacccag
TABLE-US-00030 TABLE 25 Amino acid sequence of MSP1T5 (SEQ ID NO:
92) MGHHHHHHHDYDIPTTENLYFQGKETEGLRQEMSKDLEEVKAKVQPYLDD
FQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRAR
AHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTL
SEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ
TABLE-US-00031 TABLE 26 DNA sequence encoding MSP1T6 (SEQ ID NO:
102)
atgggtcatcatcatcatcatcatcacgattatgatattcctactactgagaatttgtattttcagggtaaaga-
tttagaagaggt
gaaggccaaggttcagccatatctcgatgactttcagaaaaaatggcaggaagagatggaattatatcgtcaaa-
aggtgg
aaccgctgcgtgcggaactgcaagagggggcacgccaaaaactccatgagctccaagagaagctcagcccatta-
ggc
gaagaaatgcgcgatcgcgcccgtgcacatgttgatgcactccggactcatttggcgccgtattcggatgaact-
tcgccag
cgtttggccgcacgtctcgaggcgctgaaagaaaacgggggtgcccgcttggctgagtaccacgcgaaagcgac-
agaa
cacctgagcaccttgagcgaaaaagcgaaaccggcgctggaagatctacgccagggcttattgcctgttcttga-
gagcttt aaagtcagttttctgtcagctctggaagaatatactaaaaagctgaatacccag
TABLE-US-00032 TABLE 27 Amino acid sequence of MSP1T6 (SEQ ID NO:
93) MGHHHHHHHDYDIPTTENLYFQGKDLEEVKAKVQPYLDDFQKKWQEEMEL
YRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLA
PYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDL
RQGLLPVLESFKVSFLSALEEYTKKLNTQ
[0195] MSP1T5 and MSP1T6 discs preps are not homogeneous under all
assembly conditions. The results are highly dependent on the
particular assembly conditions.
[0196] In the following MSP construct (MSP1N1), H10 is not
included, and two H4 motifs are inserted. The coding and amino acid
sequences are given in Tables 28 and 29, respectively. This MSP is
designed to increase the number of possible salt bridges on the
interhelical interface.
TABLE-US-00033 TABLE 28 DNA sequence encoding MSP1N1 (SEQ ID NO:
103)
atgggtcatcatcatcatcatcatcacgattatgatattcctactactgagaatttgtattttcagggttccgt-
gacgcaggaattc
tgggacaacctggaaaaagaaaccgagggactgcgtcaggaaatgtccaaagatttagaagaggtgaaggccaa-
ggt
tcagccatatctcgatgactttcagaaaaaatggcaggaagagatggaattatatcgtcaaaaggtggaaccat-
atctcga
tgactttcagaaaaaatggcaggaagagatggaattatatcgtcaaaaggtggaaccgctgcgtgcggaactgc-
aagag
ggggcacgccaaaaactccatgagctccaagagaagctcagcccattaggcgaagaaatgcgcgatcgcgcccg-
tgc
acatgttgatgcactccggactcatttggcgccgtattcggatgaacttcgccagcgtttggccgcacgtctcg-
aggcgctga
aagaaaacgggggtgcccgcttggctgagtaccacgcgaaagcgacagaacacctgagcaccttgagcgaaaaa-
gc gaaaccggcgctggaagatctacgccagggcttattg
TABLE-US-00034 TABLE 29 Amino acid sequence of MSP1N1 (SEQ ID NO:
97) MGHHHHHHHDYDIPTTENLYFQGSVTQEFWDNLEKETEGLRQEMSKDLEE
VKAKVQPYLDDFQKKWQEEMELYRQKVEPYLDDFQKKWQEEMELYRQKVE
PLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDEL
RQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLL
[0197] The following extended MSPs incorporate a cleavable His-tag
and use a TEV protease recognition site.
TABLE-US-00035 TABLE 30 DNA sequence encoding MSP1E3TEV
(HisTev-H1-H2-H3-H4-H5-H6- H4-H5-H6-H7-H8-H9-H10) (SEQ ID NO: 105)
atgggtcatcatcatcatcatcatcacgattatgatattcctactactgagaatttgtattttcagggtctgaa-
gctgttggacaat
tgggactctgttacgtctaccttcagtaaacttcgcgaacaactgggccccgtgacgcaggaattctgggacaa-
cctggaa
aaagaaaccgagggactgcgtcaggaaatgtccaaagatttagaagaggtgaaggccaaggttcagccatatct-
cgat
gactttcagaaaaaatggcaggaagagatggaattatatcgtcaaaaggtggaaccgctgcgtgcggaactgca-
agag
ggggcacgccaaaaactccatgagctccaagagaagctcagcccattaggcgaagaaatgcgcgatcgcgcccg-
tgc
acatgttgatgcactccggactcatttggcgccatatctcgatgactttcagaaaaaatggcaggaagagatgg-
aattatat
cgtcaaaaggtggaaccgctgcgtgcggaactgcaagagggggcacgccaaaaactccatgagctccaagagaa-
gc
tcagcccattaggcgaagaaatgcgcgatcgcgcccgtgcacatgttgatgcactccggactcatttggcgccg-
tattcgg
atgaacttcgccagcgtttggccgcacgtctcgaggcgctgaaagaaaacgggggtgcccgcttggctgagtac-
cacgc
gaaagcgacagaacacctgagcaccttgagcgaaaaagcgaaaccggcgctggaagatctacgccagggcttat-
tgc
ctgttcttgagagctttaaagtcagttttctgtcagctctggaagaatatactaaaaagctgaatacccag
TABLE-US-00036 TABLE 31 Amino acid sequence of MSP1E3TEV (SEQ ID
NO: 94) MGHHHHHHHDYDIPTTENLYFQGLKLLDNWDSVTSTFSKLREQLGPVTQE
FWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVE
PLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYLDDF
QKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARA
HVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLS
EKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ
TABLE-US-00037 TABLE 32 DNA sequence encoding MSP1E3D1 (SEQ ID NO:
106) (HisTev-H0.5-H2-H3-H4-H5-H6-H4-H5-H6-H7-H8-H9-H10)
atgggtcatcatcatcatcatcatcacgattatgatattcctactactgagaatttgtattttcagggttctac-
cttcagtaaacttc
gcgaacaactgggccccgtgacgcaggaattctgggacaacctggaaaaagaaaccgagggactgcgtcaggaa-
at
gtccaaagatttagaagaggtgaaggccaaggttcagccatatctcgatgactttcagaaaaaatggcaggaag-
agatg
gaattatatcgtcaaaaggtggaaccgctgcgtgcggaactgcaagagggggcacgccaaaaactccatgagct-
ccaa
gagaagctcagcccattaggcgaagaaatgcgcgatcgcgcccgtgcacatgttgatgcactccggactcattt-
ggcgcc
atatctcgatgactttcagaaaaaatggcaggaagagatggaattatatcgtcaaaaggtggaaccgctgcgtg-
cggaac
tgcaagagggggcacgccaaaaactccatgagctccaagagaagctcagcccattaggcgaagaaatgcgcgat-
cg
cgcccgtgcacatgttgatgcactccggactcatttggcgccgtattcggatgaacttcgccagcgtttggccg-
cacgtctcg
aggcgctgaaagaaaacgggggtgcccgcttggctgagtaccacgcgaaagcgacagaacacctgagcaccttg-
agc
gaaaaagcgaaaccggcgctggaagatctacgccagggcttattgcctgttcttgagagctttaaagtcagttt-
tctgtcagc tctggaagaatatactaaaaagctgaatacccag
TABLE-US-00038 TABLE 33 Amino acid sequence of MSP1E3D1 (SEQ ID NO:
95) MGHHHHHHHDYDIPTTENLYFQGSTFSKLREQLGPVTQEFWDNLEKETEG
LRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGAR
QKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYLDDFQKKWQEEMELY
RQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAP
YSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLR
QGLLPVLESFKVSFLSALEEYTKKLNTQ
[0198] A protein corresponding to MSP2 with a N-terminal TEV
cleavable His-tag has been designed. The coding and amino acid
sequences are given in Tables 34 and 35, respectively.
TABLE-US-00039 TABLE 34 DNA sequence encoding MSP2TEV
(HisTev-H1-H2-H3-H4-H5-H6-H7-H8-H9-H10-
GT-H1-H2-H3-H4-H5-H6-H7-H8-H9-H10) (SEQ ID NO: 107)
atgggtcatcatcatcatcatcatcacgattatgatattcctactactgagaatttgtattttcagggtctaaa-
gctccttgacaac
tgggacagcgtgacctccaccttcagcaagctgcgcgaacagctcggccctgtgacccaggagttctgggataa-
cctgg
aaaaggagacagagggcctgaggcaggagatgagcaaggatctggaggaggtgaaggccaaggtgcagccctac-
c
tggacgacttccagaagaagtggcaggaggagatggagctctaccgccagaaggtggagccgctgcgcgcagag-
ctc
caagagggcgcgcgccagaagctgcacgagctgcaagagaagctgagcccactgggcgaggagatgcgcgaccg-
c
gcgcgcgcccatgtggacgcgctgcgcacgcatctggccccctacagcgacgagctgcgccagcgcttggccgc-
gcgc
cttgaggctctcaaggagaacggcggcgccagactggccgagtaccacgccaaggccaccgagcatctgagcac-
gct
cagcgagaaggccaagcccgcgctcgaggacctccgccaaggcctgctgcccgtgctggagagcttcaaggtca-
gctt
cctgagcgctctcgaggagtacactaagaagctcaacacccagggtaccctaaagctccttgacaactgggaca-
gcgtg
acctccaccttcagcaagctgcgcgaacagctcggccctgtgacccaggagttctgggataacctggaaaagga-
gaca
gagggcctgaggcaggagatgagcaaggatctggaggaggtgaaggccaaggtgcagccctacctggacgactt-
cca
gaagaagtggcaggaggagatggagctctaccgccagaaggtggagccgctgcgcgcagagctccaagagggcg-
c
gcgccagaagctgcacgagctgcaagagaagctgagcccactgggcgaggagatgcgcgaccgcgcgcgcgccc-
at
gtggacgcgctgcgcacgcatctggccccctacagcgacgagctgcgccagcgcttggccgcgcgccttgaggc-
tctca
aggagaacggcggcgccagactggccgagtaccacgccaaggccaccgagcatctgagcacgctcagcgagaag-
g
ccaagcccgcgctcgaggacctccgccaaggcctgctgcccgtgctggagagcttcaaggtcagcttcctgagc-
gctctc gaggagtacactaagaagctcaacacccag
TABLE-US-00040 TABLE 35 Amino acid sequence of HisTEV-MSP2 (SEQ ID
NO: 96) MGHHHHHHHDYDIPTTENLYFQGLKLLDNWDSVTSTFSKLREQLGPVTQE
FWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVE
PLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDEL
RQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLP
VLESFKVSFLSALEYTKKLNTQGTLKLLDNWDSVTSTFSKLREQLGPVTQ
EFWDNLEKETEGLRQEMKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVE
PLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDEL
RQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLP
VLESFKVSFLSALEEYTKKLNTQ
[0199] New constructs have been designed to produce a which make a
linear dimer to generate Nanodiscs with only a single polypeptide
sequence. These are fusions that make use of our knowledge of the
parts of the MSP1 sequences which are important and are thus are
MSP2 derivatives. All have the TEV protease-cleavage His-tag.
TABLE-US-00041 TABLE 36 DNA sequence encoding MSP2N1
(HisTev-H0.5-H2-H3-H4-H5-H6-
H7-H8-H9-H10-GT-H1/2-H2-H3-H4-H5-H6-H7-H8-H9-H10) (SEQ ID NO: 108)
atgggtcatcatcatcatcatcatcacgattatgatattcctactactgagaatttgtattttcagggttctac-
cttcagtaaacttc
gcgaacaactgggccccgtgacgcaggaattctgggacaacctggaaaaagaaaccgagggactgcgtcaggaa-
at
gtccaaagatttagaagaggtgaaggccaaggttcagccatatctcgatgactttcagaaaaaatggcaggaag-
agatg
gaattatatcgtcaaaaggtggaaccgctgcgtgcggaactgcaagagggggcacgccaaaaactccatgagct-
ccaa
gagaagctcagcccattaggcgaagaaatgcgcgatcgcgcccgtgcacatgttgatgcactccggactcattt-
ggcgcc
gtattcggatgaacttcgccagcgtttggccgcacgtctcgaggcgctgaaagaaaacgggggtgcccgcttgg-
ctgagt
accacgcgaaagcgacagaacacctgagcaccttgagcgaaaaagcgaaaccggcgctggaagatctacgccag-
g
gcttattgcctgttcttgagagctttaaagtcagttttctgtcagctctggaagaatatactaaaaagctgaat-
acccagggtac
cttcagtaaacttcgcgaacaactgggccccgtgacgcaggaattctgggacaacctggaaaaagaaaccgagg-
gact
gcgtcaggaaatgtccaaagatttagaagaggtgaaggccaaggttcagccatatctcgatgactttcagaaaa-
aatggc
aggaagagatggaattatatcgtcaaaaggtggaaccgctgcgtgcggaactgcaagagggggcacgccaaaaa-
ctc
catgagctccaagagaagctcagcccattaggcgaagaaatgcgcgatcgcgcccgtgcacatgttgatgcact-
ccgga
ctcatttggcgccgtattcggatgaacttcgccagcgtttggccgcacgtctcgaggcgctgaaagaaaacggg-
ggtgccc
gcttggctgagtaccacgcgaaagcgacagaacacctgagcaccttgagcgaaaaagcgaaaccggcgctggaa-
ga
tctacgccagggcttattgcctgttcttgagagctttaaagtcagttttctgtcagctctggaagaatatacta-
aaaagctgaat acccag
TABLE-US-00042 TABLE 37 Amino acid sequence of MSP2N1 (SEQ ID NO:
98) MGHHHHHHHDYDIPTTENLYFQGSTFSKLREQLGPVTQEFWDNLEKETEG
LRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGAR
QKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEAL
KENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLS
ALEEYTKKLNTQGTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEE
VKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLS
PLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEY
HAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNT Q
TABLE-US-00043 TABLE 38 DNA sequence encoding MSP2N2 (SEQ ID NO:
109) (HisTev-H0.5-H2-H3-H4-H5-H6-H7-H8-H9-H10-GT-
H2-H3-H4-H5-H6-H7-H8-H9-H10)
atgggtcatcatcatcatcatcatcacgattatgatattcctactactga
gaatttgtattttcagggttctaccttcagtaaacttcgcgaacaactgg
gccccgtgacgcaggaattctgggacaacctggaaaaagaaaccgaggga
ctgcgtcaggaaatgtccaaagatttagaagaggtgaaggccaaggttca
gccatatctcgatgactttcagaaaaaatggcaggaagagatggaattat
atcgtcaaaaggtggaaccgctgcgtgcggaactgcaagagggggcacgc
caaaaactccatgagctccaagagaagctcagcccattaggcgaagaaat
gcgcgatcgcgcccgtgcacatgttgatgcactccggactcatttggcgc
cgtattcggatgaacttcgccagcgtttggccgcacgtctcgaggcgctg
aaagaaaacgggggtgcccgcttggctgagtaccacgcgaaagcgacaga
acacctgagcaccttgagcgaaaaagcgaaaccggcgctggaagatctac
gccagggcttattgcctgttcttgagagctttaaagtcagttttctgtca
gctctggaagaatatactaaaaagctgaatacccagggtacccccgtgac
gcaggaattctgggacaacctggaaaaagaaaccgagggactgcgtcagg
aaatgtccaaagatttagaagaggtgaaggccaaggttcagccatatctc
gatgactttcagaaaaaatggcaggaagagatggaattatatcgtcaaaa
ggtggaaccgctgcgtgcggaactgcaagagggggcacgccaaaaactcc
atgagctccaagagaagctcagcccattaggcgaagaaatgcgcgatcgc
gcccgtgcacatgttgatgcactccggactcatttggcgccgtattcgga
tgaacttcgccagcgtttggccgcacgtctcgaggcgctgaaagaaaacg
ggggtgcccgcttggctgagtaccacgcgaaagcgacagaacacctgagc
accttgagcgaaaaagcgaaaccggcgctggaagatctacgccagggctt
attgcctgttcttgagagctttaaagtcagttttctgtcagctctggaag
aatatactaaaaagctgaatacccag
TABLE-US-00044 TABLE 39 Amino acid sequence of MSP2N2 (SEQ ID NO:
99) MGHHHHHHHDYDIPTTENLYFQGSTFSKLREQLGPVTQEFWDNLEKETEG
LRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGAR
QKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEAL
KENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLS
ALEEYTKKLNTQGTPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYL
DDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDR
ARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLS
TLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ
[0200] A further MSP2 derivative (MSP2N3 has been designed to
include helices 2-10 following the linker part of the H1 helix
sequence. The DNA coding and amino acid sequences are given in
Tables 40 and 41, respectively.
TABLE-US-00045 TABLE 40 DNA sequence encoding MSP2N3
(HisTev-H0.5-H2-H3-H4-H5-H6-H7-H8-H9-H10-GTREQLG-
H2-H3-H4-H5-H6-H7-H8-H9-H10) (SEQ ID NO: 110)
Atgggtcatcatcatcatcatcatcacgattatgatattcctactactga
gaatttgtattttcagggttctaccttcagtaaacttcgcgaacaactgg
gccccgtgacgcaggaattctgggacaacctggaaaaagaaaccgaggga
ctgcgtcaggaaatgtccaaagatttagaagaggtgaaggccaaggttca
gccatatctcgatgactttcagaaaaaatggcaggaagagatggaattat
atcgtcaaaaggtggaaccgctgcgtgcggaactgcaagagggggcacgc
caaaaactccatgagctccaagagaagctcagcccattaggcgaagaaat
gcgcgatcgcgcccgtgcacatgttgatgcactccggactcatttggcgc
cgtattcggatgaacttcgccagcgtttggccgcacgtctcgaggcgctg
aaagaaaacgggggtgcccgcttggctgagtaccacgcgaaagcgacaga
acacctgagcaccttgagcgaaaaagcgaaaccggcgctggaagatctac
gccagggcttattgcctgttcttgagagctttaaagtcagttttctgtca
gctctggaagaatatactaaaaagctgaatacccagggtacccgcgaaca
actgggccccgtgacgcaggaattctgggacaacctggaaaaagaaaccg
agggactgcgtcaggaaatgtccaaagatttagaagaggtgaaggccaag
gttcagccatatctcgatgactttcagaaaaaatggcaggaagagatgga
attatatcgtcaaaaggtggaaccgctgcgtgcggaactgcaagaggggg
cacgccaaaaactccatgagctccaagagaagctcagcccattaggcgaa
gaaatgcgcgatcgcgcccgtgcacatgttgatgcactccggactcattt
ggcgccgtattcggatgaacttcgccagcgtttggccgcacgtctcgagg
cgctgaaagaaaacgggggtgcccgcttggctgagtaccacgcgaaagcg
acagaacacctgagcaccttgagcgaaaaagcgaaaccggcgctggaaga
tctacgccagggcttattgcctgttcttgagagctttaaagtcagttttc
tgtcagctctggaagaatatactaaaaagctgaatacccagtaagctt
TABLE-US-00046 TABLE 41 Amino acid sequence of MSP2N3 (SEQ ID NO:
111) MGHHHHHHHDYDIPTTENLYFQGSTFSKLREQLGPVTQEFWDNLEKETEG
LRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGAR
QKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEAL
KENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLS
ALEEYTKKLNTQGTREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAK
VQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGE
EMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKA
TEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ
[0201] Unlike MSP2 and MSP2TEV these proteins self-assemble with
lipids at 300:1 to 400:1 molar ratios with preferable formation of
significantly bigger particles (Stokes diameter approximately 15.5
nm, corresponding to a calculated diameter assuming discoidal shape
of about 17 nm).
[0202] New dimer sequences (i.e., tandem repeat MSP) have been
designed with the fusion region to be composed of two different
linkers which have high propensity to form beta-turns (Creighton,
Proteins, p. 226). These scaffold proteins are specifically
designed to promote the anti-parallel helix-turn-helix structure in
Nanodiscs. The constituent scaffold proteins include MSP1T3, as
well as the specially designed new scaffold proteins as described
herein, MSP1N1 and the circularly permuted MSP2N5 which has a
modified sequence of amphipathic helices to optimize the salt
bridges formed between two scaffold proteins in the antiparallel
helix-turn-helix structure.
[0203] The general scheme for a tandem repeat MSP is
MSP-Linker-MSP, where linker may be either the Linker 1 or Linker 2
sequence defined below and MSP may be any of the monomeric membrane
scaffold proteins previously defined. Linker 1 (Lb1) is composed of
4 amino acids, preferably the sequence Asn-Pro-Gly-Thr (SEQ ID
NO:104). Linker 2 (Lb2) is composed of 6 amino acids with one
additional residue on both ends to provide more flexibility,
preferably the sequence Ser-Asn-Pro-Gly-Thr-Gln (SEQ ID
NO:136).
TABLE-US-00047 TABLE 42 DNA sequence encoding MSP2N4 (His-TEV
BH2S-H3-H4-H5-H6-H7-H8-H9-H10-NPGT-H2-H3- H4-H5-H6-H7-H8-H9-H10)
(SEQ ID NO: 112) atgggtcatcatcatcatcatcatcacgattatgatattcctactactga
gaatttgtattttcagggttccgtgacgcaggaattctgggacaacctgg
aaaaagaaaccgagggactgcgtcaggaaatgtccaaagatttagaagag
gtgaaggccaaggttcagccatatctcgatgactttcagaaaaaatggca
ggaagagatggaattatatcgtcaaaaggtggaaccgctgcgtgcggaac
tgcaagagggggcacgccaaaaactccatgagctccaagagaagctcagc
ccattaggcgaagaaatgcgcgatcgcgcccgtgcacatgttgatgcact
ccggactcatttggcgccgtattcggatgaacttcgccagcgtttggccg
cacgtctcgaggcgctgaaagaaaacgggggtgcccgcttggctgagtac
cacgcgaaagcgacagaacacctgagcaccttgagcgaaaaagcgaaacc
ggcgctggaagatctacgccagggcttattgcctgttcttgagagcttta
aagtcagttttctgtcagctctggaagaatatactaaaaagctgaatacc
cagaatccaggtacccccgtgacgcaggaattctgggacaacctggaaaa
agaaaccgagggactgcgtcaggaaatgtccaaagatttagaagaggtga
aggccaaggttcagccatatctcgatgactttcagaaaaaatggcaggaa
gagatggaattatatcgtcaaaaggtggaaccgctgcgtgcggaactgca
agagggggcacgccaaaaactccatgagctccaagagaagctcagcccat
taggcgaagaaatgcgcgatcgcgcccgtgcacatgttgatgcactccgg
actcatttggcgccgtattcggatgaacttcgccagcgtttggccgcacg
tctcgaggcgctgaaagaaaacgggggtgcccgcttggctgagtaccacg
cgaaagcgacagaacacctgagcaccttgagcgaaaaagcgaaaccggcg
ctggaagatctacgccagggcttattgcctgttcttgagagctttaaagt
cagttttctgtcagctctggaagaatatactaaaaagctgaatacccag
TABLE-US-00048 TABLE 43 Amino acid sequence of MSP2N4 (SEQ ID NO:
113) MGHHHHHHHDYDIPTTENLYFQGSVTQEFWDNLEKETEGLRQEMSKDLEE
VKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLS
PLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEY
HAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNT
QNPGTPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQE
EMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALR
THLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPA
LEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ
TABLE-US-00049 TABLE 44 DNA sequence encoding MSP2N5
(His-TEVBH2S-H3-H4-H4-H5-H6-H7-H8-H9-NPGT-H3-H4-
H4-H5-H6-H7-H8-H9-H2) (SEQ ID NO: 114)
atgggtcatcatcatcatcatcatcacgattatgatattcctactactga
gaatttgtattttcagggttccgtgacgcaggaattctgggacaacctgg
aaaaagaaaccgagggactgcgtcaggaaatgtccaaagatttagaagag
gtgaaggccaaggttcagccatatctcgatgactttcagaaaaaatggca
ggaagagatggaattatatcgtcaaaaggtggaaccatatctcgatgact
ttcagaaaaaatggcaggaagagatggaattatatcgtcaaaaggtggaa
ccgctgcgtgcggaactgcaagagggggcacgccaaaaactccatgagct
ccaagagaagctcagcccattaggcgaagaaatgcgcgatcgcgcccgtg
cacatgttgatgcactccggactcatttggcgccgtattcggatgaactt
cgccagcgtttggccgcacgtctcgaggcgctgaaagaaaacgggggtgc
ccgcttggctgagtaccacgcgaaagcgacagaacacctgagcaccttga
gcgaaaaagcgaaaccggcgctggaagatctacgccagggcttattgaat
ccaggtaccaaagatttagaagaggtgaaggccaaggttcagccatatct
cgatgactttcagaaaaaatggcaggaagagatggaattatatcgtcaaa
aggtggaaccatatctcgatgactttcagaaaaaatggcaggaagagatg
gaattatatcgtcaaaaggtggaaccgctgcgtgcggaactgcaagaggg
ggcacgccaaaaactccatgagctccaagagaagctcagcccattaggcg
aagaaatgcgcgatcgcgcccgtgcacatgttgatgcactccggactcat
ttggcgccgtattcggatgaacttcgccagcgtttggccgcacgtctcga
ggcgctgaaagaaaacgggggtgcccgcttggctgagtaccacgcgaaag
cgacagaacacctgagcaccttgagcgaaaaagcgaaaccggcgctggaa
gatctacgccagggcttattgcccgtgacgcaggaattctgggacaacct
ggaaaaagaaaccgagggactgcgtcaggaaatgtcc
TABLE-US-00050 TABLE 45 Amino acid sequence of MSP2N5 (SEQ ID NO:
115) MGHHHHHHHDYDIPTTENLYFQGSVTQEFWDNLEKETEGLRQEMSKDLEE
VKAKVQPYLDDFQKKWQEEMELYRQKVEPYLDDFQKKWQEEMELYRQKVE
PLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDEL
RQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLN
PGTKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPYLDDFQKKWQEEM
ELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTH
LAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALE
DLRQGLLPVTQEFWDNLEKETEGLRQEMS
TABLE-US-00051 TABLE 46 DNA sequence encoding MSP2N6
(His-TEVBH2S-H3-H4-H4-H5-H6-H7-H8-H9-SNPGTQ-
H3-H4-H4-H5-H6-H7-H8-H9-H2) (SEQ ID NO: 116)
atgggtcatcatcatcatcatcatcacgattatgatattcctactactga
gaatttgtattttcagggttccgtgacgcaggaattctgggacaacctgg
aaaaagaaaccgagggactgcgtcaggaaatgtccaaagatttagaagag
gtgaaggccaaggttcagccatatctcgatgactttcagaaaaaatggca
ggaagagatggaattatatcgtcaaaaggtggaaccatatctcgatgact
ttcagaaaaaatggcaggaagagatggaattatatcgtcaaaaggtggaa
ccgctgcgtgcggaactgcaagagggggcacgccaaaaactccatgagct
ccaagagaagctcagcccattaggcgaagaaatgcgcgatcgcgcccgtg
cacatgttgatgcactccggactcatttggcgccgtattcggatgaactt
cgccagcgtttggccgcacgtctcgaggcgctgaaagaaaacgggggtgc
ccgcttggctgagtaccacgcgaaagcgacagaacacctgagcaccttga
gcgaaaaagcgaaaccggcgctggaagatctacgccagggcttattgtcc
aatccaggtacccaaaaagatttagaagaggtgaaggccaaggttcagcc
atatctcgatgactttcagaaaaaatggcaggaagagatggaattatatc
gtcaaaaggtggaaccatatctcgatgactttcagaaaaaatggcaggaa
gagatggaattatatcgtcaaaaggtggaaccgctgcgtgcggaactgca
agagggggcacgccaaaaactccatgagctccaagagaagctcagcccat
taggcgaagaaatgcgcgatcgcgcccgtgcacatgttgatgcactccgg
actcatttggcgccgtattcggatgaacttcgccagcgtttggccgcacg
tctcgaggcgctgaaagaaaacgggggtgcccgcttggctgagtaccacg
cgaaagcgacagaacacctgagcaccttgagcgaaaaagcgaaaccggcg
ctggaagatctacgccagggcttattgcccgtgacgcaggaattctggga
caacctggaaaaagaaaccgagggactgcgtcaggaaatgtcc
TABLE-US-00052 TABLE 47 Amino acid sequence MSP2N6 (SEQ ID NO: 117)
MGHHHHHHHDYDIPTTENLYFQGSVTQEFWDNLEKETEGLRQEMSKDLEE
VKAKVQPYLDDFQKKWQEEMELYRQKVEPYLDDFQKKWQEEMELYRQKVE
PLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDEL
RQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLS
NPGTQKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPYLDDFQKKWQE
EMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALR
THLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPA
LEDLRQGLLPVTQEFWDNLEKETEGLRQEMS
[0204] Fusion constructs of membrane scaffold proteins have been
constructed with other proteins and peptides. Fusions with
cytochrome P450 reductase (CPR) include the following:
TABLE-US-00053 TABLE 48 DNA sequence encoding MSP2CPR
(MSP2-linker-CPR, linker amino acid sequence is VD and CPR is the
rat cytochrome P450 reductase complete sequence) (SEQ ID NO: 118)
atgggtcatcatcatcatcatcacattgagggacgtctgaagctgttgga
caattgggactctgttacgtctaccttcagtaaacttcgcgaacaactgg
gccccgtgacgcaggaattctgggacaacctggaaaaagaaaccgaggga
ctgcgtcaggaaatgtccaaagatttagaagaggtgaaggccaaggttca
gccatatctcgatgactttcagaaaaaatggcaggaagagatggaattat
atcgtcaaaaggtggaaccgctgcgtgcggaactgcaagagggggcacgc
caaaaactccatgagctccaagagaagctcagcccattaggcgaagaaat
gcgcgatcgcgcccgtgcacatgttgatgcactccggactcatttggcgc
cgtattcggatgaacttcgccagcgtttggccgcacgtctcgaggcgctg
aaagaaaacgggggtgcccgcttggctgagtaccacgcgaaagcgacaga
acacctgagcaccttgagcgaaaaagcgaaaccggcgctggaagatctac
gccagggcttattgcctgttcttgagagctttaaagtcagttttctgtca
gctctggaagaatatactaaaaagctgaatacccagggtaccctgaagct
gttggacaattgggactctgttacgtctaccttcagtaaacttcgcgaac
aactgggccccgtgacgcaggaattctgggacaacctggaaaaagaaacc
gagggactgcgtcaggaaatgtccaaagatttagaagaggtgaaggccaa
ggttcagccatatctcgatgactttcagaaaaaatggcaggaagagatgg
aattatatcgtcaaaaggtggaaccgctgcgtgcggaactgcaagagggg
gcacgccaaaaactccatgagctccaagagaagctcagcccattaggcga
agaaatgcgcgatcgcgcccgtgcacatgttgatgcactccggactcatt
tggcgccgtattcggatgaacttcgccagcgtttggccgcacgtctcgag
gcgctgaaagaaaacgggggtgcccgcttggctgagtaccacgcgaaagc
gacagaacacctgagcaccttgagcgaaaaagcgaaaccggcgctggaag
atctacgccagggcttattgcctgttcttgagagctttaaagtcagtttt
ctgtcagctctggaagaatatactaaaaagctgaatacccagtcgaccat
gggagactctcacgaagacaccagtgccaccatgcctgaggccgtggctg
aagaagtgtctctattcagcacgacggacatggttctgttttctctcatc
gtgggggtcctgacctactggttcatctttagaaagaagaaagaagagat
accggagttcagcaagatccaaacaacggccccacccgtcaaagagagca
gcttcgtggaaaagatgaagaaaacgggaaggaacattatcgtattctat
ggctcccagacgggaaccgctgaggagtttgccaaccggctgtccaagga
tgcccaccgctacgggatgcggggcatgtccgcagaccctgaagagtatg
acttggccgacctgagcagcctgcctgagatcgacaagtccctggtagtc
ttctgcatggccacatacggagagggcgaccccacggacaatgcgcagga
cttctatgactggctgcaggagactgacgtggacctcactggggtcaagt
ttgctgtatttggtcttgggaacaagacctatgagcacttcaatgccatg
ggcaagtatgtggaccagcggctggagcagcttggcgcccagcgcatctt
tgagttgggccttggtgatgatgacgggaacttggaagaggatttcatca
cgtggagggagcagttctggccagctgtgtgcgagttctttggggtagaa
gccactggggaggagtcgagcattcgccagtatgagctcgtggtccacga
agacatggacgtagccaaggtgtacacgggtgagatgggccgtctgaaga
gctacgagaaccagaaaccccccttcgatgctaagaatccattcctggct
gctgtcaccgccaaccggaagctgaaccaaggcactgagcggcatctaat
gcacctggagttggacatctcagactccaagatcaggtatgaatctggag
atcacgtggctgtgtacccagccaatgactcagccctggtcaaccagatt
ggggagatcctgggagctgacctggatgtcatcatgtctctaaacaatct
cgatgaggagtcaaacaagaagcatccgttcccctgccccaccacctacc
gcacggccctcacctactacctggacatcactaacccgccacgcaccaat
gtgctctacgaactggcacagtacgcctcagagccctcggagcaggagca
cctgcacaagatggcgtcatcctcaggcgagggcaaggagctgtacctga
gctgggtggtggaagcccggaggcacatcctagccatcctccaagactac
ccatcactgcggccacccatcgaccacctgtgtgagctgctgccacgcct
gcaggcccgatactactccattgcctcatcctccaaggtccaccccaact
ccgtgcacatctgtgccgtggccgtggagtacgaagcgaagtctggccga
gtgaacaagggggtggccactagctggcttcgggccaaggaaccagcagg
cgagaatggcggccgcgccctggtacccatgttcgtgcgcaaatctcagt
tccgcttgcctttcaagtccaccacacctgtcatcatggtgggccccggc
actgggattgcccctttcatgggcttcatccaggaacgagcttggcttcg
agagcaaggcaaggaggtgggagagacgctgctatactatggctgccggc
gctcggatgaggactatctgtaccgtgaagagctagcccgcttccacaag
gacggtgccctcacgcagcttaatgtggccttttcccgggagcaggccca
caaggtctatgtccagcaccttctgaagagagacagggaacacctgtgga
agctgatccacgagggcggtgcccacatctatgtgtgcggggatgctcga
aatatggccaaagatgtgcaaaacacattctatgacattgtggctgagtt
cgggcccatggagcacacccaggctgtggactatgttaagaagctgatga
ccaagggccgctactcactagatgtgtggagc
TABLE-US-00054 TABLE 49 Amino acid sequence of MSP2CPR (SEQ ID NO:
119) MGHHHHHHIEGRLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEG
LRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGAR
QKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEAL
KENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLS
ALEEYTKKLNTQGTLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKET
EGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEG
ARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLE
ALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSF
LSALEEYTKKLNTQSTMGDSHEDTSATMPEAVAEEVSLFSTTDMVLFSLI
VGVLTYWFIFRKKKEEIPEFSKIQTTAPPVKESSFVEKMKKTGRNIIVFY
GSQTGTAEEFANRLSKDAHRYGMRGMSADPEEYDLADLSSLPEIDKSLVV
FCMATYGEGDPTDNAQDFYDWLQETDVDLTGVKFAVFGLGNKTYEHFNAM
GKYVDQRLEQLGAQRIFELGLGDDDGNLEEDFITWREQFWPAVCEFFGVE
ATGEESSIRQYELVVHEDMDVAKVYTGEMGRLKSYENQKPPFDAKNPFLA
AVTANRKLNQGTERHLMHLELDISDSKIRYESGDHVAVYPANDSALVNQI
GEILGADLDVIMSLNNLDEESNKKHPFPCPTTYRTALTYYLDITNPPRTN
VLYELAQYASEPSEQEHLHKMASSSGEGKELYLSWVVEARRHILAILQDY
PSLRPPIDHLCELLPRLQARYYSIASSSKVHPNSVHICAVAVEYEAKSGR
VNKGVATSWLRAKEPAGENGGRALVPMFVRKSQFRLPFKSTTPVIMVGPG
TGIAPFMGFIQERAWLREQGKEVGETLLYYGCRRSDEDYLYREELARFHK
DGALTQLNVAFSREQAHKVYVQHLLKRDREHLWKLIHEGGAHIYVCGDAR
NMAKDVQNTFYDIVAEFGPMEHTQAVDYVKKLMTKGRYSLDVWS
Fusions have been prepared with fluorescent proteins (FP) and MSP
sequences. All constructs of the form His-TEV2-(FP)-MSP1 T2 or
His-TEV-MSP1T2-GT-(FP), where (FP) is the enhanced green
fluorescent protein (EGFP), the enhanced yellow fluorescent protein
(EYFP) or cyan fluorescent protein (CFP).
[0205] The overall N-terminal sequences are of the form: His-TEV2
(which have been modified to incorporate a BamH1 restriction site
into the sequence). The modified His-TEV2 DNA sequence is
TABLE-US-00055 (SEQ ID NO: 120)
atgggtcatcatcatcatcatcatcacgattatgatattcctactactga
gaatttgtattttcagggatcc,
and the modified His-TEV2 Protein sequence is
TABLE-US-00056 MGHHHHHHHDYDIPTTENLYFQGS. (SEQ ID NO: 121)
[0206] The fluorescent proteins have the following DNA and protein
sequences:
TABLE-US-00057 TABLE 50 DNA sequence encoding EGFP (SEQ ID NO: 122)
gtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcga
gctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcg
agggcgatgccacctacggcaagctgaccctgaagttcatctgcaccacc
ggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacgg
cgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttct
tcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttc
aaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcga
caccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacg
gcaacatcctggggcacaagctggagtacaactacaacagccacaacgtc
tatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagat
ccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagc
agaacacccccatcggcgacggccccgtgctgctgcccgacaaccactac
ctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatca
catggtcctgctggagttcgtgaccgccgccgggatcactctcggcatgg
acgagctgtacaag
TABLE-US-00058 TABLE 51 Amino acid sequence of EGFP (SEQ ID NO:
123) VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTT
GKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFF
KDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNV
YIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHY
LSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK
TABLE-US-00059 TABLE 52 DNA sequence encoding EYFP (SEQ ID NO: 124)
gtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcga
gctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcg
agggcgatgccacctacggcaagctgaccctgaagttcatctgcaccacc
ggcaagctgcccgtgccctggcccaccctcgtgaccaccttcggctacgg
cctgcagtgcttcgcccgctaccccgaccacatgaagcagcacgacttct
tcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttc
aaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcga
caccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacg
gcaacatcctggggcacaagctggagtacaactacaacagccacaacgtc
tatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagat
ccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagc
agaacacccccatcggcgacggccccgtgctgctgcccgacaaccactac
ctgagctaccagtccgccctgagcaaagaccccaacgagaagcgcgatca
catggtcctgctggagttcgtgaccgccgccgggatcactctcggcatgg
acgagctgtacaag
TABLE-US-00060 TABLE 53 Amino acid sequence of EYFP (SEQ ID NO:
125) VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTT
GKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFF
KDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNV
YIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHY
LSYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK
TABLE-US-00061 TABLE 54 DNA sequence encoding ECFP (SEQ ID NO: 126)
gtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcga
gctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcg
agggcgatgccacctacggcaagctgaccctgaagttcatctgcaccacc
ggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctgggg
cgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttct
tcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttc
aaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcga
caccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacg
gcaacatcctggggcacaagctggagtacaactacatcagccacaacgtc
tatatcaccgccgacaagcagaagaacggcatcaaggccaacttcaagat
ccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagc
agaacacccccatcggcgacggccccgtgctgctgcccgacaaccactac
ctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatca
catggtcctgctggagttcgtgaccgccgccgggatcactctcggcatgg
acgagctgtacaagtaa
TABLE-US-00062 TABLE 55 Amino acid sequence of ECFP (SEQ ID NO:
127) VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTT
GKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFF
KDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNV
YITADKQKNGIKANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHY
LSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK
TABLE-US-00063 TABLE 56 DNA sequence encoding His-TEV-MSP1T2-GT
(SEQ ID NO: 128) atgggtcatcatcatcatcatcatcacgattatgatattcctactactga
gaatttgtattttcagggttctaccttcagtaaacttcgcgaacaactgg
gccccgtgacgcaggaattctgggacaacctggaaaaagaaaccgaggga
ctgcgtcaggaaatgtccaaagatttagaagaggtgaaggccaaggttca
gccatatctcgatgactttcagaaaaaatggcaggaagagatggaattat
atcgtcaaaaggtggaaccgctgcgtgcggaactgcaagagggggcacgc
caaaaactccatgagctccaagagaagctcagcccattaggcgaagaaat
gcgcgatcgcgcccgtgcacatgttgatgcactccggactcatttggcgc
cgtattcggatgaacttcgccagcgtttggccgcacgtctcgaggcgctg
aaagaaaacgggggtgcccgcttggctgagtaccacgcgaaagcgacaga
acacctgagcaccttgagcgaaaaagcgaaaccggcgctggaagatctac
gccagggcttattgcctgttcttgagagctttaaagtcagttttctgtca
gctctggaagaatatactaaaaagctgaatacccagggtacc
TABLE-US-00064 TABLE 57 Amino acid sequence of His-TEV-MSP1T2-GT
(SEQ ID NO: 129) MGHHHHHHHDYDIPTTENLYFQGSTFSKLREQLGPVTQEFWDNLEKETEG
LRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGAR
QKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEAL
KENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLS
ALEEYTKKLNTQGT
MSP derivatives have been prepared with the incorporation of
cysteine residues into the scaffold proteins by point mutation. DNA
coding and amino acid sequences are given in Tables 58 and 59,
respectively. In MSP1RC12=a cysteine residue is incorporated at the
last residue in the Factor X recognition site. This mutant is used
to prepare fluorescently labeled discs and attach to surfaces or
matrices. In MSP1K90C, Lysine90 is replaced by a cysteine. See
Tables 60 and 61 for coding and amino acid sequences respectively.
In MSP1K152C, Lysine 152 is replaced by cysteine; see Tables 62 and
63.
TABLE-US-00065 TABLE 58 DNA sequence encoding MSP1RC12= (SEQ ID NO:
130) Atgggtcatcatcatcatcatcacattgagggatgtctgaagctgttgga
caattgggactctgttacgtctaccttcagtaaacttcgcgaacaactgg
gccccgtgacgcaggaattctgggacaacctggaaaaagaaaccgaggga
ctgcgtcaggaaatgtccaaagatttagaagaggtgaaggccaaggttca
gccatatctcgatgactttcagaaaaaatggcaggaagagatggaattat
atcgtcaaaaggtggaaccgctgcgtgcggaactgcaagagggggcacgc
caaaaactccatgagctccaagagaagctcagcccattaggcgaagaaat
gcgcgatcgcgcccgtgcacatgttgatgcactccggactcatttggcgc
cgtattcggatgaacttcgccagcgtttggccgcacgtctcgaggcgctg
aaagaaaacgggggtgcccgcttggctgagtaccacgcgaaagcgacaga
acacctgagcaccttgagcgaaaaagcgaaaccggcgctggaagatctac
gccagggcttattgcctgttcttgagagctttaaagtcagttttctgtca
gctctggaagaatatactaaaaagctgaatacccag
TABLE-US-00066 TABLE 59 MSP1RC12= Protein Sequence (SEQ ID NO: 131)
MGHHHHHHIEGCLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEG
LRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGAR
QKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEAL
KENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLS ALEEYTKKLNTQ
TABLE-US-00067 TABLE 60 DNA sequence encoding MSP1K90C (SEQ ID NO:
132) atgggtcatcatcatcatcatcacattgagggacgtctgaagctgttgga
caattgggactctgttacgtctaccttcagtaaacttcgcgaacaactgg
gccccgtgacgcaggaattctgggacaacctggaaaaagaaaccgaggga
ctgcgtcaggaaatgtccaaagatttagaagaggtgaaggccaaggttca
gccatatctcgatgactttcagaaaaaatggcaggaagagatggaattat
atcgtcaaaaggtggaaccgctgcgtgcggaactgcaagagggggcacgc
caatgtctccatgagctccaagagaagctcagcccattaggcgaagaaat
gcgcgatcgcgcccgtgcacatgttgatgcactccggactcatttggcgc
cgtattcggatgaacttcgccagcgtttggccgcacgtctcgaggcgctg
aaagaaaacgggggtgcccgcttggctgagtaccacgcgaaagcgacaga
acacctgagcaccttgagcgaaaaagcgaaaccggcgctggaagatctac
gccagggcttattgcctgttcttgagagctttaaagtcagttttctgtca
gctctggaagaatatactaaaaagctgaatacccag
TABLE-US-00068 TABLE 61 MSP1K90C Protein sequence (SEQ ID NO: 133)
MGHHHHHHIEGRLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEG
LRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGAR
QCLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEAL
KENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLS ALEEYTKKLNTQ
TABLE-US-00069 TABLE 62 DNA sequence encoding MSP1K152C (SEQ ID NO:
134) atgggtcatcatcatcatcatcacattgagggacgtctgaagctgttgga
caattgggactctgttacgtctaccttcagtaaacttcgcgaacaactgg
gccccgtgacgcaggaattctgggacaacctggaaaaagaaaccgaggga
ctgcgtcaggaaatgtccaaagatttagaagaggtgaaggccaaggttca
gccatatctcgatgactttcagaaaaaatggcaggaagagatggaattat
atcgtcaaaaggtggaaccgctgcgtgcggaactgcaagagggggcacgc
caaaaactccatgagctccaagagaagctcagcccattaggcgaagaaat
gcgcgatcgcgcccgtgcacatgttgatgcactccggactcatttggcgc
cgtattcggatgaacttcgccagcgtttggccgcacgtctcgaggcgctg
aaagaaaacgggggtgcccgcttggctgagtaccacgcatgcgcgacaga
acacctgagcaccttgagcgaaaaagcgaaaccggcgctggaagatctac
gccagggcttattgcctgttcttgagagctttaaagtcagttttctgtca
gctctggaagaatatactaaaaagctgaatacccag
TABLE-US-00070 TABLE 63 MSP1K152C Protein sequence (SEQ ID NO: 135)
MGHHHHHHIEGRLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEG
LRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGAR
QKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEAL
KENGGARLAEYHACATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLS ALEEYTKKLNTQ
[0207] The mutations in MSP1K90C and in MSP1K152C are located on
inter-helical interfaces. Discs were formed in the presence of DTT.
The discs are more stable toward temperature-induced irreversible
degradation. These are our variants of the "Milano" mutations.
[0208] In addition to these sequences, there are two fusion protein
constructs of reference. These are composed of two MSP1 constructs
linked by a Gly-Ser linker:
TABLE-US-00071 MSP2 (MSP1BGLyBThrBMSP1, SEQ ID NO: 17) and MSP2D1D1
(MSP1T3BGlyBThrB H2-H3-H4-H5-H6-H7-H8-H9- H10, SEQ ID NO: 86).
[0209] Other constructs that can be readily produced include
permutations of the above, i.e. MSP1 or MSP2 or MSP2a with any
combination of the following: hinge deletion, hinge replacement,
half-repeat deletion, histidine tag, different linkers for MSP2
analogs.
Example 3
Expression of Recombinant MSPs
[0210] To express MSP proteins, the nucleic acid constructs were
inserted between the NcoI and HindIII sites in the pET28 expression
vector and transformed into E. coli BL21 (DE3). Transformants were
grown on LB plates using kanamycin for selection. Colonies were
used to inoculate 5 ml starter cultures grown in LB broth
containing 30 .mu.g/ml kanamycin. For overexpression, cultures were
inoculated by adding 1 volume overnight culture to 100 volumes LB
broth containing 30 .mu.g/ml kanamycin and grown in shaker flasks
at 37 C. When the optical density at 600 nm reached 0.6-0.8,
isopropyl-.beta.-D-thiogalactopyranoside (IPTG) was added to a
concentration of 1 mM to induce expression and cells were grown 3-4
hours longer before harvesting by centrifugation. Cell pellets were
flash frozen and stored at -80 C.
Example 4
Purification of Recombinant MSPs
[0211] Purification of histidine-tagged MSPs was carried out as
follows. A frozen cell pellet from 1 liter of expression culture
was resuspended in 25 milliliters of 20 mM Tris HCl pH 7.5
containing 1 mM phenylmethylsulfonyl fluoride. Triton X-100
(t-octylphenoxypolyethoxyethanol) was added from a 10% (w/v) stock
in distilled H20 to a final concentration of 1%. The resuspended
cells were sonicated on ice at 50% duty cycle at a power setting of
5 for four cycles of 1 minute on, 5 minutes off with a Branson
probe sonifier. The resulting lysate was centrifuged for 30 minutes
at 30,000 rpm in a Beckman Ti 45 rotor in a ultracentrifuge. The
resulting supernatant was filtered through a 0.22 .mu.m nylon
syringe filter. The salt concentration was adjusted to 0.5 M from a
4 M NaCl stock in water and applied to a 5 ml Hi-Trap nickel loaded
column (Pharmacia, Piscataway, N.J.).
[0212] For His-tagged-MSP1, the column is washed with 20 ml buffer
(10 mM Tris pH 8, 0.5 M NaCl) containing 1% Triton X-100, followed
by 20 ml buffer+50 mM sodium cholate, and then 20 ml buffer and 20
ml 100 mM imidazole in buffer. The His-tagged polypeptide is eluted
with 15 ml 0.5 M imidazole in buffer.
[0213] For His-tagged-MSP2, the column is washed with 20 ml buffer
(10 mM Tris pH 8, 0.5 M NaCl) containing 1% Triton X-100; 20 ml
buffer+50 mM cholate; 20 ml buffer; 20 ml 35 mM imidazole in
buffer. The His-tagged polypeptide is then eluted with 15 ml 0.5 M
imidazole in buffer, and the purified protein is dialyzed against
10 mM Tris pH 8, 0.15 M NaCl using a 10,000 MW cutoff cellulose
dialysis membrane.
Example 5
Production of MSP-containing Nanoscale Particles
[0214] To reconstitute MSP proteins of the present invention with
lipid, purified MSP was concentrated in a pressurized
ultrafiltration device (Amicon) using a 10,000 MW cutoff filter to
.about.2-6 mg protein/ml. Concentration of protein was determined
by bicinchoninic acid assay (Pierce Chemical, Rockford, Ill.) or
measurement of A280 using theoretical absorption coefficient.
Phospholipid (dipalmitoyl phosphatidylcholine in this case, however
different phosphatidylcholines and mixtures of phosphatidylcholine
and other lipids can be used) in chloroform stock solution was
dried under a stream of nitrogen and placed in vacuo overnight.
Phosphate analysis was performed to determine the concentration of
chloroform stock solutions. The dried lipid film was resuspended in
buffer 10 mM Tris HCl pH 8.0 or pH 7.5 containing 0.15 M NaCl and
50 mM sodium cholate to give a final lipid concentration of 25 mM.
The suspension was vortexed and heated to 50 C to obtain a clear
solution. Phospholipid solution was added to solution of MSP (2-6
mg/ml protein) to give molar ratios for MSP1:lipid of 2:200 and for
MSP2 of 1:200. The mixture was incubated overnight at 37 C and then
dialyzed against 1000 volumes of buffer without cholate with 4
changes of buffer over 2-3 days.
Example 6
Tissue Factor Incorporation
[0215] Tissue Factor (TF) is a representative membrane protein. In
order to demonstrate the value of MSP technology for a tethered
membrane protein, recombinant human TF was incorporated into
MSP-supported Nanodiscs. The recombinant protein consists of an
extracellular domain, the transmembrane anchor and a truncated
cytosolic domain. The truncation increases the homogeneity of the
protein by removing the C-terminal portions of the protein which
are subject to proteolysis by bacterial enzymes. This modification
does not affect TF activity. Additional modifications to the
protein include an N-terminal trafficking peptide and an HPC4
epitope tag. The trafficking peptide directs the expressed protein
to the intermembrane space of the recombinant E. coli host cell, in
which space the peptide sequence is cleaved. The HPC4 epitope
allows for affinity purification with Ca.sup.2+ dependent antibody
(Rezaie et al., 1992) and does not affect TF activity.
[0216] A 25 mM lipid mixture containing 80% phosphatidyl choline
and 20% phosphatidyl serine was solubilized with 50 mM cholate in
10 mM Tris Cl, 150 mM NaCl at pH 8.0. TF, MSP1 and lipid (in a
ratio of 1:10:1000) were combined and incubated overnight at 37 C.
The sample was then dialyzed at 37 C (10,000 dalton molecular
weight cutoff membrane) against buffer containing 10 mM Tris Cl,
150 mM NaCl at pH 8.0 (lacking cholate) for 2 hours. Dialysis was
then continued at 4 C for an additional 6 hours with buffer changes
every 2 hours. The approximately 1 ml sample was then concentrated
to <250 .mu.l using a YM-10 centrifuge concentrator and injected
into a Pharmacia 10/30 Superdex 200 HR gel filtration column.
Samples were eluted with buffer identical to that described above
(no cholate) at 0.5 ml per minute. Fractions from chromatography
were run on an 8-25% gradient SDS polyacrylamide gel to determine
apparent size and then checked for coagulation activity. The
chromatogram showing elution of TF incorporated into an excess
population of MSP1 Nanodiscs is shown in FIG. 16A-16B.
[0217] The activity of TF in several disk fractions was determined
by coagulation assays with human serum. Activity was monitored in
fractions 25-28 as the inverse of coagulation time. Activity was
highest in fraction 25 at 40 hr.sup.-1 and decreased through
fraction 28 at 30 hr.sup.-1. This is expected from the size
chromatogram in that the leading edge of the Nanodisc peak has a
larger effective mass due to the incorporation of TF in the
MSP-supported bilayer. This assay thus demonstrates that TF is
incorporated into Nanodiscs in an active conformation and that the
membrane environment of the Nanodisc closely mimics that of the
native membrane system.
[0218] Cytochrome b5 is a membrane anchored heme protein having a
single membrane anchor domain that penetrates the membrane bilayer.
Cytochrome b5 solubilized from its native membrane exists as large
aggregates in the absence of detergent and appears as a smear
rather than a discrete band on native polyacrylamide gel
electrophoresis. Formation of Nanodiscs through a self-assembly
process wherein cytochrome b5 is added to the preparation of MSP
and phospholipid results in the incorporation of cytochrome b5 into
Nanodisc structures. This is verified by the intense heme staining
of the band corresponding to Nanodiscs. The data show that
cytochrome b5 can be successfully solubilized using MSP technology
and that disc complexes containing cytochrome b5 can be
chromatographically separated and purified away from the undesired
aggregated material. The optical absorption properties of the heme
chromophore of the purified material demonstrate that the heme
active site in a native conformation.
[0219] Nanodiscs can also be formed by mixing 20 .mu.l of MSP1 (10
mg/ml), 6.6 .mu.l cytochrome b5 (0.5 mM) and 50 .mu.l egg
phosphatidylcholine/sodium cholate (11.2 egg PC, 6.2 mg/ml sodium
cholate), incubating overnight at 4 C, followed by dialysis to
remove cholate. Purification was accomplished using a Pharmacia
MonoQ FPLC anion exchange column equilibrated in 25 mM Tris Cl, pH
8.0. A linear gradient was run at 0.5 ml/min from 0-1 M NaCl in 20
min.
[0220] As an alternative to incorporating tethered membrane
proteins into Nanodiscs from solubilized, purified proteins, the
tethered membrane proteins can be incorporated into Nanodiscs with
MSPs using membrane or membrane fragment preparations containing
those tethered membrane proteins of interest.
Example 7
Embedded Membrane Protein Incorporation
[0221] Cytochrome P450 2B4 from rabbit liver microsomes, cytochrome
P450 3A4 found in nature in human liver microsomes and cytochrome
P450 6B1 from insect microsomes are representative of embedded
membrane proteins.
[0222] Cytochrome P450 2B4 was isolated from rabbit liver
microsomes after induction with phenobarbital. Formation of 2B4
Nanodiscs is as follows. Cytochrome P450 2B4 was reconstituted into
disks by the detergent dialysis method. The buffer consisted of 10
mM Tris-HCl pH 8.0, 0.1 M NaCl, 10% (v/v) glycerol. The mixture of
apo A-I, cholate and phospholipid (1:220:110 mole ratio) was
incubated for 8 hours at 37.degree. C. followed by addition of P450
(1:0.5, apo A-I:P450 mole ratio) and incubation overnight at room
temperature. The mixture was dialyzed using a 10,000 MW cutoff
slide-a-lyzer (Pierce Chemical Co., Rockford, Ill.) at room
temperature for two hours followed by a change of buffer and
continued dialysis at 4.degree. C. It was found that 82% of the
P450 content could be recovered under these conditions. After
dialysis, the mixture was injected onto a Superdex 200 HR10/30 gel
filtration column (Pharmacia, Uppsala, S E) equilibrated in
reconstitution buffer at room temperature at a flow rate of 0.25
ml/minute with collection of 0.5 ml fractions. Fractions were
assayed using native polyacrylamide gradient gel electrophoresis on
8-25% gradient native gels and Coomassie staining using the
Phastgel system (Pharmacia, Uppsala, Sweden).
[0223] Human cytochrome P450 3A4, normally from liver microsomes,
has also been cloned, expressed in E. coli, purified and
incorporated into MSP-supported bilayer Nanodiscs. Ten nanomoles of
MSP2, one micromole of lipid, five nanomoles of cytochrome P450 3A4
protein and two micromoles cholic acid were incubated together at
37 C for 2 hours. The incubated mixture was then dialyzed in a 10K
Slide-A-lyzer Dialysis Cassette (Pierce Chemical Co., Rockford,
Ill.). The dialysis was carried out with 10 mM potassium phosphate
(pH 7.4) 150 mM NaCl buffer. The sample was dialyzed at 37 C for 6
hours followed by a buffer change, and dialysis continued at 4 C
with two buffer changes at 12 hour intervals. The samples were then
fractionated on a Superdex 200 HR 10/30 column (Pharmacia, Uppsala,
S E) equilibrated in dialysis buffer at room temperature at a flow
rate of 0.5 ml/min.
[0224] Cytochrome P450 6B1 is another model embedded membrane
protein; it has been isolated from Papilio polyxenes, the black
swallowtail. These butterflies feed exclusively on plants producing
furanocoumarins, plant metabolites that are phototoxic to most
organisms. Cytochrome 6B1 catalyzes the detoxification of
furanocoumarins.
[0225] In order to show the utility of the MSP methodology of the
present invention, we demonstrated that isolated membranes
containing their repertoire of membrane proteins and natural lipids
could be used as a source for incorporating membrane proteins into
Nanodiscs. An important illustrative embodiment is the use of the
common insect cell (Sf9)-baculovirus expression system which is
used widely as a heterologous expression system. Thus, we used an
insect cell line co-infected such that a microsomal preparation
containing overexpressed insect CYP6B1 and also overexpressed
insect NADPH cytochrome P450 reductase. In these experiments we not
only demonstrate that MSP Nanodiscs can be used to incorporate
another cytochrome P450 system into soluble monodisperse particles
but also that the source of this P450 could be simply whole
membranes containing this protein.
[0226] A standard baculovirus expression system was used to obtain
microsomal preparations with overexpressed insect cytochrome CYP6B1
and insect NADPH P450 reductase. Construction of the recombinant
CYP6B1 baculovirus expression vector and infection of Spodoptera
frugiperda (Sf9) was performed as previously described (Chen et
al., 2002). Typically, 32 plates containing 6.times.10.sup.7
baculovirus-infected cells each (MOI of 2) were collected 72 hours
post-infection. Microsomal membranes were homogenized in 2 ml
grinding buffer (pH 7.8) composed of 0.1 M sodium phosphate buffer
(pH 7.8), 1.1 mM EDTA, 20% glycerol, 0.5 mM PMSF, 0.1 mM DTT, and 5
.mu.g/ml (w/v) leupeptin. Membranes were frozen in liquid nitrogen
and stored at 80 C.
[0227] To assemble Nanodiscs comprising CYP6B1 from the microsomal
membrane preparation, the protein concentration of the membranes
was determined using a BCA protein assay kit from Pierce (Rockford,
Ill.). We assumed a 1:1 mass relationship of protein: lipid in the
membranes and an average molecular weight of phospholipids of 750
grams/mole. The membranes were detergent-solubilized with 0.5 M
cholic acid (neutralized) and mixed with MSP in the approximate
ratio of 1:25:50 to 1:2000:1000, preferably 1:75:150 in at least
some cases, for MSP:lipid:detergent. Typically, reconstitution
samples include approximately 100 nmol scaffold protein, 10 .mu.mol
lipid, and 20 .mu.mol neutralized cholic acid and were
pre-incubated for 1.5 hours at 4 C. The temperature chosen is
higher than the phase transition temperature for the lipids.
Detergent was removed by incubating with Biobeads SM-2 Adsorbent
from BioRad Laboratories (Hercules, Calif.) (0.4 grams Biobeads per
1 ml of reconstitution mixture) for 1.5 hours at 4 C followed by
centrifugation at 11,750.times.g for 5 minutes. His6-tagged MSP
particles were purified by incubating with 1 ml of Ni-NTA agarose
from Qiagen, Inc. (Valencia, Calif.) per 7.5 mg of His6-tagged MSP
for 1 hour at 4 C, followed by centrifugation at 11,750.times.g for
5 minutes. MSP particles bound to the Ni-NTA agarose were washed
with three sequential resin volumes of 0.1 M sodium phosphate
buffer (pH 7.4) containing 0.3 M NaCl, 0.15 M NaCl, and no NaCl,
respectively. To maintain the integrity of the CYP6B1 protein, MSP
particles were eluted with 0.1 M sodium phosphate buffer (pH 7.4)
containing 0.25 M EDTA (to chelate trace metal ions) rather than
the 50 mM imidazole used in previous MSP purifications.
[0228] Based on the lipid concentration contained in the microsomal
preparations, MSP technology was used to assemble microsomal
proteins into nanoparticle discs using a ratio of 110:1:220
lipid:MSP1:cholate. The microsomal sample was detergent solubilized
with neutralized cholate and mixed with MSP1. The sample was
incubated at 4 C for 2 hours. The detergent can be removed by
dialysis or adsorption to hydrophobic beads. In this experiment
Biobeads (hydrophobic beads, trademark of BioRad, Hercules, Calif.)
were added in excess (0.25 g per 1 ml disc mixture) and incubated
for 2 hours at 4 C for 2 hours to remove detergent. The sample was
removed from the beads and the His.sub.6-tagged MSP was isolated by
using a batch purification method with Ni.sup.2+ resin. The MSP
disks were then isolated by Superdex sizing column chromatography
(FIG. 9). Incorporation of P450 into the His.sub.6-tagged discs was
followed by CO difference spectroscopy of nickel affinity column
purified and sizing column-purified fractions (FIG. 10). SDS-PAGE
was performed using 8-25% gradient gels stained with Coomassie blue
to verify incorporation of cytochrome P450 6B1 into discs (FIG.
10).
[0229] The endogenous (natural) ratio of cytochrome P450 to
reductase is about 10-20. To obtain activity of the cytochrome P450
6B1 after reconstitution into discs, it is preferred that an excess
of reductase be added to the reconstitution mixture, such that a
P450 molecule and reductase molecule both partition into a single
disc. Supplementation of the microsomal preparation with
exogenously added reductase has been successfully demonstrated.
[0230] The protocol for making discs using microsomal preparations
was used with one modification. Exogenous rat reductase was added
after the solubilization step of the microsomal preparation with
sodium cholate and before the addition of MSP1. Otherwise identical
disc assembly and purification procedures were followed. The sample
was separated by a Superdex sizing column, where absorbance at 280
nm indicates the presence of MSP1, absorbance at 420 and 456 nm
indicates the presence of ferric species, and absorbance at 456 nm
also indicates presence of reductase. A ratio plot of 456 to 420 nm
was made; it showed positions on the chromatogram where the
absorbance at 456 nm was above that associated with cytochrome P450
6B1 and, therefore, could be attributed to absorbance by reductase.
Retention times reflected the presence of 10 nm particles
containing cytochrome P450 6B1 and reductase (FIG. 13).
[0231] MSP-supported Nanodiscs with purified proteins, membrane
fragments or disrupted membranes can be used in high throughput
screening ventures, for example, to identify new pharmaceuticals
and other biologically active molecules.
Example 8
Integral Membrane Protein Incorporation
[0232] Bacteriorhodopsin (BR) is a model integral membrane protein,
and a model seven transmembrane domain protein. BR was incorporated
into nanoscale structures using the following procedure, which is a
protocol useful for other proteins as well. BR was obtained as
lyophilized purple membrane from Sigma (St. Louis, Mo.). 1 mg BR
was suspended in 1 ml 25 mM potassium phosphate, pH 6.9. 1 ml 90 mM
n-octyl .beta.-D-glucopyranoside in the same buffer was added and
the sample placed in the dark at 24 C overnight. This treatment
produces a detergent-solubilized monomeric form (Dencher et al.,
1982). BR was quantitated assuming a molar extinction coefficient
at 550 nm of 63,000. BR (7.8 .mu.M) was mixed with MSP1 (97 .mu.M)
or MSP2 (110 .mu.M) and cholate (50 mM) to give final molar ratios
of MSP1:BR of 10:1 or MSP2:BR of 5:1 and a cholate concentration of
approximately 8 mM. For reconstitution with phospholipid, the lipid
is solubilized as above in the presence of 50 mM cholate and mixed
with MSP1 at a mole ratio of 1 MSP1:110 lipids:0.1 BR. The mixture
was incubated at room temperature for .about.3 hours followed by
dialysis overnight against 1000 volumes of buffer using 10,000 MW
cutoff dialysis devices (Slide-a-lyzer, Pierce Chemical). Dialysis
was continued at 4 degrees for 2 days with several changes of
buffer. 10 mM HEPES, pH 7.5, 0.15 M NaCl buffer can be used. Tris
buffer pH 7.5 or pH 8 has also been used successfully.
[0233] The human 5-hydroxytryptamine 1A G protein coupled receptor
has been incorporated into MSP-containing nanoparticles. A
commercially available insect cell expression system that provides
a membrane fraction containing the human 5-hydroxytryptamine 1A
(5-HT-1A) GPCR was used as a source of this GPCR to prepare
Nanodiscs. Briefly, the 5-HT-1A receptor containing membrane
preparation was mixed with phospholipids (phosphatidyl choline,
phosphatidylethanolamine, phosphatidyl serine) at a ratio of
45:45:10, MSP1 and cholate (neutralized cholic acid).
[0234] 5-HT-1A receptors overexpressed in a commercially available
Sf9 insect cell membrane preparation (Sigma Chemical Co., St.
Louis, Mo.) were solubilized using the following protocol. POPC,
POPS and POPE (Avanti Phospholipids) in chloroform were mixed in a
45:10:45 mole ratio and dried down under a stream of nitrogen, then
placed under vacuum for several hours to remove residual solvent.
The phospholipids were dispersed in 50 mM Tris pH 7.4, 0.2 M NaCl,
50 mM sodium cholate buffer at a concentration of 25 mM
phospholipid. Five microliters of the Sf9 membrane preparation (0.2
mg/ml protein), 1.62 microliters of phospholipid in buffer, 2.4
microliters of MSP1 (4.2 mg/ml) and 0.28 microliters 4 M NaCl were
mixed and left for 1 hour on ice. The mixture was diluted to 100
microliters total volume with 50 mM Tris pH 7.4 and dialyzed in a
mini slide-a-lyzer (Pierce Chemical) against 50 mM Tris pH 7.4 at
4.degree. C. (two one-liter changes of buffer).
[0235] To determine the amount of 5-HT-1A receptor associated with
Nanodiscs, a radiolabeled ligand was bound to the receptor and
disk-receptor-ligand complexes were isolated using the 6-histidine
tag present in the MSP1 according to the following protocol. After
dialysis, the mixture was diluted to 200 microliters total volume
with 50 mM Tris pH 7.4. Ninety-five microliters of the diluted
mixture were placed into each of two tubes. One hundred five
microliters of stock reagent were added to give final
concentrations of 50 mM Tris pH 7.4, 10 mM MgSO.sub.4, 0.5 mM EDTA,
0.1% ascorbic acid in a final volume of 200 microliters.
Tritium-labeled 8-hydroxy-DAT (specific activity 135000 Ci/mole)
was added to each tube to give a concentration of 1.5 nM. As a
control, unlabeled metergoline (final concentration 100 micromolar)
was added to one of the tubes as a competitive ligand. After 1 hour
on ice, the mixture was applied to 200 microliters of Ni-chelating
resin to specifically bind receptor associated with His-tagged MSP1
disks. The resin was washed three times with 0.5 ml of cold 50 mM
Tris pH 7.4 to remove non-specifically bound ligand. Specifically
bound radiolabeled 8-hydroxy-DAT bound to receptor/disk complexes
was eluted with 0.5 ml 0.5 M imidazole in 10 mM Tris pH 7.4, 0.5 M
NaCl. Scintillation cocktail was mixed with the eluate and
specifically bound radioligand was determined by scintillation
counting. Between five and fifteen percent of the receptor
initially present in the Sf9 membrane was found to bind ligand in
receptor associated with MSP1 Nanodiscs.
[0236] The particles into which the 5-HT-1A GPCR had incorporated
were dialyzed. Functionality (in terms of ligand binding) was
tested by incubation with buffer containing tritiated 8-OH-DAT, an
agonist of this receptor. The particles were then run over a Ni-NTA
column to bind via the histidine tag on the MSP1 and to separate
the particles from 8-OH-DAT which had not bound to the particles,
and the material bound to the column was then eluted. Association
of the tritium labeled agonist was demonstrated, showing that the
incorporated GPCR retained its ability to bind agonist.
[0237] As discussed above for the tethered membrane proteins, the
integral and embedded membrane proteins can be incorporated into
Nanodiscs using MSPs and solubilized membrane preparations, rather
than purified, solubilized proteins. The naturalistic presentation
of the proteins within the Nanodiscs is maintained, regardless of
whether the proteins were purified or whether they were directly
derived from membrane preparations.
Example 9
Analysis of MSP-Supported Nanodisc Phospholipid Assemblies
[0238] The particles resulting from self-assembly of membrane
scaffold proteins and phospholipids, either with or without an
additional target protein, were analyzed as follows.
[0239] Bacteriorhodopsin-containing particles were dialyzed, and
the resulting mixture was injected onto a Superdex 200 HR10/30 gel
filtration column (Pharmacia) and eluted with buffer at 0.5 ml/min
at room temperature. Absorbance was monitored at 280 nm for protein
and 550 nm for BR. 0.5 ml fractions were collected. The column was
calibrated using a mixture of thyroglobulin (669 kDa, Stoke=s
diameter 170 A), ferritin (440 kDa, Stoke=s diameter 122 A),
catalase (232 kDa, Stoke=s diameter 92 A), lactate dehydrogenase
(140 kDa, Stoke=s diameter 82 A), bovine serum albumin (66 kDa,
Stoke=s diameter 71 A), and horse heart cytochrome c (12.4 kDa,
Stoke=s diameter 35.6 A).
[0240] Atomic Force Microscopy was performed with a Digital
Instruments Nanoscope IIIa in contact mode with sharpened silicon
nitride probes under buffer. MSP1 and MSP2 dipalmitoyl
phosphatidylcholine particles were treated with 1:50 Factor Xa:MSP
protein by mass in 10 mM Tris pH 8, 0.15 M NaCl, 2 mM CaCl.sub.2
for 8 hours. 2-10 ml sample was placed on a freshly cleaved mica
surface along with 20 ml imaging buffer (10 mM Tris pH 8, 0.15 M
NaCl, 10 mM MgCl.sub.2) and incubated for 30 minutes or longer
before mounting sample in the fluid cell. Several milliliters of
buffer were flushed through the fluid cell to remove unadsorbed
material.
[0241] Phosphate analysis of the nanoscale particles was carried
out as follows. Phosphate assay procedures were adapted from Chen
et al. (1956) and Fiske and Subbarow (1925). Samples containing
roughly 40 nmoles lipid phosphate were dried down in glass tubes.
75 .mu.l 8.9 N H.sub.2SO.sub.4 was added to each tube and heated to
210 C for 30 minutes. 1 drop 30% H.sub.2O.sub.2 was added to each
tube and heated for 30 minutes. Tubes were cooled, 0.65 ml H.sub.2O
was added followed by 83.3 .mu.l 2.5% w/v ammonium molybdate
tetrahydrate followed by vortexing and the addition of 83.3 .mu.l
10% w/v ascorbic acid. After mixing, the tubes were placed in a
boiling water bath for 7 minutes. Absorbance was read at 820 nm.
Absorbance was calibrated using potassium phosphate standards from
0 to 100 nmol phosphate. Buffer blanks from column chromatography
were included for MSP proteins.
Example 10
MSP-Supported Structures on Surfaces
[0242] Nanodiscs comprising MSPs and a protein of interest can be
assembled onto a gold surface or other solid surface (solid
support). The utility of this relates to the resulting epitaxial
presentation of a target incorporated into a Nanodisc assembly to
the solution. This offers an ideal system for quantitating binding
of other macromolecules or small molecules tagged with dielectric
contrast agents to the target protein. A common method of
accomplishing such measurements uses surface plasmon resonance
(SPR) technology. SPR is a common technique used to monitor
biomolecular interactions at surfaces. The ability of SPR to
rapidly detect and quantitate unlabeled protein interactions on
gold surfaces is useful for creating high through put chip assays
for diverse membrane proteins (embedded and solubilized) on
discs.
[0243] Discs consisting of the phospholipid DPPC either with or
without an additional thiolated lipid and MSP1 protein were
prepared as follows. A 25 mM lipid mixture containing
phosphatidylcholine was solubilized with 50 mM cholate in 10 mM
Tris Cl, 150 mM NaCl at pH 8.0 were combined and incubated
overnight at 37 C. For thiolated discs, 90% phosphatidylcholine and
10% thiolated lipid (ATA-TEG-DSPA, Northern Lipids, Vancouver, BC,
CA) was solubilized in 3.3 mM Tris Cl, 66.7 mM borate, 150 mM NaCl
at pH 9.0 in order to unmask the thiols in the thiolated lipids.
MSP1 and lipid (1:100) were combined and incubated overnight at 37
C. The sample was then dialyzed at 37 C (10,000 MW cutoff membrane)
against buffer containing 10 mM Tris Cl, 150 mM NaCl at pH 8.0
without cholate for 2 hours. Dialysis was then continued at 4 C for
an additional 6 hours with buffer changes every 2 hours. The
approximately 1 ml sample was concentrated to <250 .mu.l using a
YM-10 centrifuge concentrator and injected onto a Pharmacia 10/30
Superdex 200 HR gel filtration column. Samples were eluted from the
column using the stated buffer without cholate at flow rates of 0.5
ml/min. Fractions from chromatography were analyzed by
polyacrylamide gel electrophoresis using 8-25% gradient
polyacrylamide gel to determine apparent size.
[0244] The Nanodisc samples (3-20 .mu.M) prepared as described were
injected into an SPR instrument to determine if the discs would
bind to the gold surface. Both the DPPC and 10% thiolated lipid
discs adsorbed to a gold surface and a modified gold surface
covered with a monolayer of methyl terminated thiol (nonanethiol)
or carboxyl terminated thiol (11-mercaptoundecanoic acid).
Thiolated discs were injected using a buffer consisting of 3.3 mM
Tris, 66.7 mM borate, 150 mM NaCl, pH 9.0. DPPC discs were injected
using a buffer of 10 mM Tris, 150 mM NaCl, pH 7.5 or pH 8.0. In all
cases, the discs could not be removed even under harsh conditions
(0.5 M HCl). Surface coverage was shown to increase with increasing
concentration of discs injected (3 .mu.M vs. 19 .mu.M). Discs do
not form perfectly packed monolayers; accordingly, surface coverage
is limited by the jamming limit (theoretical maximum coverage based
on random sequential absorption to the surface modeling discs as
identical non-overlapping hard spheres) of 0.547. The coverage for
a full monolayer of discs was calculated based on an assumption of
disc height of 5.5 nm and a refractive index between 1.45 and 1.5.
The full monolayer values were multiplied by the jamming limit to
determine the maximum coverage that was then used to determine
percent coverage based on experimental values. When the disc
concentration was at least 10 .mu.M, the estimated coverages were
between about 62 and about 103%. The resultant SPR trace
demonstrating association of the Nanodiscs to the gold surface is
shown in FIG. 14.
[0245] Nanodiscs comprising MSPs and a protein of interest can be
attached to a solid support via the His tag on the MSP where the
support is coated with Ni-NTA or a His tag-specific antibody,
commercially available from BD Biosciences Clontech, Palo Alto,
Calif., for example, or to Ni-NTA agarose beads, commercially
available from Qiagen, Valencia, Calif., for example, or other
solid support, including beads, chips, plates and microtiter
dishes.
Example 11
General Techniques
[0246] For SDS-PAGE, microliter samples were separated on 8-25%
gradient polyacrylamide gels (Pharmacia) and stained with Coomassie
blue.
[0247] Sizing column chromatography purification was carried out as
follows. The nickel affinity-purified sample mixture was injected
onto a Superdex (Trademark of Pharmacia, Piscataway, N.J.) 200
HR10/30 gel filtration column (Pharmacia) equilibrated in 0.1M
sodium phosphate buffer (pH 7.4) at a flow rate of 0.5 ml/min.
Fractions containing CYP6B1 were concentrated using a Centricon
YM-30 centrifugal filter device (Millipore Corporation, Billerica,
Mass.) and re-injected onto the Superdex 200 HR10/30 gel filtration
column under the same buffer conditions.
[0248] Lipids were extracted by the Folch method (Folch-Pi et al.
(1957)), where the sample was homogenized with 2:1
chloroform-methanol (v/v) and washed with volume 0.88% KCl in
water. The solution was mixed vigorously and the phases were
completely separated by centrifugation (3,000.times.g) for 5
minutes.
[0249] Nanodisc assembly is generally carried out as follows. The
protein concentration of the membranes was determined using a BCA
(bicinchoninic acid) protein assay kit from Pierce (Rockford,
Ill.). We assumed a 1:1 mass relationship of protein: lipid in the
membranes with an average molecular weight of phospholipids of 750
grams/mole. The membranes were detergent solubilized with 0.5 M
cholic acid (neutralized) and mixed with MSP in the approximate
ratio of 1:25:50 to 1:2000:1000 with 1:75:150 preferable. The
membranes were detergent solubilized with 0.5 M cholic acid
(neutralized) and mixed with MSP in the approximate ratio of
1:100:200 for MSP: lipid:detergent. Typically, reconstitution
samples include approximately 100 nmol membrane scaffold protein,
10 .mu.mol lipid, and 20 .mu.mol cholate and were pre-incubated for
1.5 hours at 4 C. Detergent was removed by incubating with Biobeads
SM-2 Adsorbent from BioRad Laboratories (Hercules, Calif.) (0.4
grams Biobeads per 1 ml of reconstitution mixture) for 1.5 hours at
4 C followed by centrifugation at 11,750.times.g for 5 minutes.
His6-tagged MSP particles were purified by incubating with 1 ml of
Ni-NTA agarose from Qiagen, Inc. (Valencia, Calif.) per 7.5
milligrams of His6-tagged MSP for 1 hour at 4 C, followed by
centrifugation at 11,750.times.g for 5 minutes. MSP particles bound
to the Ni-NTA agarose were washed with three sequential resin
volumes of 0.1 M sodium phosphate buffer (pH 7.4) containing 0.3 M
NaCl, 0.15 M NaCl, and no NaCl, respectively. To maintain the
integrity of the CYP6B1 protein, MSP particles were eluted with 0.1
M sodium phosphate buffer (pH 7.4) containing 0.25 M EDTA rather
than the 50 mM imidazole used in previous MSP purifications.
[0250] Thin-Layer Chromatography (TLC) is carried out as follows.
Samples were spotted onto preparative silica gel stationary phase
TLC plates purchased from EM Science (Hawthorne, N.Y.) alongside
phospholipid standards purchased from Avant (Alabaster, Ala.) and
developed using a mobile phase of chloroform/methanol/ammonium
hydroxide (65:25:4). TLC plates were exposed to iodine vapor for
visualization, scanned using a Hewlett Packard ScanJet, and
quantified on a Macintosh computer using the public domain NIH
Image program developed at the U.S. National Institutes of Health
(available on the internet at the website entitled
rsb.info.nih.gov/nih-image).
Example 12
Substrate Binding
[0251] The CYP6B1-containing population of Nanodiscs collected
after Superdex size fractionation was concentrated to an enzyme
concentration of 50 nM. A microtiter plate was arranged with wells
A1-A5 and wells B1-B5 each containing 200 .mu.l Nanodisc samples
and wells C1-C5 each containing 200 .mu.l buffer (0.1 M sodium
phosphate, pH 7.4). To rows A and C, a 20 mM stock concentration of
xanthotoxin (Sigma Chemical Co.) in methanol was added to yield
final concentrations of 0 .mu.M (column 1), 10 .mu.M (column 2), 20
.mu.M (column 3), 50 .mu.M (column 4), and 150 .mu.M (column 5).
This dilution was such that the total organic solvent content did
not exceed 1% when added to the Nanodisc samples. To row B, 0
.mu.l, 0.1 .mu.l, 0.2 .mu.l, 0.5 .mu.l, and 1.5 .mu.l methanol were
added.
[0252] The contents of each microtiter well were scanned at 1 nm
increments using a SpectraMAX Plus microplate spectrophotometer
(Molecular Devices, Sunnyvale, Calif.) and were corrected for the
background buffer absorbance (defined in row C) and Nanodisc
absorbance (well A1).
Example 13
Nanodiscs with Larger MSPs
[0253] The relatively larger Nanodiscs (the extended membrane
scaffold protein sequences) are useful in controlling the
oligomerization state of 7-Tm receptors or other hydrophobic or
partially hydrophobic proteins which are particularly large or
which tend to oligomerize incorporated into Nanodiscs. As
specifically exemplified, a bacteriorhodopsin trimer is
self-assembled in larger nanodiscs using the longer MSPs.
[0254] Purple membrane was isolated from Halobacterium salinarum
JW-3 cultures as described (Oesterhelt and Stoeckenius 1974).
Sucrose was removed by centrifugation at 35,000 rpm in a Beckman
Ti-45 rotor for 15 minutes followed by resuspension in water. This
process was repeated three times, the sample was aliquoted,
lyophilized and stored at -20.degree. C. Concentrations of MSPs
were determined from absorbance at 280 nm using extinction
coefficients of 24740 M.sup.-1 cm.sup.-1 for MSP1 and 31720
M.sup.-1 cm.sup.-1 for the other MSPs based on calculated
extinction coefficient (Gill and von Hippel 1989). The extinction
coefficient in nanodisc buffer for MSP1E1, MSP1E2 and MSP1E3 was
found to be equal to the calculated value in 20 mM phosphate
buffer, 6 M guanidine HCl pH 6.5. DMPC was obtained from Avanti
Polar Lipids, Inc., dissolved in chloroform and quantitated by
phosphate analysis (Chen, Toribara et al. 1956). Buffer consisted
of 10 mM Tris HCl pH 7.4, 0.1 M NaCl, 0.01% NaN.sub.3 unless stated
otherwise. Water was purified with a Milli-Q system (Millipore).
All other materials were high-quality reagents.
[0255] To self-assemble nanodiscs with bacteriorhodopsin and
extended MSPs, bacteriorhodopsin was initially solubilized with 4%
w/v Triton X-100 as described (Dencher and Heyn 1978). MSP stock
solutions (200-400 .mu.M) and a DMPC/cholate mixture (200/400 mM in
buffer) were added to bR in eppendorf tubes or Falcon tubes
(typically about 190 .mu.M) to give MSP to bR molar ratios of 2:3
and different phospholipid ratios. After one hour at room
temperature, detergent was removed by treatment for 3-4 hours with
400 mg wet Biobeads SM-2 (BioRad) per ml of solution, with gentle
agitation to keep the beads suspended (Levy, Bluzat et al. 1990).
Beads were removed by centrifuging the suspension through a pinhole
made in the bottom of the tubes.
[0256] Self-assembled Nanodisc mixtures were filtered through 0.22
micron filters and injected onto a size exclusion chromatography
column (Superdex 200 HR 10/30 column) run at 0.5 ml/min at room
temperature with collection of one minute fractions. Peak elution
was monitored at 280 and 560 nm.
[0257] Nanodiscs were analyzed by SDS-PAGE, protein was quantified,
and lipid stoichiometry was determined. Samples containing MSP1E3
and different amounts of bR as calibration standards were separated
on 20% SDS-PAGE using a Phastgel system (Pharmacia) along with
gel-filtration purified samples of MSP1E3-bR nanodiscs. After
staining with Brilliant blue R-250, gels were scanned and bands
quantitated using the computer program NIH image to determine the
ratio of bR to MSP1E3 in nanodiscs. The amount of lipid per bR in
MSP1E3 disks was determined using the extinction coefficient
.di-elect cons..sub.560=56,600.A-inverted.1200 M.sup.-1 cm.sup.-1
for bR in MSP1E3 nanodiscs measured by the method of retinal
titration and phospholipid content was determined by phosphate
analysis (Chen, Toribara et al. 1956; Rehorek and Heyn 1979).
Circular dichroism spectra were measured with a Jasco J-720
spectrapolarimeter at ambient temperature at a sample OD of
approximately 2.
[0258] bR-MSP mixtures at a 3 to 2 ratio were titrated with lipid
to determine optimal ratios for bR solubilization as assessed by
gel filtration chromatography. The optimal ratio is chosen as the
ratio at which the main peak of solubilized bR is the major
component with a minimum amount of larger species. At less than
optimal ratios, species of smaller size appear. The optimal ratios
for MSP1, E1, E2, and E3 determined in this manner are 10:1, 10:1,
55:1 and 80:1, respectively with main peak being approximately 80%
of total bR injected. The results of reinjection of the pooled main
peaks are shown in FIG. 19. The sizes based on calibration of the
column with a set of standard proteins are 11, 11.4, 12.2, 12.8 nm
in diameter for MSP1EI, MSP1E2, and MSP1E3, respectively.
[0259] bR in purple membrane exhibits excitonic interactions
between bR retinal chromophores in trimers which give rise to a
positive and a negative peak in the CD spectrum. The monomeric
forms of bR show a single positive peak arising from interaction of
retinal with the protein environment. CD spectra of bR solubilized
by MSP=s at the optimal lipid ratios are shown in FIG. 20. Only
MSP1E2 and MSP1E3 have a negative peak at 600 nm, indicating
assembly of a trimeric form of bR in the nanoscale discoidal
particles.
Example 14
Amphotericin B-Loaded Nanodiscs
[0260] Two Nanodisc preparations, one containing Amphotericin B
(AmB) and one without (as control) are made. The ratio of
MSP1T2/POPC/AmB in AmB particles is 2:130:1, and the ratio of
MSP1T2/POPC in the control particle preparation is 1:65.
[0261] Synthetic 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine
(POPC) is obtained from Avanti Polar Lipids (Alabaster, Ala.) and
prepared as a 75 mM (a concentration within the range of 70 to 80
mM is acceptable) stock in chloroform and stored at -20.degree. C.
The lipid concentration is determined by quantifying total
phosphorus using the method of Chen et al. (1956). Amphotericin B
(AmB) powder is obtained from Sigma (St. Louis, Mo.) and prepared
as 2 mM stock in DMSO, protected from light and stored at
-20.degree. C. MSP1T2 is expressed and purified as described by
Denisov et al. (2004).
[0262] AmB is added to POPC lipid solution to give a lipid:AmB
molar ratio of 65:1. The solution is dried under a stream of
nitrogen and placed under vacuum overnight to remove residual
solvent. Manipulations of samples containing AmB were protected
from light whenever possible. The mixture is resuspended by the
addition of 100 mM cholate in standard buffer (10 mM Tris-HCl (pH
7.4), 0.1 M NaCl, 1 mM EDTA) to yield a lipid concentration of 50
mM. The tubes are vortexed, sonicated, and heated briefly in a
37.degree. C. water bath, until the mixture is completely
solubilized.
[0263] MSP1T2 protein is added to the lipid/AmB/cholate solution to
give a protein/lipid/AmB ratio of 2:130:1. The final lipid
concentration is approximately 15 mM. The mixture is incubated for
2 h at 4.degree. C., near the phase transition temperature for
POPC. BioBeads are added to remove cholate, and the sample is
incubated for an additional 2 h at 4.degree. C. Samples are
separated by size exclusion chromatography on a Superdex 200 HR
10/30 column (Amersham Biosciences, Piscataway, N.J.), and the 10
nm fractions were retained. Concentration of Amphotericin B is
determined by comparing the A.sub.405 to a standard curve of
Amphotericin B constructed from 1 to 20 .mu.g/ml. Nanodisc
concentration is determined by measuring A.sub.280 (MSP1T2
.di-elect cons..sub.280=21,000 M.sup.-1 cm.sup.-1).
Example 15
Ketoconazole-Loaded Nanodiscs
[0264] Nanodiscs containing the small molecule antifungal
ketoconazole were prepared by incubating MSP1T2, ketoconazole,
DMPC, and cholate at a molar ratio of 1:10:80:160, respectively.
The mixture was incubated at 25.degree. C. for 45 min, BioBeads
were added (50% w/v), and incubation was continued for an
additional 45 min. Incubation with BioBeads removed cholate, and
resulted in the self assembly of Nanodiscs and the partitioning of
lipophilic ketoconazole to the bilayer environment of nascent
Nanodiscs. The ketoconazole containing Nanodiscs were purified by
nickel affinity chromatography and the column eluate was
concentrated by diafiltration with Standard Buffer (20 mM Tris-HCl
(pH 7.4), 0.1 M NaCl, 0.5 mM EDTA).
[0265] Antifungal activity of the ketoconazole-containing Nanodisc
preparation was qualitatively assayed against a lawn of Candida
albicans grown on Yeast Potato Dextrose (YPD) agar. A colony of
freshly grown C. albicans was mixed in sterile water, and 1 ml of
the cell suspension was evenly applied to the surface of a YPD-agar
plate. The excess cell liquid was decanted and 20 .mu.l aliquots of
Nanodiscs containing ketoconazole, Nanodiscs prepared without
ketoconazole (empty Nanodiscs), and 13 .mu.g/ml ketoconazole
solution in 1% DMSO were spotted separately onto the surface of the
plate (FIG. 21A). The plate was incubated for 18 hr at 35.degree.
C. Nanodiscs containing ketoconazole inhibited the growth of the C.
albicans in a manner consistent with the ketoconazole control,
whereas empty Nanodiscs showed no effect of fungal growth. This
result demonstrates that the ketoconazole antifungal activity
co-purified with the Nanodiscs and indicates that ketoconazole was
associated with the Nanodiscs following self-assembly. Application
of 20 .mu.l aliquots of Standard Buffer or 1% DMSO showed no
antifungal activity (FIG. 21B). Addition of 20 .mu.l of a 0.13
.mu.g/ml solution of ketoconazole did not visibly inhibit growth,
indicating that the application of this 100-fold lower
concentration of the drug was too dilute to affect the fungi at the
cell density present on the plate.
Example 16
Gadolinium-Containing Nanodiscs
[0266] Nanodiscs containing an amphiphilic gadolinium chelate can
be made by incorporating phospholipid or other type of lipid having
a chelating group as the polar headgroup portion of the amphiphilic
lipid. One such phospholipid is synthesized by reacting
phosphatidylethanolamine with the dianhydride of
diethylenetriaminepentaacetic acid (DTPA) to yield a tetradentate
chelating phospholipid which can be loaded with Gd.sup.3+ either
before or after assembly of nanodiscs. The chelating lipid with or
without bound Gd.sup.3+ can be mixed with or without other types of
phospholipid in organic solution followed by removal of solvent and
formation of nanodiscs by the usual methods.
[0267] Another chelating agent suitable for gadolinium cations is
1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-diethylenetriaminepent-
aacetate (DPPE-DTPA). An ethylenediaminetetraacetate (EDTA)
derivative can be used in place of the DTPA group. Dimyristoyl and
distearoyl can substitute for the dipalmitoyl moieties. The
relatively long chain fatty acids attached to the remainder of the
chelating molecule facilitate uptake of the gadolinium or other ion
of interest (including but not limited to trivalent cations of
iridium, technecium, and lanthanides in general) into the Nanodisc
particles. DTPA phospholipids are useful. See Urizzi et al. (1996)
Tetrahedron Lett. 37:4685-4688 for a discussion of lipophilic
chelating agents.
Example 17
Nanodiscs Containing Photodynamic Compounds
[0268] Nanodiscs containing a photodynamic compound, especially a
therapeutic photodynamic compound are prepared essentially as
described herein above for Amphotericin B and ketoconazole but with
the use of the photodynamic compound, such as a psoralen,
phthalacyanin or porphyrin, with the modification that stock
solutions, assembly reactions and Nanodisc preparations are
protected from light.
Example 18
Fluorescently Labeled Nanodiscs
[0269] The methodology for preparation of Nanodiscs containing
small organic molecules, in particular fluorescent labels is as
follows.
[0270] All glassware involved in this procedure is to be washed
with 1M KOH and sonicated for 15 minutes when possible.
[0271] Two Nanodisc preparations, one labeled and one unlabeled are
described. The ratio of MSP1/DPPC/DiI in the labeled prep is
1/100/0.05 with 0.5 mg of MSP1 used, and the ratio of MSP1/DPPC in
the unlabeled prep is 1/100 with 2 mg of MSP1 used. DPPC is
obtained from stock solutions dissolved in chloroform. Appropriate
amounts of DPPC are delivered to two glass tubes. DiI in ethanol is
added to the labeled disc prep tube. Solvent is dried down using
nitrogen, and samples are placed in a vacuum dessicator overnight
to remove residual solvent.
[0272] 50 mM cholate in standard buffer (10 mM Tris-HCl pH 7.4,
0.1M NaCl, 1 mM EDTA, 0.01% NaN.sub.3) is added to the dried lipid
samples to yield 25 mM final lipid concentration. The tubes are
vortexed, heated, and sonicated until lipid is completely in
solution. 0.5 mg of MSP1 in buffer at 37.degree. C. is added to the
sample to be labeled, and 2 mg of MSP1 in buffer at 37.degree. C.
is added to the unlabeled sample. The samples are incubated at
37.degree. C. for 4 hours. Dialysis is then performed to remove the
sodium cholate. Dialysis is done at 37.degree. C. in standard
buffer for 24 hours with 3 buffer changes.
[0273] Both samples are concentrated to about 0.3 ml, filtered, and
subjected to size exclusion chromatography on a Superdex column.
Fractions are collected, combining and saving those which contain
particles with a diameter of about 10 nm. Concentration of the
Nanodiscs is determined by measuring absorption at 280 nm
(A.sub.280 of 1 mg/ml MSP1 is 1.0). Absorption of DiI at 280 nm is
assumed to be negligible in the labeled disc sample.
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Sequence CWU 1
1
1361762DNAHomo sapiens 1ccatggccca tttctggcag caagatgaac ccccccagag
cccctgggat cgagtgaagg 60acctggccac tgtgtacgtg gatgtgctca aagacagcgg
cagagactat gtgtcccagt 120ttgaaggctc cgccttggga aaacagctaa
acctaaagct ccttgacaac tgggacagcg 180tgacctccac cttcagcaag
ctgcgcgaac agctcggccc tgtgacccag gagttctggg 240ataacctgga
aaaggagaca gagggcctga ggcaagagat gagcaaggat ctggaggagg
300tgaaggccaa ggtgcagccc tacctggacg acttccagaa gaagtggcag
gaggagatgg 360agctctaccg ccagaaggtg gagccgctgc gcgcagagct
ccaagagggc gcgcgccaga 420agctgcacga gctgcaagag aagctgagcc
cactgggcga ggagatgcgc gaccgcgcgc 480gcgcccatgt ggacgcgctg
cgcacgcatc tggcccccta cagcgacgag ctgcgccagc 540gcttggccgc
gcgccttgag gctctcaagg agaacggcgg cgccagactg gccgagtacc
600acgccaaggc caccgagcat ctgagcacgc tcagcgagaa ggccaagccc
gcgctcgagg 660acctccgcca aggcctgctg cccgtgctgg agagcttcaa
ggtcagcttc ctgagcgctc 720tcgaggagta cactaagaag ctcaacaccc
agtaataagc tt 7622250PRTHomo sapiens 2Met Ala His Phe Trp Gln Gln
Asp Glu Pro Pro Gln Ser Pro Trp Asp1 5 10 15Arg Val Lys Asp Leu Ala
Thr Val Tyr Val Asp Val Leu Lys Asp Ser20 25 30Gly Arg Asp Tyr Val
Ser Gln Phe Glu Gly Ser Ala Leu Gly Lys Gln35 40 45Leu Asn Leu Lys
Leu Leu Asp Asn Trp Asp Ser Val Thr Ser Thr Phe50 55 60Ser Lys Leu
Arg Glu Gln Leu Gly Pro Val Thr Gln Glu Phe Trp Asp65 70 75 80Asn
Leu Glu Lys Glu Thr Glu Gly Leu Arg Gln Glu Met Ser Lys Asp85 90
95Leu Glu Glu Val Lys Ala Lys Val Gln Pro Tyr Leu Asp Asp Phe
Gln100 105 110Lys Lys Trp Gln Glu Glu Met Glu Leu Tyr Arg Gln Lys
Val Glu Pro115 120 125Leu Arg Ala Glu Leu Gln Glu Gly Ala Arg Gln
Lys Leu His Glu Leu130 135 140Gln Glu Lys Leu Ser Pro Leu Gly Glu
Glu Met Arg Asp Arg Ala Arg145 150 155 160Ala His Val Asp Ala Leu
Arg Thr His Leu Ala Pro Tyr Ser Asp Glu165 170 175Leu Arg Gln Arg
Leu Ala Ala Arg Leu Glu Ala Leu Lys Glu Asn Gly180 185 190Gly Ala
Arg Leu Ala Glu Tyr His Ala Lys Ala Thr Glu His Leu Ser195 200
205Thr Leu Ser Glu Lys Ala Lys Pro Ala Leu Glu Asp Leu Arg Gln
Gly210 215 220Leu Leu Pro Val Leu Glu Ser Phe Lys Val Ser Phe Leu
Ser Ala Leu225 230 235 240Glu Glu Tyr Thr Lys Lys Leu Asn Thr
Gln245 250361DNAArtificial Sequenceoligonucleotide primer
3tataccatgg gccatcatca tcatcatcat atagaaggaa gactaaagct ccttgacaac
60t 61430DNAArtificial Sequenceoligonucleotide primer 4gcaagcttat
tactgggtgt tgagcttctt 305654DNAArtificial Sequencenucleotide
sequence encoding HIS-tagged MSP1 5tataccatgg gccatcatca tcatcatcat
atagaaggaa gactaaagct ccttgacaac 60tgggacagcg tgacctccac cttcagcaag
ctgcgcgaac agctcggccc tgtgacccag 120gagttctggg ataacctgga
aaaggagaca gagggcctga ggcaggagat gagcaaggat 180ctggaggagg
tgaaggccaa ggtgcagccc tacctggacg acttccagaa gaagtggcag
240gaggagatgg agctctaccg ccagaaggtg gagccgctgc gcgcagagct
ccaagagggc 300gcgcgccaga agctgcacga gctgcaagag aagttgagcc
cactgggcga ggagatgcgc 360gaccgcgcgc gcgcccatgt ggacgcgctg
cgcacgcatc tggcccccta cagcgacgag 420ctgcgccagc gcttggccgc
gcgccttgag gctctcaagg agaacggcgg cgccagactg 480gccgagtacc
acgccaaggc caccgagcat ctgagcacgc tcagcgagaa ggccaaaccc
540gcgctcgagg acctccgcca aggcctgctg cccgtgctgg agagcttcaa
ggtcagcttc 600ctgagcgctc tcgaggagta cactaagaag ctcaacaccc
agtaataagc ttgc 6546212PRTArtificial SequenceHIS-tagged MSP1 6Met
Gly His His His His His His Ile Glu Gly Arg Leu Lys Leu Leu1 5 10
15Asp Asn Trp Asp Ser Val Thr Ser Thr Phe Ser Lys Leu Arg Glu Gln20
25 30Leu Gly Pro Val Thr Gln Glu Phe Trp Asp Asn Leu Glu Lys Glu
Thr35 40 45Glu Gly Leu Arg Gln Glu Met Ser Lys Asp Leu Glu Glu Val
Lys Ala50 55 60Lys Val Gln Pro Tyr Leu Asp Asp Phe Gln Lys Lys Trp
Gln Glu Glu65 70 75 80Met Glu Leu Tyr Arg Gln Lys Val Glu Pro Leu
Arg Ala Glu Leu Gln85 90 95Glu Gly Ala Arg Gln Lys Leu His Glu Leu
Gln Glu Lys Leu Ser Pro100 105 110Leu Gly Glu Glu Met Arg Asp Arg
Ala Arg Ala His Val Asp Ala Leu115 120 125Arg Thr His Leu Ala Pro
Tyr Ser Asp Glu Leu Arg Gln Arg Leu Ala130 135 140Ala Arg Leu Glu
Ala Leu Lys Glu Asn Gly Gly Ala Arg Leu Ala Glu145 150 155 160Tyr
His Ala Lys Ala Thr Glu His Leu Ser Thr Leu Ser Glu Lys Ala165 170
175Lys Pro Ala Leu Glu Asp Leu Arg Gln Gly Leu Leu Pro Val Leu
Glu180 185 190Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr
Thr Lys Lys195 200 205Leu Asn Thr Gln210727DNAArtificial
Sequenceoligonucleotide primer 7taccatggca aagctccttg acaactg
278619DNAArtificial Sequencenucleotide sequence encoding MSP1
without His-tag 8taccatggca aagctccttg acaactggga cagcgtgacc
tccaccttca gcaagctgcg 60cgaacagctc ggccctgtga cccaggagtt ctgggataac
ctggaaaagg agacagaggg 120cctgaggcag gagatgagca aggatctgga
ggaggtgaag gccaaggtgc agccctacct 180ggacgacttc cagaagaagt
ggcaggagga gatggagctc taccgccaga aggtggagcc 240gctgcgcgca
gagctccaag agggcgcgcg ccagaagctg cacgagctgc aagagaagtt
300gagcccactg ggcgaggaga tgcgcgaccg cgcgcgcgcc catgtggacg
cgctgcgcac 360gcatctggcc ccctacagcg acgagctgcg ccagcgcttg
gccgcgcgcc ttgaggctct 420caaggagaac ggcggcgcca gactggccga
gtaccacgcc aaggccaccg agcatctgag 480cacgctcagc gagaaggcca
aacccgcgct cgaggacctc cgccaaggcc tgctgcccgt 540gctggagagc
ttcaaggtca gcttcctgag cgctctcgag gagtacacta agaagctcaa
600cacccagtaa taagcttgc 6199201PRTArtificial SequenceMSP1 without
His-tag 9Met Ala Lys Leu Leu Asp Asn Trp Asp Ser Val Thr Ser Thr
Phe Ser1 5 10 15Lys Leu Arg Glu Gln Leu Gly Pro Val Thr Gln Glu Phe
Trp Asp Asn20 25 30Leu Glu Lys Glu Thr Glu Gly Leu Arg Gln Glu Met
Ser Lys Asp Leu35 40 45Glu Glu Val Lys Ala Lys Val Gln Pro Tyr Leu
Asp Asp Phe Gln Lys50 55 60Lys Trp Gln Glu Glu Met Glu Leu Tyr Arg
Gln Lys Val Glu Pro Leu65 70 75 80Arg Ala Glu Leu Gln Glu Gly Ala
Arg Gln Lys Leu His Glu Leu Gln85 90 95Glu Lys Leu Ser Pro Leu Gly
Glu Glu Met Arg Asp Arg Ala Arg Ala100 105 110His Val Asp Ala Leu
Arg Thr His Leu Ala Pro Tyr Ser Asp Glu Leu115 120 125Arg Gln Arg
Leu Ala Ala Arg Leu Glu Ala Leu Lys Glu Asn Gly Gly130 135 140Ala
Arg Leu Ala Glu Tyr His Ala Lys Ala Thr Glu His Leu Ser Thr145 150
155 160Leu Ser Glu Lys Ala Lys Pro Ala Leu Glu Asp Leu Arg Gln Gly
Leu165 170 175Leu Pro Val Leu Glu Ser Phe Lys Val Ser Phe Leu Ser
Ala Leu Glu180 185 190Glu Tyr Thr Lys Lys Leu Asn Thr Gln195
2001027DNAArtificial Sequenceoligonucleotide primer 10taccatggca
aagctccttg acaactg 271161DNAArtificial Sequenceoligonucleotide
primer 11tataccatgg gccatcatca tcatcatcat atagaaggaa gactaaagct
ccttgacaac 60t 611252DNAArtificial Sequenceoligonucleotide primer
12taagaagctc aacacccagg gtaccggtgg aggtagtgga ggtggtaccc ta
521350DNAArtificial Sequenceoligonucleotide primer 13cagggtaccg
gtggaggtag tggaggtggt accctaaagc tccttgacaa 501430DNAArtificial
Sequenceoligonucleotide primer 14gcaagcttat tactgggtgt tgagcttctt
30159PRTartificial sequencesynthetic peptide of linker 15Gly Thr
Gly Gly Gly Ser Gly Gly Thr1 5161260DNAArtificial
Sequencenucleotide sequence encoding His-tagged MSP2 16tataccatgg
gccatcatca tcatcatcat atagaaggaa gactaaagct ccttgacaac 60tgggacagcg
tgacctccac cttcagcaag ctgcgcgaac agctcggccc tgtgacccag
120gagttctggg ataacctgga aaaggagaca gagggcctga ggcaggagat
gagcaaggat 180ctggaggagg tgaaggccaa ggtgcagccc tacctggacg
acttccagaa gaagtggcag 240gaggagatgg agctctaccg ccagaaggtg
gagccgctgc gcgcagagct ccaagagggc 300gcgcgccaga agctgcacga
gctgcaagag aagctgagcc cactgggcga ggagatgcgc 360gaccgcgcgc
gcgcccatgt ggacgcgctg cgcacgcatc tggcccccta cagcgacgag
420ctgcgccagc gcttggccgc gcgccttgag gctctcaagg agaacggcgg
cgccagactg 480gccgagtacc acgccaaggc caccgagcat ctgagcacgc
tcagcgagaa ggccaagccc 540gcgctcgagg acctccgcca aggcctgctg
cccgtgctgg agagcttcaa ggtcagcttc 600ctgagcgctc tcgaggagta
cactaagaag ctcaacaccc agggtaccct aaagctcctt 660gacaactggg
acagcgtgac ctccaccttc agcaagctgc gcgaacagct cggccctgtg
720acccaggagt tctgggataa cctggaaaag gagacagagg gcctgaggca
ggagatgagc 780aaggatctgg aggaggtgaa ggccaaggtg cagccctacc
tggacgactt ccagaagaag 840tggcaggagg agatggagct ctaccgccag
aaggtggagc cgctgcgcgc agagctccaa 900gagggcgcgc gccagaagct
gcacgagctg caagagaagc tgagcccact gggcgaggag 960atgcgcgacc
gcgcgcgcgc ccatgtggac gcgctgcgca cgcatctggc cccctacagc
1020gacgagctgc gccagcgctt ggccgcgcgc cttgaggctc tcaaggagaa
cggcggcgcc 1080agactggccg agtaccacgc caaggccacc gagcatctga
gcacgctcag cgagaaggcc 1140aagcccgcgc tcgaggacct ccgccaaggc
ctgctgcccg tgctggagag cttcaaggtc 1200agcttcctga gcgctctcga
ggagtacact aagaagctca acacccagta ataagcttgc 126017414PRTArtificial
SequenceHis-tagged MSP2 17Met Gly His His His His His His Ile Glu
Gly Arg Leu Lys Leu Leu1 5 10 15Asp Asn Trp Asp Ser Val Thr Ser Thr
Phe Ser Lys Leu Arg Glu Gln20 25 30Leu Gly Pro Val Thr Gln Glu Phe
Trp Asp Asn Leu Glu Lys Glu Thr35 40 45Glu Gly Leu Arg Gln Glu Met
Ser Lys Asp Leu Glu Glu Val Lys Ala50 55 60Lys Val Gln Pro Tyr Leu
Asp Asp Phe Gln Lys Lys Trp Gln Glu Glu65 70 75 80Met Glu Leu Tyr
Arg Gln Lys Val Glu Pro Leu Arg Ala Glu Leu Gln85 90 95Glu Gly Ala
Arg Gln Lys Leu His Glu Leu Gln Glu Lys Leu Ser Pro100 105 110Leu
Gly Glu Glu Met Arg Asp Arg Ala Arg Ala His Val Asp Ala Leu115 120
125Arg Thr His Leu Ala Pro Tyr Ser Asp Glu Leu Arg Gln Arg Leu
Ala130 135 140Ala Arg Leu Glu Ala Leu Lys Glu Asn Gly Gly Ala Arg
Leu Ala Glu145 150 155 160Tyr His Ala Lys Ala Thr Glu His Leu Ser
Thr Leu Ser Glu Lys Ala165 170 175Lys Pro Ala Leu Glu Asp Leu Arg
Gln Gly Leu Leu Pro Val Leu Glu180 185 190Ser Phe Lys Val Ser Phe
Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys195 200 205Leu Asn Thr Gln
Gly Thr Leu Lys Leu Leu Asp Asn Trp Asp Ser Val210 215 220Thr Ser
Thr Phe Ser Lys Leu Arg Glu Gln Leu Gly Pro Val Thr Gln225 230 235
240Glu Phe Trp Asp Asn Leu Glu Lys Glu Thr Glu Gly Leu Arg Gln
Glu245 250 255Met Ser Lys Asp Leu Glu Glu Val Lys Ala Lys Val Gln
Pro Tyr Leu260 265 270Asp Asp Phe Gln Lys Lys Trp Gln Glu Glu Met
Glu Leu Tyr Arg Gln275 280 285Lys Val Glu Pro Leu Arg Ala Glu Leu
Gln Glu Gly Ala Arg Gln Lys290 295 300Leu His Glu Leu Gln Glu Lys
Leu Ser Pro Leu Gly Glu Glu Met Arg305 310 315 320Asp Arg Ala Arg
Ala His Val Asp Ala Leu Arg Thr His Leu Ala Pro325 330 335Tyr Ser
Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu Ala Leu340 345
350Lys Glu Asn Gly Gly Ala Arg Leu Ala Glu Tyr His Ala Lys Ala
Thr355 360 365Glu His Leu Ser Thr Leu Ser Glu Lys Ala Lys Pro Ala
Leu Glu Asp370 375 380Leu Arg Gln Gly Leu Leu Pro Val Leu Glu Ser
Phe Lys Val Ser Phe385 390 395 400Leu Ser Ala Leu Glu Glu Tyr Thr
Lys Lys Leu Asn Thr Gln405 410181282DNAArtificial
Sequencenucleotide sequence encoding His-tagged MSP2L 18taccatgggc
catcatcatc atcatcatat agaaggaaga ctaaagctcc ttgacaactg 60ggacagcgtg
acctccacct tcagcaagct gcgcgaacag ctcggccctg tgacccagga
120gttctgggat aacctggaaa aggagacaga gggcctgagg caggagatga
gcaaggatct 180ggaggaggtg aaggccaagg tgcagcccta cctggacgac
ttccagaaga agtggcagga 240ggagatggag ctctaccgcc agaaggtgga
gccgctgcgc gcagagctcc aagagggcgc 300gcgccagaag ctgcacgagc
tgcaagagaa gctgagccca ctgggcgagg agatgcgcga 360ccgcgcgcgc
gcccatgtgg acgcgctgcg cacgcatctg gccccctaca gcgacgagct
420gcgccagcgc ttggccgcgc gccttgaggc tctcaaggag aacggcggcg
ccagactggc 480cgagtaccac gccaaggcca ccgagcatct gagcacgctc
agcgagaagg ccaagcccgc 540gctcgaggac ctccgccaag gcctgctgcc
cgtgctggag agcttcaagg tcagcttcct 600gagcgctctc gaggagtaca
ctaagaagct caacacccag ggtaccggtg gaggtagtgg 660aggtggtacc
ctaaagctcc ttgacaactg ggacagcgtg acctccacct tcagcaagct
720gcgcgaacag ctcggccctg tgacccagga gttctgggat aacctggaaa
aggagacaga 780gggcctgagg caggagatga gcaaggatct ggaggaggtg
aaggccaagg tgcagcccta 840cctggacgac ttccagaaga agtggcagga
ggagatggag ctctaccgcc agaaggtgga 900gccgctgcgc gcagagctcc
aagagggcgc gcgccagaag ctgcacgagc tgcaagagaa 960gctgagccca
ctgggcgagg agatgcgcga ccgcgcgcgc gcccatgtgg acgcgctgcg
1020cacgcatctg gccccctaca gcgacgagct gcgccagcgc ttggccgcgc
gccttgaggc 1080tctcaaggag aacggcggcg ccagactggc cgagtaccac
gccaaggcca ccgagcatct 1140gagcacgctc agcgagaagg ccaagcccgc
gctcgaggac ctccgccaag gcctgctgcc 1200cgtgctggag agcttcaagg
tcagcttcct gagcgctctc gaggagtaca ctaagaagct 1260caacacccag
taataagctt gc 128219422PRTArtificial SequenceHis-tagged MSP2L 19Met
Gly His His His His His His Ile Glu Gly Arg Leu Lys Leu Leu1 5 10
15Asp Asn Trp Asp Ser Val Thr Ser Thr Phe Ser Lys Leu Arg Glu Gln20
25 30Leu Gly Pro Val Thr Gln Glu Phe Trp Asp Asn Leu Glu Lys Glu
Thr35 40 45Glu Gly Leu Arg Gln Glu Met Ser Lys Asp Leu Glu Glu Val
Lys Ala50 55 60Lys Val Gln Pro Tyr Leu Asp Asp Phe Gln Lys Lys Trp
Gln Glu Glu65 70 75 80Met Glu Leu Tyr Arg Gln Lys Val Glu Pro Leu
Arg Ala Glu Leu Gln85 90 95Glu Gly Ala Arg Gln Lys Leu His Glu Leu
Gln Glu Lys Leu Ser Pro100 105 110Leu Gly Glu Glu Met Arg Asp Arg
Ala Arg Ala His Val Asp Ala Leu115 120 125Arg Thr His Leu Ala Pro
Tyr Ser Asp Glu Leu Arg Gln Arg Leu Ala130 135 140Ala Arg Leu Glu
Ala Leu Lys Glu Asn Gly Gly Ala Arg Leu Ala Glu145 150 155 160Tyr
His Ala Lys Ala Thr Glu His Leu Ser Thr Leu Ser Glu Lys Ala165 170
175Lys Pro Ala Leu Glu Asp Leu Arg Gln Gly Leu Leu Pro Val Leu
Glu180 185 190Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr
Thr Lys Lys195 200 205Leu Asn Thr Gln Gly Thr Gly Gly Gly Ser Gly
Gly Gly Thr Leu Lys210 215 220Leu Leu Asp Asn Trp Asp Ser Val Thr
Ser Thr Phe Ser Lys Leu Arg225 230 235 240Glu Gln Leu Gly Pro Val
Thr Gln Glu Phe Trp Asp Asn Leu Glu Lys245 250 255Glu Thr Glu Gly
Leu Arg Gln Glu Met Ser Lys Asp Leu Glu Glu Val260 265 270Lys Ala
Lys Val Gln Pro Tyr Leu Asp Asp Phe Gln Lys Lys Trp Gln275 280
285Glu Glu Met Glu Leu Tyr Arg Gln Lys Val Glu Pro Leu Arg Ala
Glu290 295 300Leu Gln Glu Gly Ala Arg Gln Lys Leu His Glu Leu Gln
Glu Lys Leu305 310 315 320Ser Pro Leu Gly Glu Glu Met Arg Asp Arg
Ala Arg Ala His Val Asp325 330 335Ala Leu Arg Thr His Leu Ala Pro
Tyr Ser Asp Glu Leu Arg Gln Arg340 345 350Leu Ala Ala Arg Leu Glu
Ala Leu Lys Glu Asn Gly Gly Ala Arg Leu355 360 365Ala Glu Tyr His
Ala Lys Ala Thr Glu His Leu Ser Thr Leu Ser Glu370 375 380Lys Ala
Lys Pro Ala Leu Glu Asp Leu Arg Gln Gly Leu Leu Pro Val385 390 395
400Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr
Thr405 410 415Lys Lys Leu Asn Thr Gln4202043DNAArtificial
Sequenceoligonucleotide primer 20tggagctcta ccgccagaag gtggagccct
acagcgacga gct 432130DNAArtificial Sequenceoligonucleotide primer
21gcaagcttat tactgggtgt tgagcttctt 3022522DNAArtificial
Sequencenucleotide sequence encoding MSP1D5D6
22tataccatgg gccatcatca tcatcatcat atagaaggaa gactaaagct ccttgacaac
60tgggacagcg tgacctccac cttcagcaag ctgcgcgaac agctcggccc tgtgacccag
120gagttctggg ataacctgga aaaggagaca gagggcctga ggcaggagat
gagcaaggat 180ctggaggagg tgaaggccaa ggtgcagccc tacctggacg
acttccagaa gaagtggcag 240gaggagatgg agctctaccg ccagaaggtg
gagccctaca gcgacgagct gcgccagcgc 300ttggccgcgc gccttgaggc
tctcaaggag aacggcggcg ccagactggc cgagtaccac 360gccaaggcca
ccgagcatct gagcacgctc agcgagaagg ccaaacccgc gctcgaggac
420ctccgccaag gcctgctgcc cgtgctggag agcttcaagg tcagcttcct
gagcgctctc 480gaggagtaca ctaagaagct caacacccag taataagctt gc
52223168PRTArtificial SequenceHis-tagged MSP1D5D6 23Met Gly His His
His His His His Ile Glu Gly Arg Leu Lys Leu Leu1 5 10 15Asp Asn Trp
Asp Ser Val Thr Ser Thr Phe Ser Lys Leu Arg Glu Gln20 25 30Leu Gly
Pro Val Thr Gln Glu Phe Trp Asp Asn Leu Glu Lys Glu Thr35 40 45Glu
Gly Leu Arg Gln Glu Met Ser Lys Asp Leu Glu Glu Val Lys Ala50 55
60Lys Val Gln Pro Tyr Leu Asp Asp Phe Gln Lys Lys Trp Gln Glu Glu65
70 75 80Met Glu Leu Tyr Arg Gln Lys Val Glu Pro Tyr Ser Asp Glu Leu
Arg85 90 95Gln Arg Leu Ala Ala Arg Leu Glu Ala Leu Lys Glu Asn Gly
Gly Ala100 105 110Arg Leu Ala Glu Tyr His Ala Lys Ala Thr Glu His
Leu Ser Thr Leu115 120 125Ser Glu Lys Ala Lys Pro Ala Leu Glu Asp
Leu Arg Gln Gly Leu Leu130 135 140Pro Val Leu Glu Ser Phe Lys Val
Ser Phe Leu Ser Ala Leu Glu Glu145 150 155 160Tyr Thr Lys Lys Leu
Asn Thr Gln1652429DNAArtificial Sequenceoligonucleotide primer
24cagaattcgc tagccgagta ccacgccaa 292530DNAArtificial
Sequenceoligonucleotide primer 25gcaagcttat tactgggtgt tgagcttctt
302630DNAArtificial Sequenceoligonucleotide primer 26ataccatggg
ccatcatcat catcatcata 302733DNAArtificial Sequenceoligonucleotide
primer 27cagaattcgc tagcctggcg ctcaacttct ctt 3328522DNAArtificial
Sequencenucleotide sequence encoding His-tagged MSP1D6 28tataccatgg
gccatcatca tcatcatcat atagaaggaa gactaaagct ccttgacaac 60tgggacagcg
tgacctccac cttcagcaag ctgcgcgaac agctcggccc tgtgacccag
120gagttctggg ataacctgga aaaggagaca gagggcctga ggcaggagat
gagcaaggat 180ctggaggagg tgaaggccaa ggtgcagccc tacctggacg
acttccagaa gaagtggcag 240gaggagatgg agctctaccg ccagaaggtg
gagccgctgc gcgcagagct ccaagagggc 300gcgcgccaga agctgcacga
gctgcaagag aagttgagcg ccaggctagc cgagtaccac 360gccaaggcca
ccgagcatct gagcacgctc agcgagaagg ccaaacccgc gctcgaggac
420ctccgccaag gcctgctgcc cgtgctggag agcttcaagg tcagcttcct
gagcgctctc 480gaggagtaca ctaagaagct caacacccag taataagctt gc
52229168PRTArtificial SequenceHis-tagged MSP1D6 29Met Gly His His
His His His His Ile Glu Gly Arg Leu Lys Leu Leu1 5 10 15Asp Asn Trp
Asp Ser Val Thr Ser Thr Phe Ser Lys Leu Arg Glu Gln20 25 30Leu Gly
Pro Val Thr Gln Glu Phe Trp Asp Asn Leu Glu Lys Glu Thr35 40 45Glu
Gly Leu Arg Gln Glu Met Ser Lys Asp Leu Glu Glu Val Lys Ala50 55
60Lys Val Gln Pro Tyr Leu Asp Asp Phe Gln Lys Lys Trp Gln Glu Glu65
70 75 80Met Glu Leu Tyr Arg Gln Lys Val Glu Pro Leu Arg Ala Glu Leu
Gln85 90 95Glu Gly Ala Arg Gln Lys Leu His Glu Leu Gln Glu Lys Leu
Ser Ala100 105 110Arg Leu Ala Glu Tyr His Ala Lys Ala Thr Glu His
Leu Ser Thr Leu115 120 125Ser Glu Lys Ala Lys Pro Ala Leu Glu Asp
Leu Arg Gln Gly Leu Leu130 135 140Pro Val Leu Glu Ser Phe Lys Val
Ser Phe Leu Ser Ala Leu Glu Glu145 150 155 160Tyr Thr Lys Lys Leu
Asn Thr Gln1653077DNAArtificial Sequencesynthetic oligonucleotide
30taccatgggt catcatcatc atcatcacat tgagggacgt ctgaagctgt tggacaattg
60ggactctgtt acgtcta 773162DNAArtificial Sequencesynthetic
oligonucleotide 31aggaattctg ggacaacctg gaaaaagaaa ccgagggact
gcgtcaggaa atgtccaaag 60at 623254DNAArtificial Sequencesynthetic
oligonucleotide 32tatctagatg actttcagaa aaaatggcag gaagagatgg
aattatatcg tcaa 543373DNAArtificial Sequencesynthetic
oligonucleotide 33atgagctcca agagaagctc agcccattag gcgaagaaat
gcgcgatcgc gcccgtgcac 60atgttgatgc act 733465DNAArtificial
Sequencesynthetic oligonucleotide 34gtctcgaggc gctgaaagaa
aacgggggtg cccgcttggc tgagtaccac gcgaaagcga 60cagaa
653556DNAArtificial Sequencesynthetic oligonucleotide 35gaagatctac
gccagggctt attgcctgtt cttgagagct ttaaagtcag ttttct
563661DNAArtificial Sequencesynthetic oligonucleotide 36cagaattcct
gcgtcacggg gcccagttgt tcgcgaagtt tactgaaggt agacgtaaca 60g
613755DNAArtificial Sequencesynthetic oligonucleotide 37tcatctagat
atggctgaac cttggccttc acctcttcta aatctttgga cattt
553880DNAArtificial Sequencesynthetic oligonucleotide 38tggagctcat
ggagtttttg gcgtgccccc tcttgcagtt ccgcacgcag cggttccacc 60ttttgacgat
ataattccat 803976DNAArtificial Sequencesynthetic oligonucleotide
39gcctcgagac gtgcggccaa acgctggcga agttcatccg aatacggcgc caaatgagtc
60cggagtgcat caacat 764061DNAArtificial Sequencesynthetic
oligonucleotide 40gtagatcttc cagcgccggt ttcgcttttt cgctcaaggt
gctcaggtgt tctgtcgctt 60t 614166DNAArtificial Sequencesynthetic
oligonucleotide 41ccaagcttat tactgggtat tcagcttttt agtatattct
tccagagctg acagaaaact 60gacttt 6642651DNAArtificial Sequencefull
synthetic sequence encoding MSP1 42accatgggtc atcatcatca tcatcacatt
gagggacgtc tgaagctgtt ggacaattgg 60gactctgtta cgtctacctt cagtaaactt
cgcgaacaac tgggccccgt gacgcaggaa 120ttctgggaca acctggaaaa
agaaaccgag ggactgcgtc aggaaatgtc caaagattta 180gaagaggtga
aggccaaggt tcagccatat ctagatgact ttcagaaaaa atggcaggaa
240gagatggaat tatatcgtca aaaggtggaa ccgctgcgtg cggaactgca
agagggggca 300cgccaaaaac tccatgagct ccaagagaag ctcagcccat
taggcgaaga aatgcgcgat 360cgcgcccgtg cacatgttga tgcactccgg
actcatttgg cgccgtattc ggatgaactt 420cgccagcgtt tggccgcacg
tctcgaggcg ctgaaagaaa acgggggtgc ccgcttggct 480gagtaccacg
cgaaagcgac agaacacctg agcaccttga gcgaaaaagc gaaaccggcg
540ctggaagatc tacgccaggg cttattgcct gttcttgaga gctttaaagt
cagttttctg 600tcagctctgg aagaatatac taaaaagctg aatacccagt
aataagcttg g 65143201PRTArtificial SequenceHis-tagged MSP1D3 43Met
Gly His His His His His His Ile Glu Gly Arg Leu Lys Leu Leu1 5 10
15Asp Asn Trp Asp Ser Val Thr Ser Thr Phe Ser Lys Leu Arg Glu Gln20
25 30Leu Gly Pro Val Thr Gln Glu Phe Trp Asp Asn Leu Glu Lys Glu
Thr35 40 45Glu Gly Leu Arg Gln Glu Met Ser Pro Tyr Leu Asp Asp Phe
Gln Lys50 55 60Lys Trp Gln Glu Glu Met Glu Leu Tyr Arg Gln Lys Val
Glu Pro Leu65 70 75 80Arg Ala Glu Leu Gln Glu Gly Ala Arg Gln Lys
Leu His Glu Leu Gln85 90 95Glu Lys Leu Ser Pro Leu Gly Glu Glu Met
Arg Asp Arg Ala Arg Ala100 105 110His Val Asp Ala Leu Arg Thr His
Leu Ala Pro Tyr Ser Asp Glu Leu115 120 125Arg Gln Arg Leu Ala Ala
Arg Leu Glu Ala Leu Lys Glu Asn Gly Gly130 135 140Ala Arg Leu Ala
Glu Tyr His Ala Lys Ala Thr Glu His Leu Ser Thr145 150 155 160Leu
Ser Glu Lys Ala Lys Pro Ala Leu Glu Asp Leu Arg Gln Gly Leu165 170
175Leu Pro Val Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu
Glu180 185 190Glu Tyr Thr Lys Lys Leu Asn Thr Gln195
20044201PRTArtificial SequenceHis-tagged MSP1D9 44Met Gly His His
His His His His Ile Glu Gly Arg Leu Lys Leu Leu1 5 10 15Asp Asn Trp
Asp Ser Val Thr Ser Thr Phe Ser Lys Leu Arg Glu Gln20 25 30Leu Gly
Pro Val Thr Gln Glu Phe Trp Asp Asn Leu Glu Lys Glu Thr35 40 45Glu
Gly Leu Arg Gln Glu Met Ser Lys Asp Leu Glu Glu Val Lys Ala50 55
60Lys Val Gln Pro Tyr Leu Asp Asp Phe Gln Lys Lys Trp Gln Glu Glu65
70 75 80Met Glu Leu Tyr Arg Gln Lys Val Glu Pro Leu Arg Ala Glu Leu
Gln85 90 95Glu Gly Ala Arg Gln Lys Leu His Glu Leu Gln Glu Lys Leu
Ser Pro100 105 110Leu Gly Glu Glu Met Arg Asp Arg Ala Arg Ala His
Val Asp Ala Leu115 120 125Arg Thr His Leu Ala Pro Tyr Ser Asp Glu
Leu Arg Gln Arg Leu Ala130 135 140Ala Arg Leu Glu Ala Leu Lys Glu
Asn Gly Gly Ala Arg Leu Ala Glu145 150 155 160Tyr His Ala Lys Ala
Thr Glu His Leu Ser Thr Leu Ser Glu Lys Ala165 170 175Lys Pro Val
Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu180 185 190Glu
Tyr Thr Lys Lys Leu Asn Thr Gln195 20045392PRTArtificial
SequenceHis-tagged MSP2 delta 1 45Met Gly His His His His His His
Ile Glu Gly Arg Leu Lys Leu Leu1 5 10 15Asp Asn Trp Asp Ser Val Thr
Ser Thr Phe Ser Lys Leu Arg Glu Gln20 25 30Leu Gly Pro Val Thr Gln
Glu Phe Trp Asp Asn Leu Glu Lys Glu Thr35 40 45Glu Gly Leu Arg Gln
Glu Met Ser Pro Tyr Leu Asp Asp Phe Gln Lys50 55 60Lys Trp Gln Glu
Glu Met Glu Leu Tyr Arg Gln Lys Val Glu Pro Leu65 70 75 80Arg Ala
Glu Leu Gln Glu Gly Ala Arg Gln Lys Leu His Glu Leu Gln85 90 95Glu
Lys Leu Ser Pro Leu Gly Glu Glu Met Arg Asp Arg Ala Arg Ala100 105
110His Val Asp Ala Leu Arg Thr His Leu Ala Pro Tyr Ser Asp Glu
Leu115 120 125Arg Gln Arg Leu Ala Ala Arg Leu Glu Ala Leu Lys Glu
Asn Gly Gly130 135 140Ala Arg Leu Ala Glu Tyr His Ala Lys Ala Thr
Glu His Leu Ser Thr145 150 155 160Leu Ser Glu Lys Ala Lys Pro Ala
Leu Glu Asp Leu Arg Gln Gly Leu165 170 175Leu Pro Val Leu Glu Ser
Phe Lys Val Ser Phe Leu Ser Ala Leu Glu180 185 190Glu Tyr Thr Lys
Lys Leu Asn Thr Gln Gly Thr Leu Lys Leu Leu Asp195 200 205Asn Trp
Asp Ser Val Thr Ser Thr Phe Ser Lys Leu Arg Glu Gln Leu210 215
220Gly Pro Val Thr Gln Glu Phe Trp Asp Asn Leu Glu Lys Glu Thr
Glu225 230 235 240Gly Leu Arg Gln Glu Met Ser Pro Tyr Leu Asp Asp
Phe Gln Lys Lys245 250 255Trp Gln Glu Glu Met Glu Leu Tyr Arg Gln
Lys Val Glu Pro Leu Arg260 265 270Ala Glu Leu Gln Glu Gly Ala Arg
Gln Lys Leu His Glu Leu Gln Glu275 280 285Lys Leu Ser Pro Leu Gly
Glu Glu Met Arg Asp Arg Ala Arg Ala His290 295 300Val Asp Ala Leu
Arg Thr His Leu Ala Pro Tyr Ser Asp Glu Leu Arg305 310 315 320Gln
Arg Leu Ala Ala Arg Leu Glu Ala Leu Lys Glu Asn Gly Gly Ala325 330
335Arg Leu Ala Glu Tyr His Ala Lys Ala Thr Glu His Leu Ser Thr
Leu340 345 350Ser Glu Lys Ala Lys Pro Ala Leu Glu Asp Leu Arg Gln
Gly Leu Leu355 360 365Pro Val Leu Glu Ser Phe Lys Val Ser Phe Leu
Ser Ala Leu Glu Glu370 375 380Tyr Thr Lys Lys Leu Asn Thr Gln385
390464PRTArtificial Sequencesynthetic peptide of linker 46Gly Gly
Gly Xaa14712PRTArtificial Sequenceartificial sequence of His-tag
47Met Gly His His His His His His Ile Glu Gly Arg1 5
104823PRTArtificial Sequenceartificial sequence of HisTEV 48Met Gly
His His His His His His His Asp Tyr Asp Ile Pro Thr Thr1 5 10 15Glu
Asn Leu Tyr Phe Gln Gly204922PRTArtificial Sequenceartificial
sequence of Helix 1 49Leu Lys Leu Leu Asp Asn Trp Asp Ser Val Thr
Ser Thr Phe Ser Lys1 5 10 15Leu Arg Glu Gln Leu
Gly205022PRTArtificial Sequenceartificial sequence of Helix 2 50Pro
Val Thr Gln Glu Phe Trp Asp Asn Leu Glu Lys Glu Thr Glu Gly1 5 10
15Leu Arg Gln Glu Met Ser205111PRTArtificial Sequenceartificial
sequence of Helix 3 51Lys Asp Leu Glu Glu Val Lys Ala Lys Val Gln1
5 105222PRTArtificial Sequenceartificial sequence of Helix 4 52Pro
Tyr Leu Asp Asp Phe Gln Lys Lys Trp Gln Glu Glu Met Glu Leu1 5 10
15Tyr Arg Gln Lys Val Glu205322PRTArtificial Sequenceartificial
sequence of Helix 5 53Pro Leu Arg Ala Glu Leu Gln Glu Gly Ala Arg
Gln Lys Leu His Glu1 5 10 15Leu Gln Glu Lys Leu
Ser205422PRTArtificial Sequenceartificial sequence of Helix 6 54Pro
Leu Gly Glu Glu Met Arg Asp Arg Ala Arg Ala His Val Asp Ala1 5 10
15Leu Arg Thr His Leu Ala205522PRTArtificial Sequenceartificial
sequence of Helix 7 55Pro Tyr Ser Asp Glu Leu Arg Gln Arg Leu Ala
Ala Arg Leu Glu Ala1 5 10 15Leu Lys Glu Asn Gly
Gly205622PRTArtificial Sequenceartificial sequence of Helix 8 56Ala
Arg Leu Ala Glu Tyr His Ala Lys Ala Thr Glu His Leu Ser Thr1 5 10
15Leu Ser Glu Lys Ala Lys205711PRTArtificial Sequenceartificial
sequence of Helix 9 57Pro Ala Leu Glu Asp Leu Arg Gln Gly Leu Leu1
5 105824PRTArtificial Sequenceartificial sequence of Helix 10 58Pro
Val Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu1 5 10
15Tyr Thr Lys Lys Leu Asn Thr Gln205911PRTArtificial
Sequenceartificial sequence of Helix 0.5 59Ser Thr Phe Ser Lys Leu
Arg Glu Gln Leu Gly1 5 106036DNAArtificial Sequencesequence
encoding His-tag 60atgggtcatc atcatcatca tcacattgag ggacgt
366169DNAArtificial Sequencesequence encoding His-TEV 61atgggtcatc
atcatcatca tcatcacgat tatgatattc ctactactga gaatttgtat 60tttcagggt
696266DNAArtificial Sequencesequence encoding Helix 1 62ctgaagctgt
tggacaattg ggactctgtt acgtctacct tcagtaaact tcgcgaacaa 60ctgggc
666366DNAArtificial Sequencesequence encoding Helix 2 63cccgtgacgc
aggaattctg ggacaacctg gaaaaagaaa ccgagggact gcgtcaggaa 60atgtcc
666433DNAArtificial Sequencesequence encoding Helix 3 64aaagatttag
aagaggtgaa ggccaaggtt cag 336566DNAArtificial Sequencesequence
encoding Helix 4 65ccatatctcg atgactttca gaaaaaatgg caggaagaga
tggaattata tcgtcaaaag 60gtggaa 666666DNAArtificial Sequencesequence
encoding Helix 5 66ccgctgcgtg cggaactgca agagggggca cgccaaaaac
tccatgagct ccaagagaag 60ctcagc 666766DNAArtificial Sequencesequence
encoding Helix 6 67ccattaggcg aagaaatgcg cgatcgcgcc cgtgcacatg
ttgatgcact ccggactcat 60ttggcg 666866DNAArtificial Sequencesequence
encoding Helix 7 68ccgtattcgg atgaacttcg ccagcgtttg gccgcacgtc
tcgaggcgct gaaagaaaac 60gggggt 666966DNAArtificial Sequencesequence
encoding Helix 8 69gcccgcttgg ctgagtacca cgcgaaagcg acagaacacc
tgagcacctt gagcgaaaaa 60gcgaaa 667033DNAArtificial Sequencesequence
encoding Helix 9 70ccggcgctgg aagatctacg ccagggctta ttg
337172DNAArtificial Sequencesequence encoding Helix 10 71cctgttcttg
agagctttaa agtcagtttt ctgtcagctc tggaagaata tactaaaaag 60ctgaataccc
ag 727233DNAArtificial Sequencesequence encoding Helix 0.5
72tctaccttca
gtaaacttcg cgaacaactg ggc 3373234PRTArtificial Sequenceartificial
sequence of His-tagged MSP1E1 73Met Gly His His His His His His Ile
Glu Gly Arg Leu Lys Leu Leu1 5 10 15Asp Asn Trp Asp Ser Val Thr Ser
Thr Phe Ser Lys Leu Arg Glu Gln20 25 30Leu Gly Pro Val Thr Gln Glu
Phe Trp Asp Asn Leu Glu Lys Glu Thr35 40 45Glu Gly Leu Arg Gln Glu
Met Ser Lys Asp Leu Glu Glu Val Lys Ala50 55 60Lys Val Gln Pro Tyr
Leu Asp Asp Phe Gln Lys Lys Trp Gln Glu Glu65 70 75 80Met Glu Leu
Tyr Arg Gln Lys Val Glu Pro Tyr Leu Asp Asp Phe Gln85 90 95Lys Lys
Trp Gln Glu Glu Met Glu Leu Tyr Arg Gln Lys Val Glu Pro100 105
110Leu Arg Ala Glu Leu Gln Glu Gly Ala Arg Gln Lys Leu His Glu
Leu115 120 125Gln Glu Lys Leu Ser Pro Leu Gly Glu Glu Met Arg Asp
Arg Ala Arg130 135 140Ala His Val Asp Ala Leu Arg Thr His Leu Ala
Pro Tyr Ser Asp Glu145 150 155 160Leu Arg Gln Arg Leu Ala Ala Arg
Leu Glu Ala Leu Lys Glu Asn Gly165 170 175Gly Ala Arg Leu Ala Glu
Tyr His Ala Lys Ala Thr Glu His Leu Ser180 185 190Thr Leu Ser Glu
Lys Ala Lys Pro Ala Leu Glu Asp Leu Arg Gln Gly195 200 205Leu Leu
Pro Val Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu210 215
220Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln225 23074256PRTArtificial
Sequenceartificial sequence of His-tagged MSP1E2 74Met Gly His His
His His His His Ile Glu Gly Arg Leu Lys Leu Leu1 5 10 15Asp Asn Trp
Asp Ser Val Thr Ser Thr Phe Ser Lys Leu Arg Glu Gln20 25 30Leu Gly
Pro Val Thr Gln Glu Phe Trp Asp Asn Leu Glu Lys Glu Thr35 40 45Glu
Gly Leu Arg Gln Glu Met Ser Lys Asp Leu Glu Glu Val Lys Ala50 55
60Lys Val Gln Pro Tyr Leu Asp Asp Phe Gln Lys Lys Trp Gln Glu Glu65
70 75 80Met Glu Leu Tyr Arg Gln Lys Val Glu Pro Tyr Leu Asp Asp Phe
Gln85 90 95Lys Lys Trp Gln Glu Glu Met Glu Leu Tyr Arg Gln Lys Val
Glu Pro100 105 110Leu Arg Ala Glu Leu Gln Glu Gly Ala Arg Gln Lys
Leu His Glu Leu115 120 125Gln Glu Lys Leu Ser Pro Leu Arg Ala Glu
Leu Gln Glu Gly Ala Arg130 135 140Gln Lys Leu His Glu Leu Gln Glu
Lys Leu Ser Pro Leu Gly Glu Glu145 150 155 160Met Arg Asp Arg Ala
Arg Ala His Val Asp Ala Leu Arg Thr His Leu165 170 175Ala Pro Tyr
Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu180 185 190Ala
Leu Lys Glu Asn Gly Gly Ala Arg Leu Ala Glu Tyr His Ala Lys195 200
205Ala Thr Glu His Leu Ser Thr Leu Ser Glu Lys Ala Lys Pro Ala
Leu210 215 220Glu Asp Leu Arg Gln Gly Leu Leu Pro Val Leu Glu Ser
Phe Lys Val225 230 235 240Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr
Lys Lys Leu Asn Thr Gln245 250 25575278PRTArtificial
Sequenceartificial sequence of His-tagged MSP1E3 75Met Gly His His
His His His His Ile Glu Gly Arg Leu Lys Leu Leu1 5 10 15Asp Asn Trp
Asp Ser Val Thr Ser Thr Phe Ser Lys Leu Arg Glu Gln20 25 30Leu Gly
Pro Val Thr Gln Glu Phe Trp Asp Asn Leu Glu Lys Glu Thr35 40 45Glu
Gly Leu Arg Gln Glu Met Ser Lys Asp Leu Glu Glu Val Lys Ala50 55
60Lys Val Gln Pro Tyr Leu Asp Asp Phe Gln Lys Lys Trp Gln Glu Glu65
70 75 80Met Glu Leu Tyr Arg Gln Lys Val Glu Pro Tyr Leu Asp Asp Phe
Gln85 90 95Lys Lys Trp Gln Glu Glu Met Glu Leu Tyr Arg Gln Lys Val
Glu Pro100 105 110Leu Arg Ala Glu Leu Gln Glu Gly Ala Arg Gln Lys
Leu His Glu Leu115 120 125Gln Glu Lys Leu Ser Pro Leu Gly Glu Glu
Met Arg Asp Arg Ala Arg130 135 140Ala His Val Asp Ala Leu Arg Thr
His Leu Ala Pro Leu Arg Ala Glu145 150 155 160Leu Gln Glu Gly Ala
Arg Gln Lys Leu His Glu Leu Gln Glu Lys Leu165 170 175Ser Pro Leu
Gly Glu Glu Met Arg Asp Arg Ala Arg Ala His Val Asp180 185 190Ala
Leu Arg Thr His Leu Ala Pro Tyr Ser Asp Glu Leu Arg Gln Arg195 200
205Leu Ala Ala Arg Leu Glu Ala Leu Lys Glu Asn Gly Gly Ala Arg
Leu210 215 220Ala Glu Tyr His Ala Lys Ala Thr Glu His Leu Ser Thr
Leu Ser Glu225 230 235 240Lys Ala Lys Pro Ala Leu Glu Asp Leu Arg
Gln Gly Leu Leu Pro Val245 250 255Leu Glu Ser Phe Lys Val Ser Phe
Leu Ser Ala Leu Glu Glu Tyr Thr260 265 270Lys Lys Leu Asn Thr
Gln27576223PRTArtificial Sequenceartificial sequence of His-tagged
MSP1TEV 76Met Gly His His His His His His His Asp Tyr Asp Ile Pro
Thr Thr1 5 10 15Glu Asn Leu Tyr Phe Gln Gly Leu Lys Leu Leu Asp Asn
Trp Asp Ser20 25 30Val Thr Ser Thr Phe Ser Lys Leu Arg Glu Gln Leu
Gly Pro Val Thr35 40 45Gln Glu Phe Trp Asp Asn Leu Glu Lys Glu Thr
Glu Gly Leu Arg Gln50 55 60Glu Met Ser Lys Asp Leu Glu Glu Val Lys
Ala Lys Val Gln Pro Tyr65 70 75 80Leu Asp Asp Phe Gln Lys Lys Trp
Gln Glu Glu Met Glu Leu Tyr Arg85 90 95Gln Lys Val Glu Pro Leu Arg
Ala Glu Leu Gln Glu Gly Ala Arg Gln100 105 110Lys Leu His Glu Leu
Gln Glu Lys Leu Ser Pro Leu Gly Glu Glu Met115 120 125Arg Asp Arg
Ala Arg Ala His Val Asp Ala Leu Arg Thr His Leu Ala130 135 140Pro
Tyr Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu Ala145 150
155 160Leu Lys Glu Asn Gly Gly Ala Arg Leu Ala Glu Tyr His Ala Lys
Ala165 170 175Thr Glu His Leu Ser Thr Leu Ser Glu Lys Ala Lys Pro
Ala Leu Glu180 185 190Asp Leu Arg Gln Gly Leu Leu Pro Val Leu Glu
Ser Phe Lys Val Ser195 200 205Phe Leu Ser Ala Leu Glu Glu Tyr Thr
Lys Lys Leu Asn Thr Gln210 215 22077200PRTArtificial
Sequenceartificial sequence of MSP1NH 77Leu Lys Leu Leu Asp Asn Trp
Asp Ser Val Thr Ser Thr Phe Ser Lys1 5 10 15Leu Arg Glu Gln Leu Gly
Pro Val Thr Gln Glu Phe Trp Asp Asn Leu20 25 30Glu Lys Glu Thr Glu
Gly Leu Arg Gln Glu Met Ser Lys Asp Leu Glu35 40 45Glu Val Lys Ala
Lys Val Gln Pro Tyr Leu Asp Asp Phe Gln Lys Lys50 55 60Trp Gln Glu
Glu Met Glu Leu Tyr Arg Gln Lys Val Glu Pro Leu Arg65 70 75 80Ala
Glu Leu Gln Glu Gly Ala Arg Gln Lys Leu His Glu Leu Gln Glu85 90
95Lys Leu Ser Pro Leu Gly Glu Glu Met Arg Asp Arg Ala Arg Ala
His100 105 110Val Asp Ala Leu Arg Thr His Leu Ala Pro Tyr Ser Asp
Glu Leu Arg115 120 125Gln Arg Leu Ala Ala Arg Leu Glu Ala Leu Lys
Glu Asn Gly Gly Ala130 135 140Arg Leu Ala Glu Tyr His Ala Lys Ala
Thr Glu His Leu Ser Thr Leu145 150 155 160Ser Glu Lys Ala Lys Pro
Ala Leu Glu Asp Leu Arg Gln Gly Leu Leu165 170 175Pro Val Leu Glu
Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu180 185 190Tyr Thr
Lys Lys Leu Asn Thr Gln195 20078212PRTArtificial Sequenceartificial
sequence of His-tagged MSP1T2 78Met Gly His His His His His His His
Asp Tyr Asp Ile Pro Thr Thr1 5 10 15Glu Asn Leu Tyr Phe Gln Gly Ser
Thr Phe Ser Lys Leu Arg Glu Gln20 25 30Leu Gly Pro Val Thr Gln Glu
Phe Trp Asp Asn Leu Glu Lys Glu Thr35 40 45Glu Gly Leu Arg Gln Glu
Met Ser Lys Asp Leu Glu Glu Val Lys Ala50 55 60Lys Val Gln Pro Tyr
Leu Asp Asp Phe Gln Lys Lys Trp Gln Glu Glu65 70 75 80Met Glu Leu
Tyr Arg Gln Lys Val Glu Pro Leu Arg Ala Glu Leu Gln85 90 95Glu Gly
Ala Arg Gln Lys Leu His Glu Leu Gln Glu Lys Leu Ser Pro100 105
110Leu Gly Glu Glu Met Arg Asp Arg Ala Arg Ala His Val Asp Ala
Leu115 120 125Arg Thr His Leu Ala Pro Tyr Ser Asp Glu Leu Arg Gln
Arg Leu Ala130 135 140Ala Arg Leu Glu Ala Leu Lys Glu Asn Gly Gly
Ala Arg Leu Ala Glu145 150 155 160Tyr His Ala Lys Ala Thr Glu His
Leu Ser Thr Leu Ser Glu Lys Ala165 170 175Lys Pro Ala Leu Glu Asp
Leu Arg Gln Gly Leu Leu Pro Val Leu Glu180 185 190Ser Phe Lys Val
Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys195 200 205Leu Asn
Thr Gln21079189PRTArtificial Sequenceartificial sequence of
MSP1T2NH 79Ser Thr Phe Ser Lys Leu Arg Glu Gln Leu Gly Pro Val Thr
Gln Glu1 5 10 15Phe Trp Asp Asn Leu Glu Lys Glu Thr Glu Gly Leu Arg
Gln Glu Met20 25 30Ser Lys Asp Leu Glu Glu Val Lys Ala Lys Val Gln
Pro Tyr Leu Asp35 40 45Asp Phe Gln Lys Lys Trp Gln Glu Glu Met Glu
Leu Tyr Arg Gln Lys50 55 60Val Glu Pro Leu Arg Ala Glu Leu Gln Glu
Gly Ala Arg Gln Lys Leu65 70 75 80His Glu Leu Gln Glu Lys Leu Ser
Pro Leu Gly Glu Glu Met Arg Asp85 90 95Arg Ala Arg Ala His Val Asp
Ala Leu Arg Thr His Leu Ala Pro Tyr100 105 110Ser Asp Glu Leu Arg
Gln Arg Leu Ala Ala Arg Leu Glu Ala Leu Lys115 120 125Glu Asn Gly
Gly Ala Arg Leu Ala Glu Tyr His Ala Lys Ala Thr Glu130 135 140His
Leu Ser Thr Leu Ser Glu Lys Ala Lys Pro Ala Leu Glu Asp Leu145 150
155 160Arg Gln Gly Leu Leu Pro Val Leu Glu Ser Phe Lys Val Ser Phe
Leu165 170 175Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr
Gln180 18580201PRTArtificial Sequenceartificial sequence of MSP1T3
80Met Gly His His His His His His His Asp Tyr Asp Ile Pro Thr Thr1
5 10 15Glu Asn Leu Tyr Phe Gln Gly Pro Val Thr Gln Glu Phe Trp Asp
Asn20 25 30Leu Glu Lys Glu Thr Glu Gly Leu Arg Gln Glu Met Ser Lys
Asp Leu35 40 45Glu Glu Val Lys Ala Lys Val Gln Pro Tyr Leu Asp Asp
Phe Gln Lys50 55 60Lys Trp Gln Glu Glu Met Glu Leu Tyr Arg Gln Lys
Val Glu Pro Leu65 70 75 80Arg Ala Glu Leu Gln Glu Gly Ala Arg Gln
Lys Leu His Glu Leu Gln85 90 95Glu Lys Leu Ser Pro Leu Gly Glu Glu
Met Arg Asp Arg Ala Arg Ala100 105 110His Val Asp Ala Leu Arg Thr
His Leu Ala Pro Tyr Ser Asp Glu Leu115 120 125Arg Gln Arg Leu Ala
Ala Arg Leu Glu Ala Leu Lys Glu Asn Gly Gly130 135 140Ala Arg Leu
Ala Glu Tyr His Ala Lys Ala Thr Glu His Leu Ser Thr145 150 155
160Leu Ser Glu Lys Ala Lys Pro Ala Leu Glu Asp Leu Arg Gln Gly
Leu165 170 175Leu Pro Val Leu Glu Ser Phe Lys Val Ser Phe Leu Ser
Ala Leu Glu180 185 190Glu Tyr Thr Lys Lys Leu Asn Thr Gln195
20081168PRTArtificial Sequenceartificial sequence of MSP1D4D5 81Met
Gly His His His His His His Ile Glu Gly Arg Leu Lys Leu Leu1 5 10
15Asp Asn Trp Asp Ser Val Thr Ser Thr Phe Ser Lys Leu Arg Glu Gln20
25 30Leu Gly Pro Val Thr Gln Glu Phe Trp Asp Asn Leu Glu Lys Glu
Thr35 40 45Glu Gly Leu Arg Gln Glu Met Ser Lys Asp Leu Glu Glu Val
Lys Ala50 55 60Lys Val Gln Pro Leu Gly Glu Glu Met Arg Asp Arg Ala
Arg Ala His65 70 75 80Val Asp Ala Leu Arg Thr His Leu Ala Pro Tyr
Ser Asp Glu Leu Arg85 90 95Gln Arg Leu Ala Ala Arg Leu Glu Ala Leu
Lys Glu Asn Gly Gly Ala100 105 110Arg Leu Ala Glu Tyr His Ala Lys
Ala Thr Glu His Leu Ser Thr Leu115 120 125Ser Glu Lys Ala Lys Pro
Ala Leu Glu Asp Leu Arg Gln Gly Leu Leu130 135 140Pro Val Leu Glu
Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu145 150 155 160Tyr
Thr Lys Lys Leu Asn Thr Gln16582168PRTArtificial SequenceHis-tagged
MSP1D6D7 82Met Gly His His His His His His Ile Glu Gly Arg Leu Lys
Leu Leu1 5 10 15Asp Asn Trp Asp Ser Val Thr Ser Thr Phe Ser Lys Leu
Arg Glu Gln20 25 30Leu Gly Pro Val Thr Gln Glu Phe Trp Asp Asn Leu
Glu Lys Glu Thr35 40 45Glu Gly Leu Arg Gln Glu Met Ser Lys Asp Leu
Glu Glu Val Lys Ala50 55 60Lys Val Gln Pro Tyr Leu Asp Asp Phe Gln
Lys Lys Trp Gln Glu Glu65 70 75 80Met Glu Leu Tyr Arg Gln Lys Val
Glu Pro Leu Arg Ala Glu Leu Gln85 90 95Glu Gly Ala Arg Gln Lys Leu
His Glu Leu Gln Glu Lys Leu Ser Ala100 105 110Arg Leu Ala Glu Tyr
His Ala Lys Ala Thr Glu His Leu Ser Thr Leu115 120 125Ser Glu Lys
Ala Lys Pro Ala Leu Glu Asp Leu Arg Gln Gly Leu Leu130 135 140Pro
Val Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu145 150
155 160Tyr Thr Lys Lys Leu Asn Thr Gln16583190PRTArtificial
SequenceHis-tagged MSP1D3D9 83Met Gly His His His His His His Ile
Glu Gly Arg Leu Lys Leu Leu1 5 10 15Asp Asn Trp Asp Ser Val Thr Ser
Thr Phe Ser Lys Leu Arg Glu Gln20 25 30Leu Gly Pro Val Thr Gln Glu
Phe Trp Asp Asn Leu Glu Lys Glu Thr35 40 45Glu Gly Leu Arg Gln Glu
Met Ser Pro Tyr Leu Asp Asp Phe Gln Lys50 55 60Lys Trp Gln Glu Glu
Met Glu Leu Tyr Arg Gln Lys Val Glu Pro Leu65 70 75 80Arg Ala Glu
Leu Gln Glu Gly Ala Arg Gln Lys Leu His Glu Leu Gln85 90 95Glu Lys
Leu Ser Pro Leu Gly Glu Glu Met Arg Asp Arg Ala Arg Ala100 105
110His Val Asp Ala Leu Arg Thr His Leu Ala Pro Tyr Ser Asp Glu
Leu115 120 125Arg Gln Arg Leu Ala Ala Arg Leu Glu Ala Leu Lys Glu
Asn Gly Gly130 135 140Ala Arg Leu Ala Glu Tyr His Ala Lys Ala Thr
Glu His Leu Ser Thr145 150 155 160Leu Ser Glu Lys Ala Lys Pro Val
Leu Glu Ser Phe Lys Val Ser Phe165 170 175Leu Ser Ala Leu Glu Glu
Tyr Thr Lys Lys Leu Asn Thr Gln180 185 19084201PRTArtificial
SequenceHis-tagged MSP1D10.5 84Met Gly His His His His His His Ile
Glu Gly Arg Leu Lys Leu Leu1 5 10 15Asp Asn Trp Asp Ser Val Thr Ser
Thr Phe Ser Lys Leu Arg Glu Gln20 25 30Leu Gly Pro Val Thr Gln Glu
Phe Trp Asp Asn Leu Glu Lys Glu Thr35 40 45Glu Gly Leu Arg Gln Glu
Met Ser Lys Asp Leu Glu Glu Val Lys Ala50 55 60Lys Val Gln Pro Tyr
Leu Asp Asp Phe Gln Lys Lys Trp Gln Glu Glu65 70 75 80Met Glu Leu
Tyr Arg Gln Lys Val Glu Pro Leu Arg Ala Glu Leu Gln85 90 95Glu Gly
Ala Arg Gln Lys Leu His Glu Leu Gln Glu Lys Leu Ser Pro100 105
110Leu Gly Glu Glu Met Arg Asp Arg Ala Arg Ala His Val Asp Ala
Leu115 120 125Arg Thr His Leu Ala Pro Tyr Ser Asp Glu Leu Arg Gln
Arg Leu Ala130 135 140Ala Arg Leu Glu Ala Leu Lys Glu Asn Gly Gly
Ala Arg Leu Ala Glu145 150 155 160Tyr His Ala Lys Ala Thr Glu His
Leu Ser Thr Leu Ser Glu Lys Ala165 170 175Lys Pro Ala Leu Glu Asp
Leu Arg Gln Gly Leu Leu Ser Ala Leu Glu180 185 190Glu Tyr Thr Lys
Lys Leu Asn Thr Gln195 20085190PRTArtificial SequenceHis-tagged
MSP1D3D10.5 85Met Gly His His His His His His Ile Glu Gly Arg Leu
Lys Leu Leu1 5
10 15Asp Asn Trp Asp Ser Val Thr Ser Thr Phe Ser Lys Leu Arg Glu
Gln20 25 30Leu Gly Pro Val Thr Gln Glu Phe Trp Asp Asn Leu Glu Lys
Glu Thr35 40 45Glu Gly Leu Arg Gln Glu Met Ser Pro Tyr Leu Asp Asp
Phe Gln Lys50 55 60Lys Trp Gln Glu Glu Met Glu Leu Tyr Arg Gln Lys
Val Glu Pro Leu65 70 75 80Arg Ala Glu Leu Gln Glu Gly Ala Arg Gln
Lys Leu His Glu Leu Gln85 90 95Glu Lys Leu Ser Pro Leu Gly Glu Glu
Met Arg Asp Arg Ala Arg Ala100 105 110His Val Asp Ala Leu Arg Thr
His Leu Ala Pro Tyr Ser Asp Glu Leu115 120 125Arg Gln Arg Leu Ala
Ala Arg Leu Glu Ala Leu Lys Glu Asn Gly Gly130 135 140Ala Arg Leu
Ala Glu Tyr His Ala Lys Ala Thr Glu His Leu Ser Thr145 150 155
160Leu Ser Glu Lys Ala Lys Pro Ala Leu Glu Asp Leu Arg Gln Gly
Leu165 170 175Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr
Gln180 185 19086381PRTArtificial SequenceHis-tagged MSP2D1D1 86Met
Gly His His His His His His His Asp Tyr Asp Ile Pro Thr Thr1 5 10
15Glu Asn Leu Tyr Phe Gln Gly Pro Val Thr Gln Glu Phe Trp Asp Asn20
25 30Leu Glu Lys Glu Thr Glu Gly Leu Arg Gln Glu Met Ser Lys Asp
Leu35 40 45Glu Glu Val Lys Ala Lys Val Gln Pro Tyr Leu Asp Asp Phe
Gln Lys50 55 60Lys Trp Gln Glu Glu Met Glu Leu Tyr Arg Gln Lys Val
Glu Pro Leu65 70 75 80Arg Ala Glu Leu Gln Glu Gly Ala Arg Gln Lys
Leu His Glu Leu Gln85 90 95Glu Lys Leu Ser Pro Leu Gly Glu Glu Met
Arg Asp Arg Ala Arg Ala100 105 110His Val Asp Ala Leu Arg Thr His
Leu Ala Pro Tyr Ser Asp Glu Leu115 120 125Arg Gln Arg Leu Ala Ala
Arg Leu Glu Ala Leu Lys Glu Asn Gly Gly130 135 140Ala Arg Leu Ala
Glu Tyr His Ala Lys Ala Thr Glu His Leu Ser Thr145 150 155 160Leu
Ser Glu Lys Ala Lys Pro Ala Leu Glu Asp Leu Arg Gln Gly Leu165 170
175Leu Pro Val Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu
Glu180 185 190Glu Tyr Thr Lys Lys Leu Asn Thr Gln Gly Thr Pro Val
Thr Gln Glu195 200 205Phe Trp Asp Asn Leu Glu Lys Glu Thr Glu Gly
Leu Arg Gln Glu Met210 215 220Ser Lys Asp Leu Glu Glu Val Lys Ala
Lys Val Gln Pro Tyr Leu Asp225 230 235 240Asp Phe Gln Lys Lys Trp
Gln Glu Glu Met Glu Leu Tyr Arg Gln Lys245 250 255Val Glu Pro Leu
Arg Ala Glu Leu Gln Glu Gly Ala Arg Gln Lys Leu260 265 270His Glu
Leu Gln Glu Lys Leu Ser Pro Leu Gly Glu Glu Met Arg Asp275 280
285Arg Ala Arg Ala His Val Asp Ala Leu Arg Thr His Leu Ala Pro
Tyr290 295 300Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu
Ala Leu Lys305 310 315 320Glu Asn Gly Gly Ala Arg Leu Ala Glu Tyr
His Ala Lys Ala Thr Glu325 330 335His Leu Ser Thr Leu Ser Glu Lys
Ala Lys Pro Ala Leu Glu Asp Leu340 345 350Arg Gln Gly Leu Leu Pro
Val Leu Glu Ser Phe Lys Val Ser Phe Leu355 360 365Ser Ala Leu Glu
Glu Tyr Thr Lys Lys Leu Asn Thr Gln370 375 3808713PRTArtificial
Sequencesequence of Helix 10.5 87Ser Ala Leu Glu Glu Tyr Thr Lys
Lys Leu Asn Thr Gln1 5 108849DNAArtificial Sequencenucleotide
sequence of Helix 10.5 88cagttttctg tcagctctgg aagaatatac
taaaaagctg aatacccag 498943PRTArtificial Sequencesequence of GLOB
89Asp Glu Pro Pro Gln Ser Pro Trp Asp Arg Val Lys Asp Leu Ala Thr1
5 10 15Val Tyr Val Asp Val Leu Lys Asp Ser Gly Arg Asp Tyr Val Ser
Gln20 25 30Phe Glu Gly Ser Ala Leu Gly Lys Gln Leu Asn35
409066DNAArtificial SequenceOligonuceotide encoding H2S
90tccgtgacgc aggaattctg ggacaacctg gaaaaagaaa ccgagggact gcgtcaggaa
60atgtcc 6691201PRTArtificial SequenceMSP1T4 91Met Gly His His His
His His His His Asp Tyr Asp Ile Pro Thr Thr1 5 10 15Glu Asn Leu Tyr
Phe Gln Gly Ser Val Thr Gln Glu Phe Trp Asp Asn20 25 30Leu Glu Lys
Glu Thr Glu Gly Leu Arg Gln Glu Met Ser Lys Asp Leu35 40 45Glu Glu
Val Lys Ala Lys Val Gln Pro Tyr Leu Asp Asp Phe Gln Lys50 55 60Lys
Trp Gln Glu Glu Met Glu Leu Tyr Arg Gln Lys Val Glu Pro Leu65 70 75
80Arg Ala Glu Leu Gln Glu Gly Ala Arg Gln Lys Leu His Glu Leu Gln85
90 95Glu Lys Leu Ser Pro Leu Gly Glu Glu Met Arg Asp Arg Ala Arg
Ala100 105 110His Val Asp Ala Leu Arg Thr His Leu Ala Pro Tyr Ser
Asp Glu Leu115 120 125Arg Gln Arg Leu Ala Ala Arg Leu Glu Ala Leu
Lys Glu Asn Gly Gly130 135 140Ala Arg Leu Ala Glu Tyr His Ala Lys
Ala Thr Glu His Leu Ser Thr145 150 155 160Leu Ser Glu Lys Ala Lys
Pro Ala Leu Glu Asp Leu Arg Gln Gly Leu165 170 175Leu Pro Val Leu
Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu180 185 190Glu Tyr
Thr Lys Lys Leu Asn Thr Gln195 20092190PRTArtificial SequenceMSP1T5
92Met Gly His His His His His His His Asp Tyr Asp Ile Pro Thr Thr1
5 10 15Glu Asn Leu Tyr Phe Gln Gly Lys Glu Thr Glu Gly Leu Arg Gln
Glu20 25 30Met Ser Lys Asp Leu Glu Glu Val Lys Ala Lys Val Gln Pro
Tyr Leu35 40 45Asp Asp Phe Gln Lys Lys Trp Gln Glu Glu Met Glu Leu
Tyr Arg Gln50 55 60Lys Val Glu Pro Leu Arg Ala Glu Leu Gln Glu Gly
Ala Arg Gln Lys65 70 75 80Leu His Glu Leu Gln Glu Lys Leu Ser Pro
Leu Gly Glu Glu Met Arg85 90 95Asp Arg Ala Arg Ala His Val Asp Ala
Leu Arg Thr His Leu Ala Pro100 105 110Tyr Ser Asp Glu Leu Arg Gln
Arg Leu Ala Ala Arg Leu Glu Ala Leu115 120 125Lys Glu Asn Gly Gly
Ala Arg Leu Ala Glu Tyr His Ala Lys Ala Thr130 135 140Glu His Leu
Ser Thr Leu Ser Glu Lys Ala Lys Pro Ala Leu Glu Asp145 150 155
160Leu Arg Gln Gly Leu Leu Pro Val Leu Glu Ser Phe Lys Val Ser
Phe165 170 175Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr
Gln180 185 19093179PRTArtificial SequenceMSP1T6 93Met Gly His His
His His His His His Asp Tyr Asp Ile Pro Thr Thr1 5 10 15Glu Asn Leu
Tyr Phe Gln Gly Lys Asp Leu Glu Glu Val Lys Ala Lys20 25 30Val Gln
Pro Tyr Leu Asp Asp Phe Gln Lys Lys Trp Gln Glu Glu Met35 40 45Glu
Leu Tyr Arg Gln Lys Val Glu Pro Leu Arg Ala Glu Leu Gln Glu50 55
60Gly Ala Arg Gln Lys Leu His Glu Leu Gln Glu Lys Leu Ser Pro Leu65
70 75 80Gly Glu Glu Met Arg Asp Arg Ala Arg Ala His Val Asp Ala Leu
Arg85 90 95Thr His Leu Ala Pro Tyr Ser Asp Glu Leu Arg Gln Arg Leu
Ala Ala100 105 110Arg Leu Glu Ala Leu Lys Glu Asn Gly Gly Ala Arg
Leu Ala Glu Tyr115 120 125His Ala Lys Ala Thr Glu His Leu Ser Thr
Leu Ser Glu Lys Ala Lys130 135 140Pro Ala Leu Glu Asp Leu Arg Gln
Gly Leu Leu Pro Val Leu Glu Ser145 150 155 160Phe Lys Val Ser Phe
Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu165 170 175Asn Thr
Gln94289PRTArtificial SequenceMSP1E3TEV 94Met Gly His His His His
His His His Asp Tyr Asp Ile Pro Thr Thr1 5 10 15Glu Asn Leu Tyr Phe
Gln Gly Leu Lys Leu Leu Asp Asn Trp Asp Ser20 25 30Val Thr Ser Thr
Phe Ser Lys Leu Arg Glu Gln Leu Gly Pro Val Thr35 40 45Gln Glu Phe
Trp Asp Asn Leu Glu Lys Glu Thr Glu Gly Leu Arg Gln50 55 60Glu Met
Ser Lys Asp Leu Glu Glu Val Lys Ala Lys Val Gln Pro Tyr65 70 75
80Leu Asp Asp Phe Gln Lys Lys Trp Gln Glu Glu Met Glu Leu Tyr Arg85
90 95Gln Lys Val Glu Pro Leu Arg Ala Glu Leu Gln Glu Gly Ala Arg
Gln100 105 110Lys Leu His Glu Leu Gln Glu Lys Leu Ser Pro Leu Gly
Glu Glu Met115 120 125Arg Asp Arg Ala Arg Ala His Val Asp Ala Leu
Arg Thr His Leu Ala130 135 140Pro Tyr Leu Asp Asp Phe Gln Lys Lys
Trp Gln Glu Glu Met Glu Leu145 150 155 160Tyr Arg Gln Lys Val Glu
Pro Leu Arg Ala Glu Leu Gln Glu Gly Ala165 170 175Arg Gln Lys Leu
His Glu Leu Gln Glu Lys Leu Ser Pro Leu Gly Glu180 185 190Glu Met
Arg Asp Arg Ala Arg Ala His Val Asp Ala Leu Arg Thr His195 200
205Leu Ala Pro Tyr Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg
Leu210 215 220Glu Ala Leu Lys Glu Asn Gly Gly Ala Arg Leu Ala Glu
Tyr His Ala225 230 235 240Lys Ala Thr Glu His Leu Ser Thr Leu Ser
Glu Lys Ala Lys Pro Ala245 250 255Leu Glu Asp Leu Arg Gln Gly Leu
Leu Pro Val Leu Glu Ser Phe Lys260 265 270Val Ser Phe Leu Ser Ala
Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr275 280
285Gln95278PRTArtificial SequenceMSP1E3D1 95Met Gly His His His His
His His His Asp Tyr Asp Ile Pro Thr Thr1 5 10 15Glu Asn Leu Tyr Phe
Gln Gly Ser Thr Phe Ser Lys Leu Arg Glu Gln20 25 30Leu Gly Pro Val
Thr Gln Glu Phe Trp Asp Asn Leu Glu Lys Glu Thr35 40 45Glu Gly Leu
Arg Gln Glu Met Ser Lys Asp Leu Glu Glu Val Lys Ala50 55 60Lys Val
Gln Pro Tyr Leu Asp Asp Phe Gln Lys Lys Trp Gln Glu Glu65 70 75
80Met Glu Leu Tyr Arg Gln Lys Val Glu Pro Leu Arg Ala Glu Leu Gln85
90 95Glu Gly Ala Arg Gln Lys Leu His Glu Leu Gln Glu Lys Leu Ser
Pro100 105 110Leu Gly Glu Glu Met Arg Asp Arg Ala Arg Ala His Val
Asp Ala Leu115 120 125Arg Thr His Leu Ala Pro Tyr Leu Asp Asp Phe
Gln Lys Lys Trp Gln130 135 140Glu Glu Met Glu Leu Tyr Arg Gln Lys
Val Glu Pro Leu Arg Ala Glu145 150 155 160Leu Gln Glu Gly Ala Arg
Gln Lys Leu His Glu Leu Gln Glu Lys Leu165 170 175Ser Pro Leu Gly
Glu Glu Met Arg Asp Arg Ala Arg Ala His Val Asp180 185 190Ala Leu
Arg Thr His Leu Ala Pro Tyr Ser Asp Glu Leu Arg Gln Arg195 200
205Leu Ala Ala Arg Leu Glu Ala Leu Lys Glu Asn Gly Gly Ala Arg
Leu210 215 220Ala Glu Tyr His Ala Lys Ala Thr Glu His Leu Ser Thr
Leu Ser Glu225 230 235 240Lys Ala Lys Pro Ala Leu Glu Asp Leu Arg
Gln Gly Leu Leu Pro Val245 250 255Leu Glu Ser Phe Lys Val Ser Phe
Leu Ser Ala Leu Glu Glu Tyr Thr260 265 270Lys Lys Leu Asn Thr
Gln27596423PRTArtificial SequenceMSP2TEV 96Met Gly His His His His
His His His Asp Tyr Asp Ile Pro Thr Thr1 5 10 15Glu Asn Leu Tyr Phe
Gln Gly Leu Lys Leu Leu Asp Asn Trp Asp Ser20 25 30Val Thr Ser Thr
Phe Ser Lys Leu Arg Glu Gln Leu Gly Pro Val Thr35 40 45Gln Glu Phe
Trp Asp Asn Leu Glu Lys Glu Thr Glu Gly Leu Arg Gln50 55 60Glu Met
Ser Lys Asp Leu Glu Glu Val Lys Ala Lys Val Gln Pro Tyr65 70 75
80Leu Asp Asp Phe Gln Lys Lys Trp Gln Glu Glu Met Glu Leu Tyr Arg85
90 95Gln Lys Val Glu Pro Leu Arg Ala Glu Leu Gln Glu Gly Ala Arg
Gln100 105 110Lys Leu His Glu Leu Gln Glu Lys Leu Ser Pro Leu Gly
Glu Glu Met115 120 125Arg Asp Arg Ala Arg Ala His Val Asp Ala Leu
Arg Thr His Leu Ala130 135 140Pro Tyr Ser Asp Glu Leu Arg Gln Arg
Leu Ala Ala Arg Leu Glu Ala145 150 155 160Leu Lys Glu Asn Gly Gly
Ala Arg Leu Ala Glu Tyr His Ala Lys Ala165 170 175Thr Glu His Leu
Ser Thr Leu Ser Glu Lys Ala Lys Pro Ala Leu Glu180 185 190Asp Leu
Arg Gln Gly Leu Leu Pro Val Leu Glu Ser Phe Lys Val Ser195 200
205Phe Leu Ser Ala Leu Glu Tyr Thr Lys Lys Leu Asn Thr Gln Gly
Thr210 215 220Leu Lys Leu Leu Asp Asn Trp Asp Ser Val Thr Ser Thr
Phe Ser Lys225 230 235 240Leu Arg Glu Gln Leu Gly Pro Val Thr Gln
Glu Phe Trp Asp Asn Leu245 250 255Glu Lys Glu Thr Glu Gly Leu Arg
Gln Glu Met Lys Asp Leu Glu Glu260 265 270Val Lys Ala Lys Val Gln
Pro Tyr Leu Asp Asp Phe Gln Lys Lys Trp275 280 285Gln Glu Glu Met
Glu Leu Tyr Arg Gln Lys Val Glu Pro Leu Arg Ala290 295 300Glu Leu
Gln Glu Gly Ala Arg Gln Lys Leu His Glu Leu Gln Glu Lys305 310 315
320Leu Ser Pro Leu Gly Glu Glu Met Arg Asp Arg Ala Arg Ala His
Val325 330 335Asp Ala Leu Arg Thr His Leu Ala Pro Tyr Ser Asp Glu
Leu Arg Gln340 345 350Arg Leu Ala Ala Arg Leu Glu Ala Leu Lys Glu
Asn Gly Gly Ala Arg355 360 365Leu Ala Glu Tyr His Ala Lys Ala Thr
Glu His Leu Ser Thr Leu Ser370 375 380Glu Lys Ala Lys Pro Ala Leu
Glu Asp Leu Arg Gln Gly Leu Leu Pro385 390 395 400Val Leu Glu Ser
Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr405 410 415Thr Lys
Lys Leu Asn Thr Gln42097199PRTArtificial SequenceMSP1N1 97Met Gly
His His His His His His His Asp Tyr Asp Ile Pro Thr Thr1 5 10 15Glu
Asn Leu Tyr Phe Gln Gly Ser Val Thr Gln Glu Phe Trp Asp Asn20 25
30Leu Glu Lys Glu Thr Glu Gly Leu Arg Gln Glu Met Ser Lys Asp Leu35
40 45Glu Glu Val Lys Ala Lys Val Gln Pro Tyr Leu Asp Asp Phe Gln
Lys50 55 60Lys Trp Gln Glu Glu Met Glu Leu Tyr Arg Gln Lys Val Glu
Pro Tyr65 70 75 80Leu Asp Asp Phe Gln Lys Lys Trp Gln Glu Glu Met
Glu Leu Tyr Arg85 90 95Gln Lys Val Glu Pro Leu Arg Ala Glu Leu Gln
Glu Gly Ala Arg Gln100 105 110Lys Leu His Glu Leu Gln Glu Lys Leu
Ser Pro Leu Gly Glu Glu Met115 120 125Arg Asp Arg Ala Arg Ala His
Val Asp Ala Leu Arg Thr His Leu Ala130 135 140Pro Tyr Ser Asp Glu
Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu Ala145 150 155 160Leu Lys
Glu Asn Gly Gly Ala Arg Leu Ala Glu Tyr His Ala Lys Ala165 170
175Thr Glu His Leu Ser Thr Leu Ser Glu Lys Ala Lys Pro Ala Leu
Glu180 185 190Asp Leu Arg Gln Gly Leu Leu19598401PRTArtificial
SequenceMSP2N1 98Met Gly His His His His His His His Asp Tyr Asp
Ile Pro Thr Thr1 5 10 15Glu Asn Leu Tyr Phe Gln Gly Ser Thr Phe Ser
Lys Leu Arg Glu Gln20 25 30Leu Gly Pro Val Thr Gln Glu Phe Trp Asp
Asn Leu Glu Lys Glu Thr35 40 45Glu Gly Leu Arg Gln Glu Met Ser Lys
Asp Leu Glu Glu Val Lys Ala50 55 60Lys Val Gln Pro Tyr Leu Asp Asp
Phe Gln Lys Lys Trp Gln Glu Glu65 70 75 80Met Glu Leu Tyr Arg Gln
Lys Val Glu Pro Leu Arg Ala Glu Leu Gln85 90 95Glu Gly Ala Arg Gln
Lys Leu His Glu Leu Gln Glu Lys Leu Ser Pro100 105 110Leu Gly Glu
Glu Met Arg Asp Arg Ala Arg Ala His Val Asp Ala Leu115 120 125Arg
Thr His Leu Ala Pro Tyr Ser Asp Glu Leu Arg Gln Arg Leu Ala130 135
140Ala Arg Leu Glu Ala Leu Lys Glu Asn Gly Gly Ala Arg Leu Ala
Glu145 150 155 160Tyr His Ala Lys Ala Thr Glu His Leu Ser Thr Leu
Ser Glu Lys Ala165
170 175Lys Pro Ala Leu Glu Asp Leu Arg Gln Gly Leu Leu Pro Val Leu
Glu180 185 190Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr
Thr Lys Lys195 200 205Leu Asn Thr Gln Gly Thr Phe Ser Lys Leu Arg
Glu Gln Leu Gly Pro210 215 220Val Thr Gln Glu Phe Trp Asp Asn Leu
Glu Lys Glu Thr Glu Gly Leu225 230 235 240Arg Gln Glu Met Ser Lys
Asp Leu Glu Glu Val Lys Ala Lys Val Gln245 250 255Pro Tyr Leu Asp
Asp Phe Gln Lys Lys Trp Gln Glu Glu Met Glu Leu260 265 270Tyr Arg
Gln Lys Val Glu Pro Leu Arg Ala Glu Leu Gln Glu Gly Ala275 280
285Arg Gln Lys Leu His Glu Leu Gln Glu Lys Leu Ser Pro Leu Gly
Glu290 295 300Glu Met Arg Asp Arg Ala Arg Ala His Val Asp Ala Leu
Arg Thr His305 310 315 320Leu Ala Pro Tyr Ser Asp Glu Leu Arg Gln
Arg Leu Ala Ala Arg Leu325 330 335Glu Ala Leu Lys Glu Asn Gly Gly
Ala Arg Leu Ala Glu Tyr His Ala340 345 350Lys Ala Thr Glu His Leu
Ser Thr Leu Ser Glu Lys Ala Lys Pro Ala355 360 365Leu Glu Asp Leu
Arg Gln Gly Leu Leu Pro Val Leu Glu Ser Phe Lys370 375 380Val Ser
Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr385 390 395
400Gln99392PRTArtificial SequenceMSP2N2 99Met Gly His His His His
His His His Asp Tyr Asp Ile Pro Thr Thr1 5 10 15Glu Asn Leu Tyr Phe
Gln Gly Ser Thr Phe Ser Lys Leu Arg Glu Gln20 25 30Leu Gly Pro Val
Thr Gln Glu Phe Trp Asp Asn Leu Glu Lys Glu Thr35 40 45Glu Gly Leu
Arg Gln Glu Met Ser Lys Asp Leu Glu Glu Val Lys Ala50 55 60Lys Val
Gln Pro Tyr Leu Asp Asp Phe Gln Lys Lys Trp Gln Glu Glu65 70 75
80Met Glu Leu Tyr Arg Gln Lys Val Glu Pro Leu Arg Ala Glu Leu Gln85
90 95Glu Gly Ala Arg Gln Lys Leu His Glu Leu Gln Glu Lys Leu Ser
Pro100 105 110Leu Gly Glu Glu Met Arg Asp Arg Ala Arg Ala His Val
Asp Ala Leu115 120 125Arg Thr His Leu Ala Pro Tyr Ser Asp Glu Leu
Arg Gln Arg Leu Ala130 135 140Ala Arg Leu Glu Ala Leu Lys Glu Asn
Gly Gly Ala Arg Leu Ala Glu145 150 155 160Tyr His Ala Lys Ala Thr
Glu His Leu Ser Thr Leu Ser Glu Lys Ala165 170 175Lys Pro Ala Leu
Glu Asp Leu Arg Gln Gly Leu Leu Pro Val Leu Glu180 185 190Ser Phe
Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys195 200
205Leu Asn Thr Gln Gly Thr Pro Val Thr Gln Glu Phe Trp Asp Asn
Leu210 215 220Glu Lys Glu Thr Glu Gly Leu Arg Gln Glu Met Ser Lys
Asp Leu Glu225 230 235 240Glu Val Lys Ala Lys Val Gln Pro Tyr Leu
Asp Asp Phe Gln Lys Lys245 250 255Trp Gln Glu Glu Met Glu Leu Tyr
Arg Gln Lys Val Glu Pro Leu Arg260 265 270Ala Glu Leu Gln Glu Gly
Ala Arg Gln Lys Leu His Glu Leu Gln Glu275 280 285Lys Leu Ser Pro
Leu Gly Glu Glu Met Arg Asp Arg Ala Arg Ala His290 295 300Val Asp
Ala Leu Arg Thr His Leu Ala Pro Tyr Ser Asp Glu Leu Arg305 310 315
320Gln Arg Leu Ala Ala Arg Leu Glu Ala Leu Lys Glu Asn Gly Gly
Ala325 330 335Arg Leu Ala Glu Tyr His Ala Lys Ala Thr Glu His Leu
Ser Thr Leu340 345 350Ser Glu Lys Ala Lys Pro Ala Leu Glu Asp Leu
Arg Gln Gly Leu Leu355 360 365Pro Val Leu Glu Ser Phe Lys Val Ser
Phe Leu Ser Ala Leu Glu Glu370 375 380Tyr Thr Lys Lys Leu Asn Thr
Gln385 390100603DNAArtificial SequenceDNA encoding MSP1T4
100atgggtcatc atcatcatca tcatcacgat tatgatattc ctactactga
gaatttgtat 60tttcagggtt ccgtgacgca ggaattctgg gacaacctgg aaaaagaaac
cgagggactg 120cgtcaggaaa tgtccaaaga tttagaagag gtgaaggcca
aggttcagcc atatctcgat 180gactttcaga aaaaatggca ggaagagatg
gaattatatc gtcaaaaggt ggaaccgctg 240cgtgcggaac tgcaagaggg
ggcacgccaa aaactccatg agctccaaga gaagctcagc 300ccattaggcg
aagaaatgcg cgatcgcgcc cgtgcacatg ttgatgcact ccggactcat
360ttggcgccgt attcggatga acttcgccag cgtttggccg cacgtctcga
ggcgctgaaa 420gaaaacgggg gtgcccgctt ggctgagtac cacgcgaaag
cgacagaaca cctgagcacc 480ttgagcgaaa aagcgaaacc ggcgctggaa
gatctacgcc agggcttatt gcctgttctt 540gagagcttta aagtcagttt
tctgtcagct ctggaagaat atactaaaaa gctgaatacc 600cag
603101570DNAArtificial SequenceDNA encoding MSP1T5 101atgggtcatc
atcatcatca tcatcacgat tatgatattc ctactactga gaatttgtat 60tttcagggta
aagaaaccga gggactgcgt caggaaatgt ccaaagattt agaagaggtg
120aaggccaagg ttcagccata tctcgatgac tttcagaaaa aatggcagga
agagatggaa 180ttatatcgtc aaaaggtgga accgctgcgt gcggaactgc
aagagggggc acgccaaaaa 240ctccatgagc tccaagagaa gctcagccca
ttaggcgaag aaatgcgcga tcgcgcccgt 300gcacatgttg atgcactccg
gactcatttg gcgccgtatt cggatgaact tcgccagcgt 360ttggccgcac
gtctcgaggc gctgaaagaa aacgggggtg cccgcttggc tgagtaccac
420gcgaaagcga cagaacacct gagcaccttg agcgaaaaag cgaaaccggc
gctggaagat 480ctacgccagg gcttattgcc tgttcttgag agctttaaag
tcagttttct gtcagctctg 540gaagaatata ctaaaaagct gaatacccag
570102537DNAArtificial SequenceDNA encoding MSP1T6 102atgggtcatc
atcatcatca tcatcacgat tatgatattc ctactactga gaatttgtat 60tttcagggta
aagatttaga agaggtgaag gccaaggttc agccatatct cgatgacttt
120cagaaaaaat ggcaggaaga gatggaatta tatcgtcaaa aggtggaacc
gctgcgtgcg 180gaactgcaag agggggcacg ccaaaaactc catgagctcc
aagagaagct cagcccatta 240ggcgaagaaa tgcgcgatcg cgcccgtgca
catgttgatg cactccggac tcatttggcg 300ccgtattcgg atgaacttcg
ccagcgtttg gccgcacgtc tcgaggcgct gaaagaaaac 360gggggtgccc
gcttggctga gtaccacgcg aaagcgacag aacacctgag caccttgagc
420gaaaaagcga aaccggcgct ggaagatcta cgccagggct tattgcctgt
tcttgagagc 480tttaaagtca gttttctgtc agctctggaa gaatatacta
aaaagctgaa tacccag 537103597DNAArtificial SequenceDNA encoding
MSP1N1 103atgggtcatc atcatcatca tcatcacgat tatgatattc ctactactga
gaatttgtat 60tttcagggtt ccgtgacgca ggaattctgg gacaacctgg aaaaagaaac
cgagggactg 120cgtcaggaaa tgtccaaaga tttagaagag gtgaaggcca
aggttcagcc atatctcgat 180gactttcaga aaaaatggca ggaagagatg
gaattatatc gtcaaaaggt ggaaccatat 240ctcgatgact ttcagaaaaa
atggcaggaa gagatggaat tatatcgtca aaaggtggaa 300ccgctgcgtg
cggaactgca agagggggca cgccaaaaac tccatgagct ccaagagaag
360ctcagcccat taggcgaaga aatgcgcgat cgcgcccgtg cacatgttga
tgcactccgg 420actcatttgg cgccgtattc ggatgaactt cgccagcgtt
tggccgcacg tctcgaggcg 480ctgaaagaaa acgggggtgc ccgcttggct
gagtaccacg cgaaagcgac agaacacctg 540agcaccttga gcgaaaaagc
gaaaccggcg ctggaagatc tacgccaggg cttattg 5971044PRTArtificial
Sequenceartificial peptide sequence 104Asn Pro Gly
Thr1105867DNAArtificial SequenceDNA encoding MSP1E3TEV
105atgggtcatc atcatcatca tcatcacgat tatgatattc ctactactga
gaatttgtat 60tttcagggtc tgaagctgtt ggacaattgg gactctgtta cgtctacctt
cagtaaactt 120cgcgaacaac tgggccccgt gacgcaggaa ttctgggaca
acctggaaaa agaaaccgag 180ggactgcgtc aggaaatgtc caaagattta
gaagaggtga aggccaaggt tcagccatat 240ctcgatgact ttcagaaaaa
atggcaggaa gagatggaat tatatcgtca aaaggtggaa 300ccgctgcgtg
cggaactgca agagggggca cgccaaaaac tccatgagct ccaagagaag
360ctcagcccat taggcgaaga aatgcgcgat cgcgcccgtg cacatgttga
tgcactccgg 420actcatttgg cgccatatct cgatgacttt cagaaaaaat
ggcaggaaga gatggaatta 480tatcgtcaaa aggtggaacc gctgcgtgcg
gaactgcaag agggggcacg ccaaaaactc 540catgagctcc aagagaagct
cagcccatta ggcgaagaaa tgcgcgatcg cgcccgtgca 600catgttgatg
cactccggac tcatttggcg ccgtattcgg atgaacttcg ccagcgtttg
660gccgcacgtc tcgaggcgct gaaagaaaac gggggtgccc gcttggctga
gtaccacgcg 720aaagcgacag aacacctgag caccttgagc gaaaaagcga
aaccggcgct ggaagatcta 780cgccagggct tattgcctgt tcttgagagc
tttaaagtca gttttctgtc agctctggaa 840gaatatacta aaaagctgaa tacccag
867106834DNAArtificial SequenceDNA encoding MSP1E3D1 106atgggtcatc
atcatcatca tcatcacgat tatgatattc ctactactga gaatttgtat 60tttcagggtt
ctaccttcag taaacttcgc gaacaactgg gccccgtgac gcaggaattc
120tgggacaacc tggaaaaaga aaccgaggga ctgcgtcagg aaatgtccaa
agatttagaa 180gaggtgaagg ccaaggttca gccatatctc gatgactttc
agaaaaaatg gcaggaagag 240atggaattat atcgtcaaaa ggtggaaccg
ctgcgtgcgg aactgcaaga gggggcacgc 300caaaaactcc atgagctcca
agagaagctc agcccattag gcgaagaaat gcgcgatcgc 360gcccgtgcac
atgttgatgc actccggact catttggcgc catatctcga tgactttcag
420aaaaaatggc aggaagagat ggaattatat cgtcaaaagg tggaaccgct
gcgtgcggaa 480ctgcaagagg gggcacgcca aaaactccat gagctccaag
agaagctcag cccattaggc 540gaagaaatgc gcgatcgcgc ccgtgcacat
gttgatgcac tccggactca tttggcgccg 600tattcggatg aacttcgcca
gcgtttggcc gcacgtctcg aggcgctgaa agaaaacggg 660ggtgcccgct
tggctgagta ccacgcgaaa gcgacagaac acctgagcac cttgagcgaa
720aaagcgaaac cggcgctgga agatctacgc cagggcttat tgcctgttct
tgagagcttt 780aaagtcagtt ttctgtcagc tctggaagaa tatactaaaa
agctgaatac ccag 8341071275DNAArtificial SequenceDNA encoding
MSP2TEV 107atgggtcatc atcatcatca tcatcacgat tatgatattc ctactactga
gaatttgtat 60tttcagggtc taaagctcct tgacaactgg gacagcgtga cctccacctt
cagcaagctg 120cgcgaacagc tcggccctgt gacccaggag ttctgggata
acctggaaaa ggagacagag 180ggcctgaggc aggagatgag caaggatctg
gaggaggtga aggccaaggt gcagccctac 240ctggacgact tccagaagaa
gtggcaggag gagatggagc tctaccgcca gaaggtggag 300ccgctgcgcg
cagagctcca agagggcgcg cgccagaagc tgcacgagct gcaagagaag
360ctgagcccac tgggcgagga gatgcgcgac cgcgcgcgcg cccatgtgga
cgcgctgcgc 420acgcatctgg ccccctacag cgacgagctg cgccagcgct
tggccgcgcg ccttgaggct 480ctcaaggaga acggcggcgc cagactggcc
gagtaccacg ccaaggccac cgagcatctg 540agcacgctca gcgagaaggc
caagcccgcg ctcgaggacc tccgccaagg cctgctgccc 600gtgctggaga
gcttcaaggt cagcttcctg agcgctctcg aggagtacac taagaagctc
660aacacccagg gtaccctaaa gctccttgac aactgggaca gcgtgacctc
caccttcagc 720aagctgcgcg aacagctcgg ccctgtgacc caggagttct
gggataacct ggaaaaggag 780acagagggcc tgaggcagga gatgagcaag
gatctggagg aggtgaaggc caaggtgcag 840ccctacctgg acgacttcca
gaagaagtgg caggaggaga tggagctcta ccgccagaag 900gtggagccgc
tgcgcgcaga gctccaagag ggcgcgcgcc agaagctgca cgagctgcaa
960gagaagctga gcccactggg cgaggagatg cgcgaccgcg cgcgcgccca
tgtggacgcg 1020ctgcgcacgc atctggcccc ctacagcgac gagctgcgcc
agcgcttggc cgcgcgcctt 1080gaggctctca aggagaacgg cggcgccaga
ctggccgagt accacgccaa ggccaccgag 1140catctgagca cgctcagcga
gaaggccaag cccgcgctcg aggacctccg ccaaggcctg 1200ctgcccgtgc
tggagagctt caaggtcagc ttcctgagcg ctctcgagga gtacactaag
1260aagctcaaca cccag 12751081203DNAArtificial SequenceMSP2N1
108atgggtcatc atcatcatca tcatcacgat tatgatattc ctactactga
gaatttgtat 60tttcagggtt ctaccttcag taaacttcgc gaacaactgg gccccgtgac
gcaggaattc 120tgggacaacc tggaaaaaga aaccgaggga ctgcgtcagg
aaatgtccaa agatttagaa 180gaggtgaagg ccaaggttca gccatatctc
gatgactttc agaaaaaatg gcaggaagag 240atggaattat atcgtcaaaa
ggtggaaccg ctgcgtgcgg aactgcaaga gggggcacgc 300caaaaactcc
atgagctcca agagaagctc agcccattag gcgaagaaat gcgcgatcgc
360gcccgtgcac atgttgatgc actccggact catttggcgc cgtattcgga
tgaacttcgc 420cagcgtttgg ccgcacgtct cgaggcgctg aaagaaaacg
ggggtgcccg cttggctgag 480taccacgcga aagcgacaga acacctgagc
accttgagcg aaaaagcgaa accggcgctg 540gaagatctac gccagggctt
attgcctgtt cttgagagct ttaaagtcag ttttctgtca 600gctctggaag
aatatactaa aaagctgaat acccagggta ccttcagtaa acttcgcgaa
660caactgggcc ccgtgacgca ggaattctgg gacaacctgg aaaaagaaac
cgagggactg 720cgtcaggaaa tgtccaaaga tttagaagag gtgaaggcca
aggttcagcc atatctcgat 780gactttcaga aaaaatggca ggaagagatg
gaattatatc gtcaaaaggt ggaaccgctg 840cgtgcggaac tgcaagaggg
ggcacgccaa aaactccatg agctccaaga gaagctcagc 900ccattaggcg
aagaaatgcg cgatcgcgcc cgtgcacatg ttgatgcact ccggactcat
960ttggcgccgt attcggatga acttcgccag cgtttggccg cacgtctcga
ggcgctgaaa 1020gaaaacgggg gtgcccgctt ggctgagtac cacgcgaaag
cgacagaaca cctgagcacc 1080ttgagcgaaa aagcgaaacc ggcgctggaa
gatctacgcc agggcttatt gcctgttctt 1140gagagcttta aagtcagttt
tctgtcagct ctggaagaat atactaaaaa gctgaatacc 1200cag
12031091176DNAArtificial SequenceDNA encoding MSP2N2 109atgggtcatc
atcatcatca tcatcacgat tatgatattc ctactactga gaatttgtat 60tttcagggtt
ctaccttcag taaacttcgc gaacaactgg gccccgtgac gcaggaattc
120tgggacaacc tggaaaaaga aaccgaggga ctgcgtcagg aaatgtccaa
agatttagaa 180gaggtgaagg ccaaggttca gccatatctc gatgactttc
agaaaaaatg gcaggaagag 240atggaattat atcgtcaaaa ggtggaaccg
ctgcgtgcgg aactgcaaga gggggcacgc 300caaaaactcc atgagctcca
agagaagctc agcccattag gcgaagaaat gcgcgatcgc 360gcccgtgcac
atgttgatgc actccggact catttggcgc cgtattcgga tgaacttcgc
420cagcgtttgg ccgcacgtct cgaggcgctg aaagaaaacg ggggtgcccg
cttggctgag 480taccacgcga aagcgacaga acacctgagc accttgagcg
aaaaagcgaa accggcgctg 540gaagatctac gccagggctt attgcctgtt
cttgagagct ttaaagtcag ttttctgtca 600gctctggaag aatatactaa
aaagctgaat acccagggta cccccgtgac gcaggaattc 660tgggacaacc
tggaaaaaga aaccgaggga ctgcgtcagg aaatgtccaa agatttagaa
720gaggtgaagg ccaaggttca gccatatctc gatgactttc agaaaaaatg
gcaggaagag 780atggaattat atcgtcaaaa ggtggaaccg ctgcgtgcgg
aactgcaaga gggggcacgc 840caaaaactcc atgagctcca agagaagctc
agcccattag gcgaagaaat gcgcgatcgc 900gcccgtgcac atgttgatgc
actccggact catttggcgc cgtattcgga tgaacttcgc 960cagcgtttgg
ccgcacgtct cgaggcgctg aaagaaaacg ggggtgcccg cttggctgag
1020taccacgcga aagcgacaga acacctgagc accttgagcg aaaaagcgaa
accggcgctg 1080gaagatctac gccagggctt attgcctgtt cttgagagct
ttaaagtcag ttttctgtca 1140gctctggaag aatatactaa aaagctgaat acccag
11761101198DNAArtificial SequenceDNA encoding MSP2N3 110atgggtcatc
atcatcatca tcatcacgat tatgatattc ctactactga gaatttgtat 60tttcagggtt
ctaccttcag taaacttcgc gaacaactgg gccccgtgac gcaggaattc
120tgggacaacc tggaaaaaga aaccgaggga ctgcgtcagg aaatgtccaa
agatttagaa 180gaggtgaagg ccaaggttca gccatatctc gatgactttc
agaaaaaatg gcaggaagag 240atggaattat atcgtcaaaa ggtggaaccg
ctgcgtgcgg aactgcaaga gggggcacgc 300caaaaactcc atgagctcca
agagaagctc agcccattag gcgaagaaat gcgcgatcgc 360gcccgtgcac
atgttgatgc actccggact catttggcgc cgtattcgga tgaacttcgc
420cagcgtttgg ccgcacgtct cgaggcgctg aaagaaaacg ggggtgcccg
cttggctgag 480taccacgcga aagcgacaga acacctgagc accttgagcg
aaaaagcgaa accggcgctg 540gaagatctac gccagggctt attgcctgtt
cttgagagct ttaaagtcag ttttctgtca 600gctctggaag aatatactaa
aaagctgaat acccagggta cccgcgaaca actgggcccc 660gtgacgcagg
aattctggga caacctggaa aaagaaaccg agggactgcg tcaggaaatg
720tccaaagatt tagaagaggt gaaggccaag gttcagccat atctcgatga
ctttcagaaa 780aaatggcagg aagagatgga attatatcgt caaaaggtgg
aaccgctgcg tgcggaactg 840caagaggggg cacgccaaaa actccatgag
ctccaagaga agctcagccc attaggcgaa 900gaaatgcgcg atcgcgcccg
tgcacatgtt gatgcactcc ggactcattt ggcgccgtat 960tcggatgaac
ttcgccagcg tttggccgca cgtctcgagg cgctgaaaga aaacgggggt
1020gcccgcttgg ctgagtacca cgcgaaagcg acagaacacc tgagcacctt
gagcgaaaaa 1080gcgaaaccgg cgctggaaga tctacgccag ggcttattgc
ctgttcttga gagctttaaa 1140gtcagttttc tgtcagctct ggaagaatat
actaaaaagc tgaataccca gtaagctt 1198111397PRTArtificial
SequenceMSP2N3 111Met Gly His His His His His His His Asp Tyr Asp
Ile Pro Thr Thr1 5 10 15Glu Asn Leu Tyr Phe Gln Gly Ser Thr Phe Ser
Lys Leu Arg Glu Gln20 25 30Leu Gly Pro Val Thr Gln Glu Phe Trp Asp
Asn Leu Glu Lys Glu Thr35 40 45Glu Gly Leu Arg Gln Glu Met Ser Lys
Asp Leu Glu Glu Val Lys Ala50 55 60Lys Val Gln Pro Tyr Leu Asp Asp
Phe Gln Lys Lys Trp Gln Glu Glu65 70 75 80Met Glu Leu Tyr Arg Gln
Lys Val Glu Pro Leu Arg Ala Glu Leu Gln85 90 95Glu Gly Ala Arg Gln
Lys Leu His Glu Leu Gln Glu Lys Leu Ser Pro100 105 110Leu Gly Glu
Glu Met Arg Asp Arg Ala Arg Ala His Val Asp Ala Leu115 120 125Arg
Thr His Leu Ala Pro Tyr Ser Asp Glu Leu Arg Gln Arg Leu Ala130 135
140Ala Arg Leu Glu Ala Leu Lys Glu Asn Gly Gly Ala Arg Leu Ala
Glu145 150 155 160Tyr His Ala Lys Ala Thr Glu His Leu Ser Thr Leu
Ser Glu Lys Ala165 170 175Lys Pro Ala Leu Glu Asp Leu Arg Gln Gly
Leu Leu Pro Val Leu Glu180 185 190Ser Phe Lys Val Ser Phe Leu Ser
Ala Leu Glu Glu Tyr Thr Lys Lys195 200 205Leu Asn Thr Gln Gly Thr
Arg Glu Gln Leu Gly Pro Val Thr Gln Glu210 215 220Phe Trp Asp Asn
Leu Glu Lys Glu Thr Glu Gly Leu Arg Gln Glu Met225 230 235 240Ser
Lys Asp Leu Glu Glu Val Lys Ala Lys Val Gln Pro Tyr Leu Asp245 250
255Asp Phe Gln Lys Lys Trp Gln Glu Glu Met Glu Leu Tyr Arg Gln
Lys260 265 270Val Glu Pro Leu Arg Ala Glu Leu Gln Glu Gly Ala Arg
Gln Lys Leu275 280 285His Glu Leu Gln Glu Lys Leu Ser Pro Leu Gly
Glu Glu Met Arg Asp290 295 300Arg Ala Arg Ala His Val Asp Ala Leu
Arg Thr
His Leu Ala Pro Tyr305 310 315 320Ser Asp Glu Leu Arg Gln Arg Leu
Ala Ala Arg Leu Glu Ala Leu Lys325 330 335Glu Asn Gly Gly Ala Arg
Leu Ala Glu Tyr His Ala Lys Ala Thr Glu340 345 350His Leu Ser Thr
Leu Ser Glu Lys Ala Lys Pro Ala Leu Glu Asp Leu355 360 365Arg Gln
Gly Leu Leu Pro Val Leu Glu Ser Phe Lys Val Ser Phe Leu370 375
380Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln385 390
3951121149DNAArtificial SequenceDNA encoding MSP2N4 112atgggtcatc
atcatcatca tcatcacgat tatgatattc ctactactga gaatttgtat 60tttcagggtt
ccgtgacgca ggaattctgg gacaacctgg aaaaagaaac cgagggactg
120cgtcaggaaa tgtccaaaga tttagaagag gtgaaggcca aggttcagcc
atatctcgat 180gactttcaga aaaaatggca ggaagagatg gaattatatc
gtcaaaaggt ggaaccgctg 240cgtgcggaac tgcaagaggg ggcacgccaa
aaactccatg agctccaaga gaagctcagc 300ccattaggcg aagaaatgcg
cgatcgcgcc cgtgcacatg ttgatgcact ccggactcat 360ttggcgccgt
attcggatga acttcgccag cgtttggccg cacgtctcga ggcgctgaaa
420gaaaacgggg gtgcccgctt ggctgagtac cacgcgaaag cgacagaaca
cctgagcacc 480ttgagcgaaa aagcgaaacc ggcgctggaa gatctacgcc
agggcttatt gcctgttctt 540gagagcttta aagtcagttt tctgtcagct
ctggaagaat atactaaaaa gctgaatacc 600cagaatccag gtacccccgt
gacgcaggaa ttctgggaca acctggaaaa agaaaccgag 660ggactgcgtc
aggaaatgtc caaagattta gaagaggtga aggccaaggt tcagccatat
720ctcgatgact ttcagaaaaa atggcaggaa gagatggaat tatatcgtca
aaaggtggaa 780ccgctgcgtg cggaactgca agagggggca cgccaaaaac
tccatgagct ccaagagaag 840ctcagcccat taggcgaaga aatgcgcgat
cgcgcccgtg cacatgttga tgcactccgg 900actcatttgg cgccgtattc
ggatgaactt cgccagcgtt tggccgcacg tctcgaggcg 960ctgaaagaaa
acgggggtgc ccgcttggct gagtaccacg cgaaagcgac agaacacctg
1020agcaccttga gcgaaaaagc gaaaccggcg ctggaagatc tacgccaggg
cttattgcct 1080gttcttgaga gctttaaagt cagttttctg tcagctctgg
aagaatatac taaaaagctg 1140aatacccag 1149113383PRTArtificial
SequenceMSP2N4 113Met Gly His His His His His His His Asp Tyr Asp
Ile Pro Thr Thr1 5 10 15Glu Asn Leu Tyr Phe Gln Gly Ser Val Thr Gln
Glu Phe Trp Asp Asn20 25 30Leu Glu Lys Glu Thr Glu Gly Leu Arg Gln
Glu Met Ser Lys Asp Leu35 40 45Glu Glu Val Lys Ala Lys Val Gln Pro
Tyr Leu Asp Asp Phe Gln Lys50 55 60Lys Trp Gln Glu Glu Met Glu Leu
Tyr Arg Gln Lys Val Glu Pro Leu65 70 75 80Arg Ala Glu Leu Gln Glu
Gly Ala Arg Gln Lys Leu His Glu Leu Gln85 90 95Glu Lys Leu Ser Pro
Leu Gly Glu Glu Met Arg Asp Arg Ala Arg Ala100 105 110His Val Asp
Ala Leu Arg Thr His Leu Ala Pro Tyr Ser Asp Glu Leu115 120 125Arg
Gln Arg Leu Ala Ala Arg Leu Glu Ala Leu Lys Glu Asn Gly Gly130 135
140Ala Arg Leu Ala Glu Tyr His Ala Lys Ala Thr Glu His Leu Ser
Thr145 150 155 160Leu Ser Glu Lys Ala Lys Pro Ala Leu Glu Asp Leu
Arg Gln Gly Leu165 170 175Leu Pro Val Leu Glu Ser Phe Lys Val Ser
Phe Leu Ser Ala Leu Glu180 185 190Glu Tyr Thr Lys Lys Leu Asn Thr
Gln Asn Pro Gly Thr Pro Val Thr195 200 205Gln Glu Phe Trp Asp Asn
Leu Glu Lys Glu Thr Glu Gly Leu Arg Gln210 215 220Glu Met Ser Lys
Asp Leu Glu Glu Val Lys Ala Lys Val Gln Pro Tyr225 230 235 240Leu
Asp Asp Phe Gln Lys Lys Trp Gln Glu Glu Met Glu Leu Tyr Arg245 250
255Gln Lys Val Glu Pro Leu Arg Ala Glu Leu Gln Glu Gly Ala Arg
Gln260 265 270Lys Leu His Glu Leu Gln Glu Lys Leu Ser Pro Leu Gly
Glu Glu Met275 280 285Arg Asp Arg Ala Arg Ala His Val Asp Ala Leu
Arg Thr His Leu Ala290 295 300Pro Tyr Ser Asp Glu Leu Arg Gln Arg
Leu Ala Ala Arg Leu Glu Ala305 310 315 320Leu Lys Glu Asn Gly Gly
Ala Arg Leu Ala Glu Tyr His Ala Lys Ala325 330 335Thr Glu His Leu
Ser Thr Leu Ser Glu Lys Ala Lys Pro Ala Leu Glu340 345 350Asp Leu
Arg Gln Gly Leu Leu Pro Val Leu Glu Ser Phe Lys Val Ser355 360
365Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln370
375 3801141137DNAArtificial SequenceDNA encoding MSP2N5
114atgggtcatc atcatcatca tcatcacgat tatgatattc ctactactga
gaatttgtat 60tttcagggtt ccgtgacgca ggaattctgg gacaacctgg aaaaagaaac
cgagggactg 120cgtcaggaaa tgtccaaaga tttagaagag gtgaaggcca
aggttcagcc atatctcgat 180gactttcaga aaaaatggca ggaagagatg
gaattatatc gtcaaaaggt ggaaccatat 240ctcgatgact ttcagaaaaa
atggcaggaa gagatggaat tatatcgtca aaaggtggaa 300ccgctgcgtg
cggaactgca agagggggca cgccaaaaac tccatgagct ccaagagaag
360ctcagcccat taggcgaaga aatgcgcgat cgcgcccgtg cacatgttga
tgcactccgg 420actcatttgg cgccgtattc ggatgaactt cgccagcgtt
tggccgcacg tctcgaggcg 480ctgaaagaaa acgggggtgc ccgcttggct
gagtaccacg cgaaagcgac agaacacctg 540agcaccttga gcgaaaaagc
gaaaccggcg ctggaagatc tacgccaggg cttattgaat 600ccaggtacca
aagatttaga agaggtgaag gccaaggttc agccatatct cgatgacttt
660cagaaaaaat ggcaggaaga gatggaatta tatcgtcaaa aggtggaacc
atatctcgat 720gactttcaga aaaaatggca ggaagagatg gaattatatc
gtcaaaaggt ggaaccgctg 780cgtgcggaac tgcaagaggg ggcacgccaa
aaactccatg agctccaaga gaagctcagc 840ccattaggcg aagaaatgcg
cgatcgcgcc cgtgcacatg ttgatgcact ccggactcat 900ttggcgccgt
attcggatga acttcgccag cgtttggccg cacgtctcga ggcgctgaaa
960gaaaacgggg gtgcccgctt ggctgagtac cacgcgaaag cgacagaaca
cctgagcacc 1020ttgagcgaaa aagcgaaacc ggcgctggaa gatctacgcc
agggcttatt gcccgtgacg 1080caggaattct gggacaacct ggaaaaagaa
accgagggac tgcgtcagga aatgtcc 1137115379PRTArtificial
SequenceMSP2n5 115Met Gly His His His His His His His Asp Tyr Asp
Ile Pro Thr Thr1 5 10 15Glu Asn Leu Tyr Phe Gln Gly Ser Val Thr Gln
Glu Phe Trp Asp Asn20 25 30Leu Glu Lys Glu Thr Glu Gly Leu Arg Gln
Glu Met Ser Lys Asp Leu35 40 45Glu Glu Val Lys Ala Lys Val Gln Pro
Tyr Leu Asp Asp Phe Gln Lys50 55 60Lys Trp Gln Glu Glu Met Glu Leu
Tyr Arg Gln Lys Val Glu Pro Tyr65 70 75 80Leu Asp Asp Phe Gln Lys
Lys Trp Gln Glu Glu Met Glu Leu Tyr Arg85 90 95Gln Lys Val Glu Pro
Leu Arg Ala Glu Leu Gln Glu Gly Ala Arg Gln100 105 110Lys Leu His
Glu Leu Gln Glu Lys Leu Ser Pro Leu Gly Glu Glu Met115 120 125Arg
Asp Arg Ala Arg Ala His Val Asp Ala Leu Arg Thr His Leu Ala130 135
140Pro Tyr Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu
Ala145 150 155 160Leu Lys Glu Asn Gly Gly Ala Arg Leu Ala Glu Tyr
His Ala Lys Ala165 170 175Thr Glu His Leu Ser Thr Leu Ser Glu Lys
Ala Lys Pro Ala Leu Glu180 185 190Asp Leu Arg Gln Gly Leu Leu Asn
Pro Gly Thr Lys Asp Leu Glu Glu195 200 205Val Lys Ala Lys Val Gln
Pro Tyr Leu Asp Asp Phe Gln Lys Lys Trp210 215 220Gln Glu Glu Met
Glu Leu Tyr Arg Gln Lys Val Glu Pro Tyr Leu Asp225 230 235 240Asp
Phe Gln Lys Lys Trp Gln Glu Glu Met Glu Leu Tyr Arg Gln Lys245 250
255Val Glu Pro Leu Arg Ala Glu Leu Gln Glu Gly Ala Arg Gln Lys
Leu260 265 270His Glu Leu Gln Glu Lys Leu Ser Pro Leu Gly Glu Glu
Met Arg Asp275 280 285Arg Ala Arg Ala His Val Asp Ala Leu Arg Thr
His Leu Ala Pro Tyr290 295 300Ser Asp Glu Leu Arg Gln Arg Leu Ala
Ala Arg Leu Glu Ala Leu Lys305 310 315 320Glu Asn Gly Gly Ala Arg
Leu Ala Glu Tyr His Ala Lys Ala Thr Glu325 330 335His Leu Ser Thr
Leu Ser Glu Lys Ala Lys Pro Ala Leu Glu Asp Leu340 345 350Arg Gln
Gly Leu Leu Pro Val Thr Gln Glu Phe Trp Asp Asn Leu Glu355 360
365Lys Glu Thr Glu Gly Leu Arg Gln Glu Met Ser370
3751161143DNAArtificial SequenceDNA encoding MSP2N6 116atgggtcatc
atcatcatca tcatcacgat tatgatattc ctactactga gaatttgtat 60tttcagggtt
ccgtgacgca ggaattctgg gacaacctgg aaaaagaaac cgagggactg
120cgtcaggaaa tgtccaaaga tttagaagag gtgaaggcca aggttcagcc
atatctcgat 180gactttcaga aaaaatggca ggaagagatg gaattatatc
gtcaaaaggt ggaaccatat 240ctcgatgact ttcagaaaaa atggcaggaa
gagatggaat tatatcgtca aaaggtggaa 300ccgctgcgtg cggaactgca
agagggggca cgccaaaaac tccatgagct ccaagagaag 360ctcagcccat
taggcgaaga aatgcgcgat cgcgcccgtg cacatgttga tgcactccgg
420actcatttgg cgccgtattc ggatgaactt cgccagcgtt tggccgcacg
tctcgaggcg 480ctgaaagaaa acgggggtgc ccgcttggct gagtaccacg
cgaaagcgac agaacacctg 540agcaccttga gcgaaaaagc gaaaccggcg
ctggaagatc tacgccaggg cttattgtcc 600aatccaggta cccaaaaaga
tttagaagag gtgaaggcca aggttcagcc atatctcgat 660gactttcaga
aaaaatggca ggaagagatg gaattatatc gtcaaaaggt ggaaccatat
720ctcgatgact ttcagaaaaa atggcaggaa gagatggaat tatatcgtca
aaaggtggaa 780ccgctgcgtg cggaactgca agagggggca cgccaaaaac
tccatgagct ccaagagaag 840ctcagcccat taggcgaaga aatgcgcgat
cgcgcccgtg cacatgttga tgcactccgg 900actcatttgg cgccgtattc
ggatgaactt cgccagcgtt tggccgcacg tctcgaggcg 960ctgaaagaaa
acgggggtgc ccgcttggct gagtaccacg cgaaagcgac agaacacctg
1020agcaccttga gcgaaaaagc gaaaccggcg ctggaagatc tacgccaggg
cttattgccc 1080gtgacgcagg aattctggga caacctggaa aaagaaaccg
agggactgcg tcaggaaatg 1140tcc 1143117381PRTArtificial
SequenceMSP2N6 117Met Gly His His His His His His His Asp Tyr Asp
Ile Pro Thr Thr1 5 10 15Glu Asn Leu Tyr Phe Gln Gly Ser Val Thr Gln
Glu Phe Trp Asp Asn20 25 30Leu Glu Lys Glu Thr Glu Gly Leu Arg Gln
Glu Met Ser Lys Asp Leu35 40 45Glu Glu Val Lys Ala Lys Val Gln Pro
Tyr Leu Asp Asp Phe Gln Lys50 55 60Lys Trp Gln Glu Glu Met Glu Leu
Tyr Arg Gln Lys Val Glu Pro Tyr65 70 75 80Leu Asp Asp Phe Gln Lys
Lys Trp Gln Glu Glu Met Glu Leu Tyr Arg85 90 95Gln Lys Val Glu Pro
Leu Arg Ala Glu Leu Gln Glu Gly Ala Arg Gln100 105 110Lys Leu His
Glu Leu Gln Glu Lys Leu Ser Pro Leu Gly Glu Glu Met115 120 125Arg
Asp Arg Ala Arg Ala His Val Asp Ala Leu Arg Thr His Leu Ala130 135
140Pro Tyr Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu
Ala145 150 155 160Leu Lys Glu Asn Gly Gly Ala Arg Leu Ala Glu Tyr
His Ala Lys Ala165 170 175Thr Glu His Leu Ser Thr Leu Ser Glu Lys
Ala Lys Pro Ala Leu Glu180 185 190Asp Leu Arg Gln Gly Leu Leu Ser
Asn Pro Gly Thr Gln Lys Asp Leu195 200 205Glu Glu Val Lys Ala Lys
Val Gln Pro Tyr Leu Asp Asp Phe Gln Lys210 215 220Lys Trp Gln Glu
Glu Met Glu Leu Tyr Arg Gln Lys Val Glu Pro Tyr225 230 235 240Leu
Asp Asp Phe Gln Lys Lys Trp Gln Glu Glu Met Glu Leu Tyr Arg245 250
255Gln Lys Val Glu Pro Leu Arg Ala Glu Leu Gln Glu Gly Ala Arg
Gln260 265 270Lys Leu His Glu Leu Gln Glu Lys Leu Ser Pro Leu Gly
Glu Glu Met275 280 285Arg Asp Arg Ala Arg Ala His Val Asp Ala Leu
Arg Thr His Leu Ala290 295 300Pro Tyr Ser Asp Glu Leu Arg Gln Arg
Leu Ala Ala Arg Leu Glu Ala305 310 315 320Leu Lys Glu Asn Gly Gly
Ala Arg Leu Ala Glu Tyr His Ala Lys Ala325 330 335Thr Glu His Leu
Ser Thr Leu Ser Glu Lys Ala Lys Pro Ala Leu Glu340 345 350Asp Leu
Arg Gln Gly Leu Leu Pro Val Thr Gln Glu Phe Trp Asp Asn355 360
365Leu Glu Lys Glu Thr Glu Gly Leu Arg Gln Glu Met Ser370 375
3801183282DNAArtificial SequenceDNA encoding MSP2CPR 118atgggtcatc
atcatcatca tcacattgag ggacgtctga agctgttgga caattgggac 60tctgttacgt
ctaccttcag taaacttcgc gaacaactgg gccccgtgac gcaggaattc
120tgggacaacc tggaaaaaga aaccgaggga ctgcgtcagg aaatgtccaa
agatttagaa 180gaggtgaagg ccaaggttca gccatatctc gatgactttc
agaaaaaatg gcaggaagag 240atggaattat atcgtcaaaa ggtggaaccg
ctgcgtgcgg aactgcaaga gggggcacgc 300caaaaactcc atgagctcca
agagaagctc agcccattag gcgaagaaat gcgcgatcgc 360gcccgtgcac
atgttgatgc actccggact catttggcgc cgtattcgga tgaacttcgc
420cagcgtttgg ccgcacgtct cgaggcgctg aaagaaaacg ggggtgcccg
cttggctgag 480taccacgcga aagcgacaga acacctgagc accttgagcg
aaaaagcgaa accggcgctg 540gaagatctac gccagggctt attgcctgtt
cttgagagct ttaaagtcag ttttctgtca 600gctctggaag aatatactaa
aaagctgaat acccagggta ccctgaagct gttggacaat 660tgggactctg
ttacgtctac cttcagtaaa cttcgcgaac aactgggccc cgtgacgcag
720gaattctggg acaacctgga aaaagaaacc gagggactgc gtcaggaaat
gtccaaagat 780ttagaagagg tgaaggccaa ggttcagcca tatctcgatg
actttcagaa aaaatggcag 840gaagagatgg aattatatcg tcaaaaggtg
gaaccgctgc gtgcggaact gcaagagggg 900gcacgccaaa aactccatga
gctccaagag aagctcagcc cattaggcga agaaatgcgc 960gatcgcgccc
gtgcacatgt tgatgcactc cggactcatt tggcgccgta ttcggatgaa
1020cttcgccagc gtttggccgc acgtctcgag gcgctgaaag aaaacggggg
tgcccgcttg 1080gctgagtacc acgcgaaagc gacagaacac ctgagcacct
tgagcgaaaa agcgaaaccg 1140gcgctggaag atctacgcca gggcttattg
cctgttcttg agagctttaa agtcagtttt 1200ctgtcagctc tggaagaata
tactaaaaag ctgaataccc agtcgaccat gggagactct 1260cacgaagaca
ccagtgccac catgcctgag gccgtggctg aagaagtgtc tctattcagc
1320acgacggaca tggttctgtt ttctctcatc gtgggggtcc tgacctactg
gttcatcttt 1380agaaagaaga aagaagagat accggagttc agcaagatcc
aaacaacggc cccacccgtc 1440aaagagagca gcttcgtgga aaagatgaag
aaaacgggaa ggaacattat cgtattctat 1500ggctcccaga cgggaaccgc
tgaggagttt gccaaccggc tgtccaagga tgcccaccgc 1560tacgggatgc
ggggcatgtc cgcagaccct gaagagtatg acttggccga cctgagcagc
1620ctgcctgaga tcgacaagtc cctggtagtc ttctgcatgg ccacatacgg
agagggcgac 1680cccacggaca atgcgcagga cttctatgac tggctgcagg
agactgacgt ggacctcact 1740ggggtcaagt ttgctgtatt tggtcttggg
aacaagacct atgagcactt caatgccatg 1800ggcaagtatg tggaccagcg
gctggagcag cttggcgccc agcgcatctt tgagttgggc 1860cttggtgatg
atgacgggaa cttggaagag gatttcatca cgtggaggga gcagttctgg
1920ccagctgtgt gcgagttctt tggggtagaa gccactgggg aggagtcgag
cattcgccag 1980tatgagctcg tggtccacga agacatggac gtagccaagg
tgtacacggg tgagatgggc 2040cgtctgaaga gctacgagaa ccagaaaccc
cccttcgatg ctaagaatcc attcctggct 2100gctgtcaccg ccaaccggaa
gctgaaccaa ggcactgagc ggcatctaat gcacctggag 2160ttggacatct
cagactccaa gatcaggtat gaatctggag atcacgtggc tgtgtaccca
2220gccaatgact cagccctggt caaccagatt ggggagatcc tgggagctga
cctggatgtc 2280atcatgtctc taaacaatct cgatgaggag tcaaacaaga
agcatccgtt cccctgcccc 2340accacctacc gcacggccct cacctactac
ctggacatca ctaacccgcc acgcaccaat 2400gtgctctacg aactggcaca
gtacgcctca gagccctcgg agcaggagca cctgcacaag 2460atggcgtcat
cctcaggcga gggcaaggag ctgtacctga gctgggtggt ggaagcccgg
2520aggcacatcc tagccatcct ccaagactac ccatcactgc ggccacccat
cgaccacctg 2580tgtgagctgc tgccacgcct gcaggcccga tactactcca
ttgcctcatc ctccaaggtc 2640caccccaact ccgtgcacat ctgtgccgtg
gccgtggagt acgaagcgaa gtctggccga 2700gtgaacaagg gggtggccac
tagctggctt cgggccaagg aaccagcagg cgagaatggc 2760ggccgcgccc
tggtacccat gttcgtgcgc aaatctcagt tccgcttgcc tttcaagtcc
2820accacacctg tcatcatggt gggccccggc actgggattg cccctttcat
gggcttcatc 2880caggaacgag cttggcttcg agagcaaggc aaggaggtgg
gagagacgct gctatactat 2940ggctgccggc gctcggatga ggactatctg
taccgtgaag agctagcccg cttccacaag 3000gacggtgccc tcacgcagct
taatgtggcc ttttcccggg agcaggccca caaggtctat 3060gtccagcacc
ttctgaagag agacagggaa cacctgtgga agctgatcca cgagggcggt
3120gcccacatct atgtgtgcgg ggatgctcga aatatggcca aagatgtgca
aaacacattc 3180tatgacattg tggctgagtt cgggcccatg gagcacaccc
aggctgtgga ctatgttaag 3240aagctgatga ccaagggccg ctactcacta
gatgtgtgga gc 32821191094PRTArtificial SequenceMSP2CPR 119Met Gly
His His His His His His Ile Glu Gly Arg Leu Lys Leu Leu1 5 10 15Asp
Asn Trp Asp Ser Val Thr Ser Thr Phe Ser Lys Leu Arg Glu Gln20 25
30Leu Gly Pro Val Thr Gln Glu Phe Trp Asp Asn Leu Glu Lys Glu Thr35
40 45Glu Gly Leu Arg Gln Glu Met Ser Lys Asp Leu Glu Glu Val Lys
Ala50 55 60Lys Val Gln Pro Tyr Leu Asp Asp Phe Gln Lys Lys Trp Gln
Glu Glu65 70 75 80Met Glu Leu Tyr Arg Gln Lys Val Glu Pro Leu Arg
Ala Glu Leu Gln85 90 95Glu Gly Ala Arg Gln Lys Leu His Glu Leu Gln
Glu Lys Leu Ser Pro100 105 110Leu Gly Glu Glu Met Arg Asp Arg Ala
Arg Ala His Val Asp Ala Leu115 120 125Arg Thr His Leu Ala Pro Tyr
Ser Asp Glu Leu Arg Gln Arg Leu Ala130 135 140Ala Arg Leu Glu Ala
Leu Lys Glu Asn Gly Gly Ala Arg Leu Ala Glu145 150 155 160Tyr
His
Ala Lys Ala Thr Glu His Leu Ser Thr Leu Ser Glu Lys Ala165 170
175Lys Pro Ala Leu Glu Asp Leu Arg Gln Gly Leu Leu Pro Val Leu
Glu180 185 190Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr
Thr Lys Lys195 200 205Leu Asn Thr Gln Gly Thr Leu Lys Leu Leu Asp
Asn Trp Asp Ser Val210 215 220Thr Ser Thr Phe Ser Lys Leu Arg Glu
Gln Leu Gly Pro Val Thr Gln225 230 235 240Glu Phe Trp Asp Asn Leu
Glu Lys Glu Thr Glu Gly Leu Arg Gln Glu245 250 255Met Ser Lys Asp
Leu Glu Glu Val Lys Ala Lys Val Gln Pro Tyr Leu260 265 270Asp Asp
Phe Gln Lys Lys Trp Gln Glu Glu Met Glu Leu Tyr Arg Gln275 280
285Lys Val Glu Pro Leu Arg Ala Glu Leu Gln Glu Gly Ala Arg Gln
Lys290 295 300Leu His Glu Leu Gln Glu Lys Leu Ser Pro Leu Gly Glu
Glu Met Arg305 310 315 320Asp Arg Ala Arg Ala His Val Asp Ala Leu
Arg Thr His Leu Ala Pro325 330 335Tyr Ser Asp Glu Leu Arg Gln Arg
Leu Ala Ala Arg Leu Glu Ala Leu340 345 350Lys Glu Asn Gly Gly Ala
Arg Leu Ala Glu Tyr His Ala Lys Ala Thr355 360 365Glu His Leu Ser
Thr Leu Ser Glu Lys Ala Lys Pro Ala Leu Glu Asp370 375 380Leu Arg
Gln Gly Leu Leu Pro Val Leu Glu Ser Phe Lys Val Ser Phe385 390 395
400Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln Ser
Thr405 410 415Met Gly Asp Ser His Glu Asp Thr Ser Ala Thr Met Pro
Glu Ala Val420 425 430Ala Glu Glu Val Ser Leu Phe Ser Thr Thr Asp
Met Val Leu Phe Ser435 440 445Leu Ile Val Gly Val Leu Thr Tyr Trp
Phe Ile Phe Arg Lys Lys Lys450 455 460Glu Glu Ile Pro Glu Phe Ser
Lys Ile Gln Thr Thr Ala Pro Pro Val465 470 475 480Lys Glu Ser Ser
Phe Val Glu Lys Met Lys Lys Thr Gly Arg Asn Ile485 490 495Ile Val
Phe Tyr Gly Ser Gln Thr Gly Thr Ala Glu Glu Phe Ala Asn500 505
510Arg Leu Ser Lys Asp Ala His Arg Tyr Gly Met Arg Gly Met Ser
Ala515 520 525Asp Pro Glu Glu Tyr Asp Leu Ala Asp Leu Ser Ser Leu
Pro Glu Ile530 535 540Asp Lys Ser Leu Val Val Phe Cys Met Ala Thr
Tyr Gly Glu Gly Asp545 550 555 560Pro Thr Asp Asn Ala Gln Asp Phe
Tyr Asp Trp Leu Gln Glu Thr Asp565 570 575Val Asp Leu Thr Gly Val
Lys Phe Ala Val Phe Gly Leu Gly Asn Lys580 585 590Thr Tyr Glu His
Phe Asn Ala Met Gly Lys Tyr Val Asp Gln Arg Leu595 600 605Glu Gln
Leu Gly Ala Gln Arg Ile Phe Glu Leu Gly Leu Gly Asp Asp610 615
620Asp Gly Asn Leu Glu Glu Asp Phe Ile Thr Trp Arg Glu Gln Phe
Trp625 630 635 640Pro Ala Val Cys Glu Phe Phe Gly Val Glu Ala Thr
Gly Glu Glu Ser645 650 655Ser Ile Arg Gln Tyr Glu Leu Val Val His
Glu Asp Met Asp Val Ala660 665 670Lys Val Tyr Thr Gly Glu Met Gly
Arg Leu Lys Ser Tyr Glu Asn Gln675 680 685Lys Pro Pro Phe Asp Ala
Lys Asn Pro Phe Leu Ala Ala Val Thr Ala690 695 700Asn Arg Lys Leu
Asn Gln Gly Thr Glu Arg His Leu Met His Leu Glu705 710 715 720Leu
Asp Ile Ser Asp Ser Lys Ile Arg Tyr Glu Ser Gly Asp His Val725 730
735Ala Val Tyr Pro Ala Asn Asp Ser Ala Leu Val Asn Gln Ile Gly
Glu740 745 750Ile Leu Gly Ala Asp Leu Asp Val Ile Met Ser Leu Asn
Asn Leu Asp755 760 765Glu Glu Ser Asn Lys Lys His Pro Phe Pro Cys
Pro Thr Thr Tyr Arg770 775 780Thr Ala Leu Thr Tyr Tyr Leu Asp Ile
Thr Asn Pro Pro Arg Thr Asn785 790 795 800Val Leu Tyr Glu Leu Ala
Gln Tyr Ala Ser Glu Pro Ser Glu Gln Glu805 810 815His Leu His Lys
Met Ala Ser Ser Ser Gly Glu Gly Lys Glu Leu Tyr820 825 830Leu Ser
Trp Val Val Glu Ala Arg Arg His Ile Leu Ala Ile Leu Gln835 840
845Asp Tyr Pro Ser Leu Arg Pro Pro Ile Asp His Leu Cys Glu Leu
Leu850 855 860Pro Arg Leu Gln Ala Arg Tyr Tyr Ser Ile Ala Ser Ser
Ser Lys Val865 870 875 880His Pro Asn Ser Val His Ile Cys Ala Val
Ala Val Glu Tyr Glu Ala885 890 895Lys Ser Gly Arg Val Asn Lys Gly
Val Ala Thr Ser Trp Leu Arg Ala900 905 910Lys Glu Pro Ala Gly Glu
Asn Gly Gly Arg Ala Leu Val Pro Met Phe915 920 925Val Arg Lys Ser
Gln Phe Arg Leu Pro Phe Lys Ser Thr Thr Pro Val930 935 940Ile Met
Val Gly Pro Gly Thr Gly Ile Ala Pro Phe Met Gly Phe Ile945 950 955
960Gln Glu Arg Ala Trp Leu Arg Glu Gln Gly Lys Glu Val Gly Glu
Thr965 970 975Leu Leu Tyr Tyr Gly Cys Arg Arg Ser Asp Glu Asp Tyr
Leu Tyr Arg980 985 990Glu Glu Leu Ala Arg Phe His Lys Asp Gly Ala
Leu Thr Gln Leu Asn995 1000 1005Val Ala Phe Ser Arg Glu Gln Ala His
Lys Val Tyr Val Gln His1010 1015 1020Leu Leu Lys Arg Asp Arg Glu
His Leu Trp Lys Leu Ile His Glu1025 1030 1035Gly Gly Ala His Ile
Tyr Val Cys Gly Asp Ala Arg Asn Met Ala1040 1045 1050Lys Asp Val
Gln Asn Thr Phe Tyr Asp Ile Val Ala Glu Phe Gly1055 1060 1065Pro
Met Glu His Thr Gln Ala Val Asp Tyr Val Lys Lys Leu Met1070 1075
1080Thr Lys Gly Arg Tyr Ser Leu Asp Val Trp Ser1085
109012072DNAArtificial SequenceDNA encoding His-TEV2 peptide
120atgggtcatc atcatcatca tcatcacgat tatgatattc ctactactga
gaatttgtat 60tttcagggat cc 7212124PRTArtificial SequenceHis-TEV2
peptide sequence 121Met Gly His His His His His His His Asp Tyr Asp
Ile Pro Thr Thr1 5 10 15Glu Asn Leu Tyr Phe Gln Gly
Ser20122714DNAArtificial SequenceDNA encoding EGFP 122gtgagcaagg
gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc 60gacgtaaacg
gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
120aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg
gcccaccctc 180gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct
accccgacca catgaagcag 240cacgacttct tcaagtccgc catgcccgaa
ggctacgtcc aggagcgcac catcttcttc 300aaggacgacg gcaactacaa
gacccgcgcc gaggtgaagt tcgagggcga caccctggtg 360aaccgcatcg
agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
420ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca
gaagaacggc 480atcaaggtga acttcaagat ccgccacaac atcgaggacg
gcagcgtgca gctcgccgac 540cactaccagc agaacacccc catcggcgac
ggccccgtgc tgctgcccga caaccactac 600ctgagcaccc agtccgccct
gagcaaagac cccaacgaga agcgcgatca catggtcctg 660ctggagttcg
tgaccgccgc cgggatcact ctcggcatgg acgagctgta caag
714123238PRTArtificial SequenceEGFP sequence 123Val Ser Lys Gly Glu
Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val1 5 10 15Glu Leu Asp Gly
Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu20 25 30Gly Glu Gly
Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys35 40 45Thr Thr
Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu50 55 60Thr
Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln65 70 75
80His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg85
90 95Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
Val100 105 110Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu
Lys Gly Ile115 120 125Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His
Lys Leu Glu Tyr Asn130 135 140Tyr Asn Ser His Asn Val Tyr Ile Met
Ala Asp Lys Gln Lys Asn Gly145 150 155 160Ile Lys Val Asn Phe Lys
Ile Arg His Asn Ile Glu Asp Gly Ser Val165 170 175Gln Leu Ala Asp
His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro180 185 190Val Leu
Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser195 200
205Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe
Val210 215 220Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr
Lys225 230 235124714DNAArtificial SequenceDNA encoding EYFP
124gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga
gctggacggc 60gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc
cacctacggc 120aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
ccgtgccctg gcccaccctc 180gtgaccacct tcggctacgg cctgcagtgc
ttcgcccgct accccgacca catgaagcag 240cacgacttct tcaagtccgc
catgcccgaa ggctacgtcc aggagcgcac catcttcttc 300aaggacgacg
gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
360aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct
ggggcacaag 420ctggagtaca actacaacag ccacaacgtc tatatcatgg
ccgacaagca gaagaacggc 480atcaaggtga acttcaagat ccgccacaac
atcgaggacg gcagcgtgca gctcgccgac 540cactaccagc agaacacccc
catcggcgac ggccccgtgc tgctgcccga caaccactac 600ctgagctacc
agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg
660ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caag
714125238PRTArtificial SequenceEYFP sequence 125Val Ser Lys Gly Glu
Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val1 5 10 15Glu Leu Asp Gly
Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu20 25 30Gly Glu Gly
Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys35 40 45Thr Thr
Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe50 55 60Gly
Tyr Gly Leu Gln Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln65 70 75
80His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg85
90 95Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
Val100 105 110Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu
Lys Gly Ile115 120 125Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His
Lys Leu Glu Tyr Asn130 135 140Tyr Asn Ser His Asn Val Tyr Ile Met
Ala Asp Lys Gln Lys Asn Gly145 150 155 160Ile Lys Val Asn Phe Lys
Ile Arg His Asn Ile Glu Asp Gly Ser Val165 170 175Gln Leu Ala Asp
His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro180 185 190Val Leu
Leu Pro Asp Asn His Tyr Leu Ser Tyr Gln Ser Ala Leu Ser195 200
205Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe
Val210 215 220Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr
Lys225 230 235126717DNAArtificial SequenceDNA encoding ECFP
126gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga
gctggacggc 60gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc
cacctacggc 120aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
ccgtgccctg gcccaccctc 180gtgaccaccc tgacctgggg cgtgcagtgc
ttcagccgct accccgacca catgaagcag 240cacgacttct tcaagtccgc
catgcccgaa ggctacgtcc aggagcgcac catcttcttc 300aaggacgacg
gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
360aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct
ggggcacaag 420ctggagtaca actacatcag ccacaacgtc tatatcaccg
ccgacaagca gaagaacggc 480atcaaggcca acttcaagat ccgccacaac
atcgaggacg gcagcgtgca gctcgccgac 540cactaccagc agaacacccc
catcggcgac ggccccgtgc tgctgcccga caaccactac 600ctgagcaccc
agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg
660ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtaa
717127238PRTArtificial SequenceECFP sequence 127Val Ser Lys Gly Glu
Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val1 5 10 15Glu Leu Asp Gly
Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu20 25 30Gly Glu Gly
Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys35 40 45Thr Thr
Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu50 55 60Thr
Trp Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln65 70 75
80His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg85
90 95Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
Val100 105 110Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu
Lys Gly Ile115 120 125Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His
Lys Leu Glu Tyr Asn130 135 140Tyr Ile Ser His Asn Val Tyr Ile Thr
Ala Asp Lys Gln Lys Asn Gly145 150 155 160Ile Lys Ala Asn Phe Lys
Ile Arg His Asn Ile Glu Asp Gly Ser Val165 170 175Gln Leu Ala Asp
His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro180 185 190Val Leu
Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser195 200
205Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe
Val210 215 220Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr
Lys225 230 235128642DNAArtificial SequenceDNA encoding MSP1T2-GT
128atgggtcatc atcatcatca tcatcacgat tatgatattc ctactactga
gaatttgtat 60tttcagggtt ctaccttcag taaacttcgc gaacaactgg gccccgtgac
gcaggaattc 120tgggacaacc tggaaaaaga aaccgaggga ctgcgtcagg
aaatgtccaa agatttagaa 180gaggtgaagg ccaaggttca gccatatctc
gatgactttc agaaaaaatg gcaggaagag 240atggaattat atcgtcaaaa
ggtggaaccg ctgcgtgcgg aactgcaaga gggggcacgc 300caaaaactcc
atgagctcca agagaagctc agcccattag gcgaagaaat gcgcgatcgc
360gcccgtgcac atgttgatgc actccggact catttggcgc cgtattcgga
tgaacttcgc 420cagcgtttgg ccgcacgtct cgaggcgctg aaagaaaacg
ggggtgcccg cttggctgag 480taccacgcga aagcgacaga acacctgagc
accttgagcg aaaaagcgaa accggcgctg 540gaagatctac gccagggctt
attgcctgtt cttgagagct ttaaagtcag ttttctgtca 600gctctggaag
aatatactaa aaagctgaat acccagggta cc 642129214PRTArtificial
SequenceMSP1T2-GT 129Met Gly His His His His His His His Asp Tyr
Asp Ile Pro Thr Thr1 5 10 15Glu Asn Leu Tyr Phe Gln Gly Ser Thr Phe
Ser Lys Leu Arg Glu Gln20 25 30Leu Gly Pro Val Thr Gln Glu Phe Trp
Asp Asn Leu Glu Lys Glu Thr35 40 45Glu Gly Leu Arg Gln Glu Met Ser
Lys Asp Leu Glu Glu Val Lys Ala50 55 60Lys Val Gln Pro Tyr Leu Asp
Asp Phe Gln Lys Lys Trp Gln Glu Glu65 70 75 80Met Glu Leu Tyr Arg
Gln Lys Val Glu Pro Leu Arg Ala Glu Leu Gln85 90 95Glu Gly Ala Arg
Gln Lys Leu His Glu Leu Gln Glu Lys Leu Ser Pro100 105 110Leu Gly
Glu Glu Met Arg Asp Arg Ala Arg Ala His Val Asp Ala Leu115 120
125Arg Thr His Leu Ala Pro Tyr Ser Asp Glu Leu Arg Gln Arg Leu
Ala130 135 140Ala Arg Leu Glu Ala Leu Lys Glu Asn Gly Gly Ala Arg
Leu Ala Glu145 150 155 160Tyr His Ala Lys Ala Thr Glu His Leu Ser
Thr Leu Ser Glu Lys Ala165 170 175Lys Pro Ala Leu Glu Asp Leu Arg
Gln Gly Leu Leu Pro Val Leu Glu180 185 190Ser Phe Lys Val Ser Phe
Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys195 200 205Leu Asn Thr Gln
Gly Thr210130636DNAArtificial SequenceDNA encoding MSP1RC12'
130atgggtcatc atcatcatca tcacattgag ggatgtctga agctgttgga
caattgggac 60tctgttacgt ctaccttcag taaacttcgc gaacaactgg gccccgtgac
gcaggaattc 120tgggacaacc tggaaaaaga aaccgaggga ctgcgtcagg
aaatgtccaa agatttagaa 180gaggtgaagg ccaaggttca gccatatctc
gatgactttc agaaaaaatg gcaggaagag 240atggaattat atcgtcaaaa
ggtggaaccg ctgcgtgcgg aactgcaaga gggggcacgc 300caaaaactcc
atgagctcca agagaagctc agcccattag gcgaagaaat gcgcgatcgc
360gcccgtgcac atgttgatgc actccggact catttggcgc cgtattcgga
tgaacttcgc 420cagcgtttgg ccgcacgtct cgaggcgctg aaagaaaacg
ggggtgcccg cttggctgag 480taccacgcga aagcgacaga acacctgagc
accttgagcg aaaaagcgaa accggcgctg 540gaagatctac gccagggctt
attgcctgtt cttgagagct ttaaagtcag ttttctgtca 600gctctggaag
aatatactaa aaagctgaat acccag 636131212PRTArtificial
SequenceMSP1RC12' 131Met Gly His His His His His His Ile Glu Gly
Cys Leu Lys Leu Leu1 5 10 15Asp Asn Trp Asp Ser Val Thr Ser Thr Phe
Ser Lys Leu Arg Glu Gln20 25 30Leu Gly Pro Val Thr Gln Glu Phe Trp
Asp Asn Leu Glu Lys Glu Thr35
40 45Glu Gly Leu Arg Gln Glu Met Ser Lys Asp Leu Glu Glu Val Lys
Ala50 55 60Lys Val Gln Pro Tyr Leu Asp Asp Phe Gln Lys Lys Trp Gln
Glu Glu65 70 75 80Met Glu Leu Tyr Arg Gln Lys Val Glu Pro Leu Arg
Ala Glu Leu Gln85 90 95Glu Gly Ala Arg Gln Lys Leu His Glu Leu Gln
Glu Lys Leu Ser Pro100 105 110Leu Gly Glu Glu Met Arg Asp Arg Ala
Arg Ala His Val Asp Ala Leu115 120 125Arg Thr His Leu Ala Pro Tyr
Ser Asp Glu Leu Arg Gln Arg Leu Ala130 135 140Ala Arg Leu Glu Ala
Leu Lys Glu Asn Gly Gly Ala Arg Leu Ala Glu145 150 155 160Tyr His
Ala Lys Ala Thr Glu His Leu Ser Thr Leu Ser Glu Lys Ala165 170
175Lys Pro Ala Leu Glu Asp Leu Arg Gln Gly Leu Leu Pro Val Leu
Glu180 185 190Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr
Thr Lys Lys195 200 205Leu Asn Thr Gln210132636DNAArtificial
SequenceDNA encoding MSP1K9EC 132atgggtcatc atcatcatca tcacattgag
ggacgtctga agctgttgga caattgggac 60tctgttacgt ctaccttcag taaacttcgc
gaacaactgg gccccgtgac gcaggaattc 120tgggacaacc tggaaaaaga
aaccgaggga ctgcgtcagg aaatgtccaa agatttagaa 180gaggtgaagg
ccaaggttca gccatatctc gatgactttc agaaaaaatg gcaggaagag
240atggaattat atcgtcaaaa ggtggaaccg ctgcgtgcgg aactgcaaga
gggggcacgc 300caatgtctcc atgagctcca agagaagctc agcccattag
gcgaagaaat gcgcgatcgc 360gcccgtgcac atgttgatgc actccggact
catttggcgc cgtattcgga tgaacttcgc 420cagcgtttgg ccgcacgtct
cgaggcgctg aaagaaaacg ggggtgcccg cttggctgag 480taccacgcga
aagcgacaga acacctgagc accttgagcg aaaaagcgaa accggcgctg
540gaagatctac gccagggctt attgcctgtt cttgagagct ttaaagtcag
ttttctgtca 600gctctggaag aatatactaa aaagctgaat acccag
636133212PRTArtificial SequenceMSP1K9EC 133Met Gly His His His His
His His Ile Glu Gly Arg Leu Lys Leu Leu1 5 10 15Asp Asn Trp Asp Ser
Val Thr Ser Thr Phe Ser Lys Leu Arg Glu Gln20 25 30Leu Gly Pro Val
Thr Gln Glu Phe Trp Asp Asn Leu Glu Lys Glu Thr35 40 45Glu Gly Leu
Arg Gln Glu Met Ser Lys Asp Leu Glu Glu Val Lys Ala50 55 60Lys Val
Gln Pro Tyr Leu Asp Asp Phe Gln Lys Lys Trp Gln Glu Glu65 70 75
80Met Glu Leu Tyr Arg Gln Lys Val Glu Pro Leu Arg Ala Glu Leu Gln85
90 95Glu Gly Ala Arg Gln Cys Leu His Glu Leu Gln Glu Lys Leu Ser
Pro100 105 110Leu Gly Glu Glu Met Arg Asp Arg Ala Arg Ala His Val
Asp Ala Leu115 120 125Arg Thr His Leu Ala Pro Tyr Ser Asp Glu Leu
Arg Gln Arg Leu Ala130 135 140Ala Arg Leu Glu Ala Leu Lys Glu Asn
Gly Gly Ala Arg Leu Ala Glu145 150 155 160Tyr His Ala Lys Ala Thr
Glu His Leu Ser Thr Leu Ser Glu Lys Ala165 170 175Lys Pro Ala Leu
Glu Asp Leu Arg Gln Gly Leu Leu Pro Val Leu Glu180 185 190Ser Phe
Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys195 200
205Leu Asn Thr Gln210134636DNAArtificial SequenceDNA encoding
MSP1D152C 134atgggtcatc atcatcatca tcacattgag ggacgtctga agctgttgga
caattgggac 60tctgttacgt ctaccttcag taaacttcgc gaacaactgg gccccgtgac
gcaggaattc 120tgggacaacc tggaaaaaga aaccgaggga ctgcgtcagg
aaatgtccaa agatttagaa 180gaggtgaagg ccaaggttca gccatatctc
gatgactttc agaaaaaatg gcaggaagag 240atggaattat atcgtcaaaa
ggtggaaccg ctgcgtgcgg aactgcaaga gggggcacgc 300caaaaactcc
atgagctcca agagaagctc agcccattag gcgaagaaat gcgcgatcgc
360gcccgtgcac atgttgatgc actccggact catttggcgc cgtattcgga
tgaacttcgc 420cagcgtttgg ccgcacgtct cgaggcgctg aaagaaaacg
ggggtgcccg cttggctgag 480taccacgcat gcgcgacaga acacctgagc
accttgagcg aaaaagcgaa accggcgctg 540gaagatctac gccagggctt
attgcctgtt cttgagagct ttaaagtcag ttttctgtca 600gctctggaag
aatatactaa aaagctgaat acccag 636135212PRTArtificial
SequenceMSP1K152C 135Met Gly His His His His His His Ile Glu Gly
Arg Leu Lys Leu Leu1 5 10 15Asp Asn Trp Asp Ser Val Thr Ser Thr Phe
Ser Lys Leu Arg Glu Gln20 25 30Leu Gly Pro Val Thr Gln Glu Phe Trp
Asp Asn Leu Glu Lys Glu Thr35 40 45Glu Gly Leu Arg Gln Glu Met Ser
Lys Asp Leu Glu Glu Val Lys Ala50 55 60Lys Val Gln Pro Tyr Leu Asp
Asp Phe Gln Lys Lys Trp Gln Glu Glu65 70 75 80Met Glu Leu Tyr Arg
Gln Lys Val Glu Pro Leu Arg Ala Glu Leu Gln85 90 95Glu Gly Ala Arg
Gln Lys Leu His Glu Leu Gln Glu Lys Leu Ser Pro100 105 110Leu Gly
Glu Glu Met Arg Asp Arg Ala Arg Ala His Val Asp Ala Leu115 120
125Arg Thr His Leu Ala Pro Tyr Ser Asp Glu Leu Arg Gln Arg Leu
Ala130 135 140Ala Arg Leu Glu Ala Leu Lys Glu Asn Gly Gly Ala Arg
Leu Ala Glu145 150 155 160Tyr His Ala Cys Ala Thr Glu His Leu Ser
Thr Leu Ser Glu Lys Ala165 170 175Lys Pro Ala Leu Glu Asp Leu Arg
Gln Gly Leu Leu Pro Val Leu Glu180 185 190Ser Phe Lys Val Ser Phe
Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys195 200 205Leu Asn Thr
Gln21013622PRTArtificial SequenceHelix 2S peptide sequence 136Pro
Val Thr Gln Glu Phe Trp Asp Asn Leu Glu Lys Glu Thr Glu Gly1 5 10
15Leu Arg Gln Glu Met Ser20
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