U.S. patent application number 12/118530 was filed with the patent office on 2009-05-28 for methods and systems for monitoring production of a target protein in a nanolipoprotein particle.
Invention is credited to Erin S. Arroyo, Jenny A. Cappuccio, Matthew A. COLEMAN, Paul D. Hoeprich.
Application Number | 20090136937 12/118530 |
Document ID | / |
Family ID | 39709214 |
Filed Date | 2009-05-28 |
United States Patent
Application |
20090136937 |
Kind Code |
A1 |
COLEMAN; Matthew A. ; et
al. |
May 28, 2009 |
METHODS AND SYSTEMS FOR MONITORING PRODUCTION OF A TARGET PROTEIN
IN A NANOLIPOPROTEIN PARTICLE
Abstract
Provided herein are methods and systems for the monitoring
production of a target protein in of a nanolipoprotein particle
(NLP) that also includes a scaffold protein and a membrane forming
lipid. The target protein is capable of assuming an active form and
an inactive form. Monitoring is performed by an indicator protein
that is capable of assuming an active form and an inactive form,
the active form associated with a detectable activity of the
indicator protein, the detectable activity further associated with
the active form of the target protein.
Inventors: |
COLEMAN; Matthew A.;
(Oakland, CA) ; Hoeprich; Paul D.; (Pleasanton,
CA) ; Arroyo; Erin S.; (Fairfax, CA) ;
Cappuccio; Jenny A.; (Livermore, CA) |
Correspondence
Address: |
LLNL/Steinfl & Bruno;John H. Lee, Assistant Laboratory Counsel
Lawrence Livermore National Laboratory, L-703, P.O. Box 808
Livermore
CA
94551
US
|
Family ID: |
39709214 |
Appl. No.: |
12/118530 |
Filed: |
May 9, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60928579 |
May 9, 2007 |
|
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|
60928573 |
May 9, 2007 |
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Current U.S.
Class: |
435/6.12 ;
422/68.1; 435/287.1; 435/287.2; 435/6.13; 435/7.9; 436/501;
436/86 |
Current CPC
Class: |
G01N 33/582 20130101;
C12P 21/02 20130101; G01N 33/54346 20130101; C07K 14/705 20130101;
G01N 33/587 20130101 |
Class at
Publication: |
435/6 ; 436/86;
436/501; 435/7.9; 422/68.1; 435/287.1; 435/287.2 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; G01N 33/68 20060101 G01N033/68; G01N 33/566 20060101
G01N033/566; C12M 1/00 20060101 C12M001/00; B01J 19/00 20060101
B01J019/00; G01N 33/53 20060101 G01N033/53 |
Goverment Interests
STATEMENT OF GOVERNMENT GRANT
[0002] The United States Government has rights in this invention
pursuant to Contract No. DE-AC52-07NA27344 between the U.S.
Department of Energy and Lawrence Livermore National Security, LLC,
for the operation of Lawrence Livermore National Security."
Claims
1. A method for monitoring production of a target protein in a
nanolipoprotein particle, the nanolipoprotein particle comprising
the target protein, a membrane forming lipid and a scaffold
protein, the target protein capable of assuming a target protein
active form and a target protein inactive form, the method
comprising: providing an indicator protein, the indicator protein
capable of assuming an indicator protein active form and an
indicator protein inactive form, the indicator protein active form
associated to an indicator protein detectable activity, the
indicator protein detectable activity associated to the target
protein active form; contacting the indicator protein with the
target protein, the membrane forming lipid and the scaffold protein
for a time and under conditions to allow assembly of the indicator
protein, the target protein, the membrane forming lipid and the
scaffold protein in the nanolipoprotein particle; and detecting the
indicator protein detectable activity from the nanolipoprotein
particle.
2. The method of claim 1, wherein the target protein is a membrane
protein and the membrane forming lipid is selected from the group
consisting of phospholipids, sphingolipids, glycolipids, ether
lipids, sterols and alkylphosphocholins.
3. The method of claim 1, wherein the target protein is selected
from the group consisting of a protein coupled receptor (GPCR), an
ion channel protein (IC) and a small multidrug resistance
transporter (SMR).
4. The method of claim 1, wherein the target protein is selected
from the group consisting of V2R, CRF, ETB, MC5R, NTR1, 5HT1A, H2,
M1, herg, .alpha.1AR, .beta.1AR, OP1R, .beta.2AR and M2.
5. The method of claim 1, wherein the indicator protein is
structurally related to the target protein so that the production
of the indicator protein in an active form can be associated with
the production of the target protein in an active form.
6. The method of claim 1 wherein the indicator protein is selected
from the group consisting of GFP, GFP-fused to a membrane protein,
cytochromes and dye labeled proteins.
7. The method of claim 1, wherein the indicator protein is selected
from the group consisting of sensory rhodopsin, proteorhodopsin,
and phytochromes.
8. The method of claim 1, wherein the indicator protein is
bacteriorhodopsin.
9. The method of claim 1, wherein detecting the indicator protein
detectable activity is performed by providing a labeled molecule
that specifically binds to the indicator protein the labeled
molecule providing a labeling signal; contacting the labeled
molecule with the nanolipoprotein particle for a time and under
condition to allow binding of the labeled molecule with the
indicator protein in the nanolipoprotein particle; and detecting
the labeling signal from the labeled molecule bound to the
indicator protein in the nanolipoprotein particle.
10. The method of claim 9, wherein the labeled molecule is selected
from the group consisting of radioactive isotopes, chemiluminescent
dyes, fluorophores, chromophores, enzymes, enzymes substrates,
enzyme cofactor, enzyme inhibitors, dyes, metal ions,
nanoparticles, metal ions and ligands.
11. A method for monitoring production of a target protein in a
nanolipoprotein particle, the nanolipoprotein particle comprising
the target protein a membrane forming lipid and a scaffold protein,
the target protein capable of assuming a target protein active form
and a target protein inactive form, the method comprising:
providing a first polynucleotide encoding for the target protein;
providing a second polynucleotide encoding for an indicator
protein, the indicator protein capable of assuming an indicator
protein active form and an indicator protein inactive form, the
indicator protein active form associated to an indicator protein
detectable activity, the indicator protein detectable activity
associated to the target protein active form; contacting the first
and second polynucleotides with the membrane forming lipid and the
scaffold protein for a time and under conditions to allow assembly
of the indicator protein, the target protein, the membrane forming
lipid and the scaffold protein in the nanolipoprotein particle; and
detecting the indicator protein detectable activity from the
nanolipoprotein particle.
12. The method of claim 11, wherein contacting the first and second
polynucleotide with the membrane forming lipid and the scaffold
protein is performed in a single reaction mixture.
13. The method of claim 11, wherein the target protein is a
membrane protein and the membrane forming lipid is selected from
the group consisting of phospholipids, sphingolipids, glycolipids,
ether lipids, sterols and alkylphosphocholins.
14. The method of claim 11, wherein the target protein is selected
from the group consisting of a protein coupled receptor (GPCR), an
ion channel protein (IC) and a small multidrug resistance
transporter (SMR).
15. The method of claim 11, wherein at least one of the first and
the second polynucleotide is an engineered polynucleotide encoding
for a chimeric product.
16. The method of claim 11, wherein the target protein is selected
from the group consisting of V2R, CRF, ETB, MC5R, NTR1, 5HT1A, H2,
M1, herg, .alpha.1AR, .beta.1AR, OP1R, .beta.2AR and M2.
17. The method of claim 11, wherein the indicator protein is
structurally related to the target protein of interest so that the
production of the indicator protein in an active form can be
associated with the production of the target protein in an active
form.
18. The method of claim 11 wherein the indicator protein is
selected from the group consisting of GFP, GFP-fused to a membrane
protein, cytochromes and dye labeled proteins.
19. The method of claim 11, wherein the indicator protein is
selected from the group consisting of sensory rhodopsin,
proteorhodopsin, and phytochromes.
20. The method of claim 11, wherein the indicator protein is
bacteriorhodopsin.
21. The method of claim 11, wherein detecting the indicator protein
detectable activity is performed by providing a labeled molecule
that specifically binds to the indicator protein the labeled
molecule providing a labeling signal; contacting the labeled
molecule with the nanolipoprotein particle for a time and under
condition to allow binding of the labeled molecule with the
indicator protein in the nanolipoprotein particle; and detecting
the labeling signal from the labeled molecule bound to the
indicator protein in the nanolipoprotein particle.
22. The method of claim 21, wherein the labeled molecule is
selected from the group consisting of radioactive isotopes,
chemiluminescent dyes, fluorophores, chromophores, enzymes, enzymes
substrates, enzyme cofactor, enzyme inhibitors, dyes, metal ions,
nanoparticles, metal ions and ligands.
23. A method for monitoring production of a target protein in a
nanolipoprotein particle, the nanolipoprotein particle comprising
the target protein, a membrane forming lipid and a scaffold
protein, the target protein capable of assuming a target protein
active form and a target protein inactive form, the method
comprising: providing a first polynucleotide encoding for the
target protein; providing a second polynucleotide encoding for an
indicator protein, the indicator protein capable of assuming an
indicator protein active form and an indicator protein inactive
form, the indicator protein active form associated to an indicator
protein detectable activity, the indicator protein detectable
activity associated to the target protein active form; providing a
third polynucleotide encoding for the scaffold protein; contacting
the first, second and third polynucleotides with the membrane
forming lipid and the scaffold protein for a time and under
conditions to allow assembly of the indicator protein, the target
protein, the membrane forming lipid and the scaffold protein in the
nanolipoprotein particle; and detecting the indicator protein
detectable activity from the nanolipoprotein particle.
24. The method of claim 23, wherein contacting the first, second
and third polynucleotided with the membrane forming lipid and the
scaffold protein is performed in a single reaction mixture.
25. The method of claim 23, wherein the target protein is a
membrane protein and the membrane forming lipid is selected from
the group consisting of phospholipids, sphingolipids, glycolipids,
ether lipids, sterols and alkylphosphocholins.
26. The method of claim 23, wherein the target protein is selected
from the group consisting of a protein coupled receptor (GPCR), an
ion channel protein (IC) and a small multidrug resistance
transporter (SMR).
27. The method of claim 23, wherein at least one of the first, the
second polynucleotide and the third polynucleotide is an engineered
polynucleotide encoding for a chimeric product.
28. The method of claim 23, wherein the target protein is selected
from the group consisting of V2R, CRF, ETB, MC5R, NTR1, 5HT1A, H2,
M1, herg, .alpha.1AR, .beta.1AR, OP1R, .beta.2AR and M2.
29. The method of claim 23, wherein the indicator protein is
structurally related to the target protein of interest so that the
production of the indicator protein in an active form can be
associated with the production of the target protein in an active
form.
30. The method of claim 23 wherein the indicator protein is
selected from the group consisting of GFP, GFP-fused to a membrane
protein, cytochromes and dye labeled proteins.
31. The method of claim 23, wherein the indicator protein is
selected from the group consisting of sensory rhodopsin,
proteorhodopsin, and phytochromes.
32. The method of claim 23, wherein the indicator protein is
bacteriorhodopsin.
33. The method of claim 23, wherein detecting the indicator protein
detectable activity is performed by providing a labeled molecule
that specifically binds to the indicator protein the labeled
molecule providing a labeling signal; contacting the labeled
molecule with the nanolipoprotein particle for a time and under
condition to allow binding of the labeled molecule with the
indicator protein in the nanolipoprotein particle; and detecting
the labeling signal from the labeled molecule bound to the
indicator protein in the nanolipoprotein particle.
34. The method of claim 33, wherein the labeled molecule is
selected from the group consisting of radioactive isotopes,
chemiluminescent dyes, fluorophores, chromophores, enzymes, enzymes
substrates, enzyme cofactor, enzyme inhibitors, dyes, metal ions,
nanoparticles, metal ions and ligands.
35. A system for monitoring production of a target protein in a
nanolipoprotein particle, the nanolipoprotein particle comprising
the target protein, a membrane forming lipid and a scaffold
protein, the target protein capable of assuming a target protein
active form and a target protein inactive form, the system
comprising: an indicator protein capable of assuming an indicator
protein active form and an indicator protein inactive form, the
indicator protein active form associated to an indicator protein
detectable activity, the indicator protein detectable activity
associated to the target protein active form, and at least one of
the target protein, the membrane forming lipid and the scaffold
protein.
36. The system of claim 35, wherein the target protein is a
membrane protein and the membrane forming lipid is selected from
the group consisting of phospholipids, sphingolipids, glycolipids,
ether lipids, sterols and alkylphosphocholins.
37. The system of claim 35, wherein the target protein is selected
from the group consisting of a protein coupled receptor (GPCR), an
ion channel protein (IC) or a small multidrug resistance
transporter (SMR).
38. The system of claim 35, wherein the target protein is selected
from the group consisting of V2R, CRF, ETB, MC5R, NTR1, 5HT1A, H2,
M1, herg, .alpha.1AR, .beta.1AR, OP1R, .beta.2AR and M2.
39. The system of claim 35, wherein the indicator protein is
structurally related to the target protein of interest so that the
production of the indicator protein in an active form can be
associated with the production of the target protein in an active
form.
40. The system of claim 35, wherein the indicator protein is
selected from the group consisting of GFP, GFP-fused to a membrane
protein, cytochromes and dye labeled proteins.
41. The system of claim 35, wherein the indicator protein is
selected from the group consisting of sensory rhodopsin,
proteorhodopsin, and phytochromes.
42. The system of claim 35, wherein the indicator protein is
bacteriorhodopsin.
43. The system of claim 35, further comprising a labeled molecule
that specifically binds to the indicator protein, the labeled
molecule providing a labeling signal.
44. The system of claim 43, wherein the labeled molecule is
selected from the group consisting of radioactive isotopes,
chemioluminescent dyes, fluorophores, chromophores, enzymes,
enzymes substrates, enzyme cofactor, enzyme inhibitors, dyes, metal
ions, nanoparticles, metal ions and ligands.
45. A system for monitoring production of a target protein in a
nanolipoprotein particle, the nanolipoprotein particle comprising
the target protein, a membrane forming lipid and a scaffold
protein, the target protein capable of assuming a target protein
active form and a target protein inactive form, the system
comprising: a first polynucleotide encoding for the target protein;
and a second polynucleotide encoding for an indicator protein, the
indicator protein capable of assuming an indicator protein active
form and an indicator protein inactive form, the indicator protein
active form associated to an indicator protein detectable activity,
the indicator protein detectable activity associated to the target
protein active form.
46. The system of claim 45, wherein the target protein is a
membrane protein and the membrane forming lipid is selected from
the group consisting of phospholipids, sphingolipids, glycolipids,
ether lipids, sterols and alkylphosphocholins.
47. The system of claim 45, wherein the target protein is selected
from the group consisting of a protein coupled receptor (GPCR), an
ion channel protein (IC) or a small multidrug resistance
transporter (SMR).
48. The system of claim 45, wherein the target protein is selected
from the group consisting of V2R, CRF, ETB, MC5R, NTR1, 5HT1A, H2,
M1, herg, .alpha.1AR, .beta.1AR, OP1R, .beta.2AR and M2.
49. The system of claim 45, wherein the indicator protein is
structurally related to the target protein of interest so that the
production of the indicator protein in an active form can be
associated with the production of the target protein in an active
form.
50. The system of claim 45, wherein the indicator protein is
selected from the group consisting of GFP, GFP-fused to a membrane
protein, cytochromes and dye labeled proteins.
51. The system of claim 45, wherein the indicator protein is
selected from the group consisting of sensory rhodopsin,
proteorhodopsin, and phytochromes.
52. The system of claim 45, wherein the indicator protein is
bacteriorhodopsin.
53. The system of claim 45, further comprising a labeled molecule
that specifically binds to the indicator protein, the labeled
molecule providing a labeling signal.
54. The system of claim 53, wherein the labeled molecule is
selected from the group consisting of radioactive isotopes,
chemiluminescent dyes, fluorophores, chromophores, enzymes, enzymes
substrates, enzyme cofactor, enzyme inhibitors, dyes, metal ions,
nanoparticles, metal ions and ligands.
55. A system for monitoring production of a target protein in a
nanolipoprotein particle, the nanolipoprotein particle comprising
the target protein, a membrane forming lipid and a scaffold
protein, the target protein capable of assuming a target protein
active form and a target protein inactive form, the system
comprising: a first polynucleotide encoding for the target protein;
a second polynucleotide encoding for an indicator protein, the
indicator protein capable of assuming an indicator protein active
form and an indicator protein inactive form, the indicator protein
active form associated to an indicator protein detectable activity,
the indicator protein detectable activity associated to the target
protein active form; and a third polynucleotide encoding for the
scaffold protein.
56. The system of claim 55, wherein the target protein is a
membrane protein and the membrane forming lipid is selected from
the group consisting of phospholipids, sphingolipids, glycolipids,
ether lipids, sterols and alkylphosphocholins.
57. The system of claim 55, wherein the target protein is selected
from the group consisting of a protein coupled receptor (GPCR), an
ion channel protein (IC) or a small multidrug resistance
transporter (SMR).
58. The system of claim 55, wherein the target protein is selected
from the group consisting of V2R, CRF, ETB, MC5R, NTR1, 5HT1A, H2,
M1, herg, .alpha.1AR, .beta.1AR, OP1R, .beta.2AR and M2.
59. The system of claim 55, wherein the indicator protein is
structurally related to the target protein of interest so that the
production of the indicator protein in an active form can be
associated with the production of the target protein in an active
form.
60. The system of claim 55, wherein the indicator protein is
selected from the group consisting of GFP, GFP-fused to a membrane
protein, cytochromes and dye labeled proteins.
61. The system of claim 55, wherein the indicator protein is
selected from the group consisting of sensory rhodopsin,
proteorhodopsin, and phytochromes.
62. The system of claim 55, wherein the indicator protein is
bacteriorhodopsin.
63. The system of claim 55, further comprising a labeled molecule
that specifically binds to the indicator protein, the labeled
molecule providing a labeling signal.
64. The system of claim 63, wherein the labeled molecule is
selected from the group consisting of radioactive isotopes,
chemiluminescent dyes, fluorophores, chromophores, enzymes, enzymes
substrates, enzyme cofactor, enzyme inhibitors, dyes, metal ions,
nanoparticles, metal ions and ligands.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application entitled "Cell-free Self Assembly of Nano-lipoprotein
Particles as a Platform for Co-expressed Membrane Proteins" Ser.
No. 60/928,579, filed on May 9, 2007 Docket No. IL-11841, and to
U.S. Provisional Application entitled "Monitoring IVT or Cell-free
Membrane Protein Expressions, Folding and Functional Using In Situ
Expression of Bacteriorhodopsin as an Internal Colorimetric" Ser.
No. 60/928,573 filed on May 9, 2007 Docket No. IL-11842, the
disclosures of which are incorporated herein by reference in their
entirety. This application may also be related to U.S. application
entitled "Methods and Systems for Producing Nanolipoprotein
Particle" filed on the same day of the present application with
Docket No. P197-US, the disclosure of which is also incorporated
herein by reference in its entirety.
TECHNICAL FIELD
[0003] The present disclosure relates to membranes and membrane
associated proteins and to complexes mimicking said membranes and
membrane associated proteins.
BACKGROUND
[0004] Membrane-associated proteins and protein complexes account
for--30% or more of the cellular proteins. Membrane proteins are
held within a bilayer structure. The basic membrane bilayer
construct consists of two opposing layers of amphiphilic molecules
know as phospholipids; each molecule has a hydrophilic moiety,
i.e., a polar phosphate group/derivative, and a hydrophobic moiety,
i.e., a long hydrocarbon chain. These molecules self-assemble in a
biological (largely aqueous) environment according to
thermodynamics associated with water exclusion or hydrophobic
association.
[0005] In order to facilitate the myriad functions of biological
membranes including the passage of nutrients, signaling molecules
and other molecules into and out of the cell, membrane proteins are
arrayed in the bilayer structure as depicted below. Note that some
proteins span the bilayer, others are anchored within the bilayer,
and still others organize "peripheral" proteins into complexes.
Many membrane bound protein complexes mediate essential cellular
processes e.g. signal transduction, transport, recognition, and
cell-cell communication. In general, this class of proteins is
challenging to study because of their insolubility and tendency to
aggregate when removed from their protein lipid bilayer
environment.
[0006] Membrane proteins are optimally folded and functional when
in a lipid bilayer, but standard protein purification methods often
remove lipids, invariably altering protein conformation and
function.
[0007] Furthermore, also non-membrane proteins (i.e. proteins that
do not exercise a biological activity in connection with a location
on a membrane) may still be desirably associated with a membrane
for the purpose of solubilization and/or transporting and
delivering to a cell.
[0008] To overcome these problems, fully functional integral
membrane proteins and additional proteins can be assembled in
lipid/protein-based particulate structures called nanolipoprotein
particles (NLPs) usually comprising membrane forming lipids and
apolipoproteins.
[0009] NLP assembly and function usually involves the association
of specific apolipoprotein and lipid molecules leading to formation
of proteolipid complexes; the latter are used to transport a
diverse array of lipid molecules within organisms.
[0010] NLPs made in the presence of a solubilized membrane protein
(target) result in a membrane protein NLP construct Accordingly,
NLP assembly can also be used for stabilization and
characterization of membrane proteins.
SUMMARY
[0011] Provided herein, are methods and systems for monitoring
production of a target protein in a NLP nanostructure. In
particular, the methods and systems herein disclosed allow
monitoring of synthesis, correct folding and incorporation of the
target protein in a NLP, following assembly of the NLP, which in
some embodiments can also occur in a single reaction.
[0012] According to a first aspect, a method for monitoring
production of a target protein in a nanolipoprotein particle is
disclosed. The nanolipoprotein particle comprises the target
protein a membrane forming lipid and a scaffold protein. The target
protein is capable of assuming a target protein active form and a
target protein inactive form. The method comprises: providing an
indicator protein, the indicator protein capable of assuming an
indicator protein active form and an indicator protein inactive
form, the indicator protein active form associated to an indicator
protein detectable activity, the indicator protein detectable
activity associated to the target protein active form. The method
further comprises contacting the indicator protein with the target
protein, the membrane forming lipid and the scaffold protein for a
time and under conditions to allow assembly of the indicator
protein, the target protein, the membrane forming lipid and the
scaffold protein in the nanolipoprotein particle. The method also
comprises detecting the indicator protein detectable activity from
the nanolipoprotein particle.
[0013] According to a second aspect, a method for monitoring
production of a target protein in a nanolipoprotein particle is
disclosed. The nanolipoprotein particle comprises the target
protein a membrane forming lipid and a scaffold protein. The target
protein is capable of assuming a target protein active form and a
target protein inactive form. The method comprises: providing a
first polynucleotide encoding for the target protein; providing a
second polynucleotide encoding for the indicator protein, the
indicator protein capable of assuming an indicator protein active
form and an indicator protein inactive form, the indicator protein
active form associated to an indicator protein detectable activity,
the indicator protein detectable activity associated to the target
protein active form. The method further comprises: contacting the
first and second polynucleotides with the membrane forming lipid
and the scaffold protein for a time and under conditions to allow
assembly of the indicator protein, the target protein, the membrane
forming lipid and the scaffold protein in the nanolipoprotein
particle. The method also comprises: detecting the indicator
protein detectable activity from the nanolipoprotein particle.
[0014] According to a third aspect, a method for monitoring
production of a target protein in a nanolipoprotein particle is
disclosed. The nanolipoprotein particle comprises the target
protein a membrane forming lipid and a scaffold protein. The target
protein is capable of assuming a target protein active form and a
target protein inactive form. The method comprises: providing a
first polynucleotide encoding for the target protein; providing a
second polynucleotide encoding for the indicator protein, the
indicator protein capable of assuming an indicator protein active
form and an indicator protein inactive form, the indicator protein
active form associated to an indicator protein detectable activity,
the indicator protein detectable activity associated to the target
protein active form; and providing a third polynucleotide encoding
for the scaffold protein. The method further comprises: contacting
the first, second and third polynucleotides with the membrane
forming lipid and the scaffold protein for a time and under
conditions to allow assembly of the indicator protein, the target
protein, the membrane forming lipid and the scaffold protein in the
nanolipoprotein particle. The method also comprises: detecting the
indicator protein detectable activity from the nanolipoprotein
particle.
[0015] According to a fourth aspect, a system for monitoring
production of a target protein in a nanolipoprotein particle is
disclosed. The nanolipoprotein particle comprises the target
protein a membrane forming lipid and a scaffold protein. The target
protein is capable of assuming a target protein active form and a
target protein inactive form. The system comprises: an indicator
protein capable of assuming an indicator protein active form and an
indicator protein inactive form, the indicator protein active form
associated to an indicator protein detectable activity, the
indicator protein detectable activity associated to the target
protein active form. The system further comprises at least one of
the target proteins the membrane forming lipid and the scaffold
protein.
[0016] According to a fifth aspect, a system for monitoring
production of a target protein in a nanolipoprotein particle is
disclosed. The nanolipoprotein particle comprises the target
protein a membrane forming lipid and a scaffold protein. The target
protein is capable of assuming a target protein active form and a
target protein inactive form. The system comprises: the system
comprising: a first polynucleotide encoding for the target protein;
and a second polynucleotide encoding for the indicator protein, the
indicator protein capable of assuming an indicator protein active
form and an indicator protein inactive form, the indicator protein
active form associated to an indicator protein detectable activity,
the indicator protein detectable activity associated to the target
protein active form.
[0017] According to a sixth aspect, a system for monitoring
production of a target protein in a nanolipoprotein particle is
disclosed. The nanolipoprotein particle comprises the target
protein a membrane forming lipid and a scaffold protein. The target
protein is capable of assuming a target protein active form and a
target protein inactive form. The system comprises: a first
polynucleotide encoding for the target protein; a second
polynucleotide encoding for the indicator protein, the indicator
protein capable of assuming an indicator protein active form and an
indicator protein inactive form, the indicator protein active form
associated to an indicator protein detectable activity, the
indicator protein detectable activity associated to the target
protein active form; and a third polynucleotide encoding for the
scaffold protein.
[0018] The methods and systems herein described can be used in
connection with the characterization and in particular the
optimization of the reaction conditions related to the production
of a target protein of interest
[0019] The methods and systems herein described can be also used to
identify proper folding parameters for general/active membrane
protein production.
[0020] The methods and systems herein described can be further used
in processes wherein the indicator protein is used as an additive
to cell free expression systems regardless of organism/system
extracts.
[0021] The methods and systems herein described can additionally be
used for the correct and efficient production of membrane proteins,
with particular reference to the membrane proteins difficult to
produce from native systems.
[0022] The methods and systems herein described can also be used in
processes for screening parameters for evaluation of production of
membrane proteins.
[0023] The methods and systems herein described can also be used in
processes for identification of novel membrane protein folding
conditions
[0024] The methods and systems herein described can also be used in
a manufacturing quality control assay for cell-free lysates.
[0025] The methods and systems herein described can also be used in
experimental quality control assay for cell-free lysates.
[0026] The methods and systems herein described can also be used in
processes screening for production parameters and successful
membrane protein folding evaluation.
[0027] The methods and systems herein described can be applied in
several fields including basic biology research, applied biology,
bio-engineering, bio-energy, medical research, medical diagnostics,
therapeutics, and bio-fuels.
[0028] The details of one or more embodiments of the disclosure are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages will be apparent from the
description and drawings, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] The accompanying drawings, which are incorporated into and
constitute a part of this specification, illustrate one or more
embodiments of the present disclosure and, together with the
detailed description, serve to explain the principles and
implementations of the disclosure.
[0030] FIG. 1 shows a schematic illustration of single step
cell-free co-expression and stabilization of integral membrane
proteins using an apolipoprotein scaffold according to an
embodiment herein described.
[0031] FIG. 2 shows single-step production, purification and
characterization of MP-NLP complexes, according to an embodiment
herein described. a. Coomassie stained SDS-PAGE gel image of Total
(T), Soluble (S) and Pellet (P) fractions from cell-free produced
bacteriorhodopsin (bR) in the presence and absence of co-expressed
apolipoprotein (.DELTA.49A1). A (+) indicates the addition of
either DMPC, .DELTA.49A1 DNA or bacteriOpsin DNA (bOp) to the
cell-free reaction, (-) denotes absence of additive. Grey arrows
indicate .DELTA.49A1 (lane 2S upper arrow and lane 3S), and black
arrows indicate bR (lane 1T and lane 2S lower arrow). Sample 1
indicates bOp & DMPC; Sample 2 indicates bOp, .DELTA.49A1
co-expressed in the presence of DMPC; Sample 3 indicates
.DELTA.49A1 & DMPC; Sample 4 indicates the control cell-free
reaction (No DNA) in the presence of DMPC only.
[0032] FIG. 3 shows solubility of cell-free produced bR-NLPs. The
(+) indicates the addition of either DMPC, .DELTA.49A1 DNA or
bacteriOpsin DNA (bOp) to the cell-free reaction, (-) denotes
absences of additive, all were expressed in the presence of 50
.mu.M all-trans-retinal. a. Solubility of bR is increased in the
presence of .DELTA.49A1 DNA. Cell-free reactions were carried out
in the presence of .sup.35S-Methionine. Grey arrows indicate
.DELTA.49A1 (Coomassie: lane 3; Autorad: lanes 3 and 8 upper
arrow), and black arrows indicate bR (Coomassie: lane 1; Autorad:
lanes 1 and 8 lower arrow--in Autorad). Left Panel, Coomassie
stained SDS-PAGE gel image of total (T) and soluble (S) fractions
from cell-free produced bR in the presence and absence of
co-expressed apolipoprotein (.DELTA.49A1) and DMPC. Right Panel,
Autoradiogram of the gel shown in left panel, illustrating the
benefit of adding the apolipoprotein for increasing the solubility
of bR.
[0033] FIG. 4 shows solubility of bacteriorhodopsin observed
directly in reaction vessel. The solubility of expressed bR is
increased by .DELTA.49A1 co-expression. A (+) indicates the
addition of either DMPC, .DELTA.49A1 DNA or bacterioOpsin DNA (bOp)
to the cell-free reaction, (-) denotes absence of additive. All
were expressed in the presence of --30-50 .mu.M all-trans-retinal.
Reaction 1, expression of bR in the presence of DMPC alone, purple
color (shown in the figure as a dark gray) has settled to bottom of
vessel. Reaction 2, expression of bR in the presence of .DELTA.49A1
co-expression, purple color (also shown as dark gray) remains
dispersed through out vessel indicating the formation of soluble
bR-NLPs.
[0034] FIG. 5 shows co-expressed Ni-NTA purified bR-NLPs have
several distinct sizes. Size exclusion chromatography of affinity
purified co-expressed bR and .DELTA.49A1. Tube 0, color of sample
before SEC. Numbered fractions collected are indicated at the
bottom of the trace. The color of corresponding concentrated
fractions (top) is shown in the numbered tubes. Purple color (shown
in the figure as various shades if gray) indicates the presence of
properly folded bR. Fraction(s) determined to contain lipid rich
constructs (vesicles or sheets), bR-NLPs, empty-NLPs and lipid poor
protein are indicated on the trace. Samples were collected using a
VP HPLC (Shimadzu) with a Superdex 200 10/300 GL column (GE
Healthcare) at a flow rate of 0.5 mL/min of 10 mM Tris pH 7.4;
0.15M NaCl; 0.25 mM EDTA, 0.005% NaN.sub.3. Fractions were
concentrated using a molecular sieve cut off filter (MWCO 50 k)
Vivaspin 2 (Sartorius) or MWCO 10 k for the free protein
fractions
[0035] FIG. 6 shows the results of an experiments related to
single-step production, purification and characterization of MP-NLP
complexes according to an embodiment herein described. Native gel
of size exclusion purified nanolipoprotein particles (NLPs)
prepared with .DELTA.49A1 with and without bR. Lane M, Molecular
weight marker; Lane 2.sub.1-3, Fractions from SEC purified
cell-free co-expressed bR-NLPs; Lane 2.sub.1, lipid rich first
fraction; Lane 2.sub.2 bR-NLPs second fraction; Lane 2.sub.3,
bR-NLPs third fraction; Lane 3, Cell-free produced "empty"-NLPs.
Lane 4, conventional assembly of "empty"-NLPs with purchased
.DELTA.1-55 apolipoprotein A1 (.DELTA.55A1).
[0036] FIG. 7 shows AFM image of lipid rich SEC fraction. An AFM
image representing the first fraction collected by size exclusion
chromatography of the cell-free co-expression of .DELTA.49A1 and bR
in the presence of DMPC. AFM image of the lipid rich fraction
displaying all three populations depicted in the top table.
Observed diameter and height measurements for the lipid rich
fraction displays three populations; Line A indicates, large lipid
complexes, liposomes or membrane sheets; Line B indicates bR-NLPs
and Line C indicates "empty"-NLPs. This analysis clearly indicates
the majority of structures in this fraction were lipid vesicles or
membrane sheets with much larger diameters and heights.
[0037] FIG. 8 shows Light/Dark adaptation of bR-NLPs. Top Dark
adapted bR-NLPs with a .lamda..sub.max=549 nm; Bottom, light
adapted bR-NLP with a .lamda..sub.max=554 nm. Arrows indicate the
maximum peak heights which differ by a 5 nm shift between light and
dark adapted.
[0038] FIG. 9 shows diagram illustrating co-expression of membrane
proteins with apolipoprotein .DELTA.49A1 in the presence of lipid
increases solubility of multiple membrane proteins according to an
embodiment herein described. The membrane protein expressed alone
is indicated in white blocks; the membrane protein expressed in
presence of DMPC vesicles is indicated in gray blocks, and the
membrane protein co-expressed with apolipoprotein (.DELTA.49ApoA1)
in the presence of DMPC vesicles are indicated with black blocks.
Membrane proteins with the number of transmembrane domain in
parentheses are (GYPE) glycophorin B (MNS blood group) (2.TM.),
(CMTM1) CKLF-like MARVEL transmembrane domain (3TM), (EmrE) E. coli
SMR efflux transporter (4TM) (5HT1A) 5-hydroxytryptamine
(serotonin) receptor (7 TM), (bR) bacteriorhodopsin (7TM).
[0039] FIG. 10 shows the result of AFM analysis confirming the
association between NLPs and bR according to an embodiment herein
disclosed. Panel A shows the AFM image of NLPs produced through
cell-free co-expression of .DELTA.49A1 and bR in the presence of
DMPC. The brighter green regions (show in the figure as light grey)
are NLPs with a higher height indicating the insertion and
plausible location of bR in the lipid bilayer. Scale bars are 50
nm; Arrow 1, indicates expression of "empty"-NLP's, while arrow 2,
indicates the bR-NLP complex. Panel B shows a height histogram of
NLPs produced through conventional assembly of (top trace)
.DELTA.49A1 with DMPC alone or (bottom trace) in the presence of
purple membrane bR and DMPC. The shaded areas indicate populations
with an increased height. Panel C shows a eight histogram of NLPs
produced through cell-free expression of (top trace) .DELTA.49A1
with DMPC alone or (bottom trace) co-expression of bR and
.DELTA.49A1 in the presence of DMPC. The shaded areas indicate
populations with an increased height. NLPs heights were analyzed
through cross-sectional analysis
[0040] FIG. 11 shows the FTIR difference spectra for the
bR.fwdarw.M transition FTIR difference spectra of (A) bR WT and (B)
bR-NLP. The largest peaks are 9.4 and 0.34 mOD, respectively. The
positive bands represent vibrations in the M state and negative
bands represent the ground state. Despite the smaller signal, the
spectrum of BR NLP clearly indicates functional protein that is
stable over .about.10.sup.4 laser flashes.
[0041] FIG. 12 shows a schematic illustration of some models of
NLPs with (Panels B and C) and without (Panel A) bacteriorhodopsin
(shown in black) according to an embodiment herein described.
[0042] FIG. 13 shows lipoproteins expressed in cell-free extracts
according to an embodiment herein described. Lipoprotein were
purified using Ni-NTA affinity chromatography and run on a SDS-PAGE
gel, stained with Coomassie Brilliant Blue (A-B and D-E) or
detected by fluorescent scanning of labeled lysine residues (C).
Arrows indicate apolipoprotein of interest. Proteins A-D and F-G
are shown with SeeBlue MW marker (Invitrogen) (A) Full-length
apolipoprotein A1 (B) MSP1 truncated form of ApoA1 (C) Full-length
Apolipoprotein E4 (D) 22kD truncated ApoE4-fusion protein (H)
Thrombin cleaved truncated ApoE422k. Other Lipoproteins produced
(not shown) include Apolipophorin III B. mori, Apolipophorin III,
M. Sexta.
[0043] FIG. 14 shows a diagram illustrating the results of size
exclusion chromatography separation of ApoE422k Nanolipoprotein
particles (NLPs) according to an embodiment herein disclosed. Free
lipid, and free protein denoted on graph are separated from the NLP
rich fraction.
[0044] FIG. 15 shows native gel electrophoresis of NLPs according
to an embodiment herein disclosed. 1) Native Mark molecular weight
marker. 2) "Empty"-NLPs 3) Membrane protein (bacteriorhodopsin)
bR-NLPs. 4-20% Tris-glycine gel, with Tris-glycine running buffer,
stained with SyproRuby Stain (BioRad) Imaged with a Typhoon
scanner.
[0045] FIG. 16 shows a protein microarray of biotinylated bR-NLPs
according to an embodiment herein disclosed. (1)
biotinylated-bacteriorhodopsin (2) negative control, native
bacteriorhodopsin (3) biotinylated-bacteriorhodopsin associated
NLPs
[0046] FIG. 17 shows a diagram illustrating the light and dark
adapted visible spectra of bacteriorhodopsin associated NLPs,
according to an embodiment herein disclosed. (Top traces) Light and
dark adapted visible spectra of bacteriorhodopsin associated NLPs
and (bottom traces) NLPs without membrane protein. Black) Dark
adapted spectra (bR Xmax=550 nm). Grey) Light adapted spectra (bR
Xmax=560 nm)
[0047] FIG. 18 shows a diagram illustrating the Atomic Force
Microscopy of nanolipoprotein particles (NLPs) according to an
embodiment herein disclosed. NLPs consisting of cell-free produced
apoE4 22K lipoprotein and DMPC. Particle dimensions are as follows;
Height: 4.94 nm, std dev: 0.369 nm Width of top: 9.72 nm, std dev:
1.50 nm, Full width at half max: 20.4 nm std dev: 3.5 nm
[0048] FIG. 19 shows the results of the electron microscopy of
nanolipoprotein particles (NLPs) according to an embodiment herein
disclosed wherein the NLPs show a discoidal shape. The
magnification is 40K
[0049] FIG. 20 shows a diagram illustrating cell-free expression of
membrane proteins in the presence of NLPs (Co-translation)
according to an embodiment herein disclosed. The membrane protein
expressed alone is indicated with black bars; the membrane protein
expressed in the presence of pre-formed ApoE4 22k NLPs
(Co-Translation) is indicated with grey bars. Membrane proteins
with the number of trans membrane domain in parentheses are (GYPE)
glycophorin B (MNS blood group) (2.TM.), (CMTM1) CKLF-like MARVEL
transmembrane domain (3.TM.), (EmrE) E. coli SMR efflux transporter
(4.TM.) (5HT1A) 5-hydroxytryptamine (serotonin) receptor (7 .TM.),
(bR) bacteriorhodopsin (7.TM.).
[0050] FIG. 21 shows the results of experiments related to cell
free production of a membrane protein in the presence of NLP
scaffold according to an embodiment herein disclosed. Panel A shows
the cell free production of bR with a cell free extract, Lane 1.
bOp+retinal, Lane 2. bOp alone Lane 3. bOp+Lipid+retinal, Lane 4.
Apolipoprotein+bOp+Lipid+Retinal 5. Apolipoprotein+bOp+Lipid, 6.
Apolipoprotein alone, 7. Apolipoprotein+bOp+Retinal. Panel B shows
the results of PAGE analysis of size exclusion purified proteins or
NLP associated with bR. Lane 1. bR, Lane 2. bR+NLP, Lane 3. NLP.
Panel C shows microarray analysis of purified bR associated with
NLPs using an anti-biotin antibody. Lane 1. bR only, Lane 2. Size
exclusion purified biotinylated bR-NLPs. Lane 3. NLPs only. Lane 4.
Biotinylated IgG.
[0051] FIG. 22 shows the results of experiments related to the cell
free production of NLPs according to an embodiment herein disclosed
illustrated by Atomic force microscopy (AFM). The AFM can resolve
membrane surface features at a lateral resolution of 0.6-1 nm and a
vertical resolution of 0.1 nm, under physiological conditions
without the need of a crystalline system. AFM of scaffolded NLPs
with and without biotinylated bR are shown. Note, homogeneous
globular structures about 5 nm in height and 20-60 nm in
diameter.
[0052] FIG. 23 shows the schematic of NLP assembly according to an
embodiment herein disclosed.
[0053] FIG. 24 is a diagram illustrating size exclusion
chromatography of a NLPs assembly reaction mixture, according to an
embodiment herein disclosed.
[0054] FIG. 25 shows a sypro RUBY-stained native PAGE indicating
molecular size and relative homogeneity of NLPs prepared from four
different apolipoproteins without cholate according to an
embodiment herein disclosed.
[0055] FIG. 26 shows a diagram illustrating ion mobility traces of
mean aerodynamic diameter size distributions for four NLP
preparations obtained according to an embodiment herein
disclosed.
[0056] FIG. 27 shows a negative stain TEM and AFM of apoE422K-NLP
preparations with (panel B, D and F) and without cholate (Panel A,
C and E) according to an embodiment herein disclosed. The scale bar
in each panels is 50 nm; insets are a higher magnification.
[0057] FIG. 28 illustrates characterization of
fluorescently-labeled NLPs showing similar structure and in
comparison with unlabeled NLPs, according to an embodiment herein
disclosed. Cy.sub.3-labeled apoE422K (50%) and DMPC containing 1%
NBD-DMPC were used to form labeled NLPs. Panel A shows the SEC
fractionation of labeled NLPs. Early eluting peaks correspond to
large DMPC vesicles and NLP fractions, later eluting peak contains
unreacted apoE422K. Panel B shows a fluorescent scan of SEC
fractions: that contain NBD-DMPC (green), Cy.sub.3-apoE422K(red),
and fluorescent NLPs (yellow). Panel C shows the native PAGE of SEC
fractions highlighting the migration and homogeneity of the NLP
peak. Panel D shows a topographical AFM image (left) and TEM image
(right) of the main NLP fraction vial, highlighting the
homogeneity, size, and structure of the fluorescent NLPs
[0058] FIG. 29 shows a schematic illustration of refolded apoE422K
proteins generated in three different forms according to an
embodiment herein disclosed. Panel A. shows a fully extended form.
Panel B shows a doubled-back/"hairpin". and Panel C shows
semi-extended/"double-hairpin" folds.
[0059] FIG. 30 shows a method of purification and characterization
of self-assembled NLPs according to an embodiment herein disclosed.
Panel A shows the size exclusion chromatography (SEC) trace of
apoE422K/DMPC NLPs. Panels B and C show AFM (Panel B) and TEM
(Panel C) images of apoE422K/DMPC NLPs after formation and
purification. Cross-sectional analysis of AFM images (white and
black arrowed line in image corresponds to top and bottom
cross-sectional traces shown to the left, respectively) was used to
measure NLP height and diameter. The circles in the traces
correspond to the FWHM points used to determine particle diameter.
Arrow heads in the AFM and TEM images (C and D) point to NLPs of
differing diameter indicating size heterogeneity (black arrow head
points to smaller NLPs and white arrow head larger NLPs). AFM scale
bar 100 nm, TEM scale bar 50 nm.
[0060] FIG. 31 shows the NLP diameter distribution for 1000 NLPs
with a bin width of 1.0 nm according to an embodiment herein
disclosed. The inset shows the AFM image of the four different
sized NLPs. The scale bar is 10 nm.
[0061] FIG. 32 shows a diagram illustrating the comparison of
diameter distributions according to an embodiment herein disclosed,
wherein the diameter is measured by AFM, IMS and TEM. A. NLP
diameters determined through AFM binned at 1 nm. B. NLP diameters
determined through IMS.C. NLP diameters determined through TEM
binned at 1.6 nm
DETAILED DESCRIPTION
[0062] Methods and systems for monitoring the production of a
target protein in a NLP are disclosed. In particular, the methods
and systems herein disclosed allow monitoring the synthesis,
correct folding and incorporation of a target protein in a NLP. The
term "nanolipoprotein particle" "nanodisc" "rHDL" or "NLP" as used
herein indicates a supramolecular complex formed by a membrane
forming lipid and a scaffold protein, that following assembly in
presence of a target protein also include the target protein. The
scaffold protein and target protein constitute protein components
of the NLP. The membrane forming lipid constitutes a lipid
component of the NLP. The term "protein" as used herein indicates a
polypeptide with a particular secondary and tertiary structure that
can participate in, but not limited to, interactions with other
biomolecules including other proteins, DNA, RNA, lipids,
metabolites, hormones, chemokines, and small molecules.
[0063] The term "polypeptide" as used herein indicates an organic
polymer composed of two or more amino acid monomers and/or analogs
thereof. Accordingly, the term "polypeptide" includes amino acid
polymers of any length including full length proteins and peptides,
as well as analogs and fragments thereof. A polypeptide of three or
more amino acids can be a protein oligomer or oligopeptide.
[0064] As used herein the term "amino acid", "amino acidic
monomer", or "amino acid residue" refers to any of the twenty
naturally occurring amino acids including synthetic amino acids
with unnatural side chains and including both D and L optical
isomers. The term "amino acid analog" refers to an amino acid in
which one or more individual atoms have been replaced, either with
a different atom, isotope, or with a different functional group but
is otherwise identical to its natural amino acid analog. The term
"scaffold protein" as used herein indicates any protein that is
capable of self assembly with an amphipatic lipid in an aqueous
environment, organizing the amphipatic lipid into a bilayer, and
include but are not limited to apolipoproteins, lipophorines,
derivatives thereof (such as truncated and tandemly arrayed
sequences) and fragments thereof (e.g. peptides), such as
apolipoprotein E4, 22K fragment, liphorin III, apolipoprotein A-1
and the like. In particular, in some embodiments rationally
designed amphipathic peptides can serve as a protein component of
the NLP. In some embodiment, the peptides are amphipatic helical
peptides that mimic the alpha helices of an apolipoprotein
component that are oriented with the long axis perpendicular to the
fatty acyl chains of the amphipatic lipid and in particular of the
phosphoplipid.
[0065] The term "target protein" as used herein indicates any
protein having a structure that is suitable for attachment to or
association with a biological membrane or biomembrane (i.e. an
enclosing or separating amphipathic layer that acts as a barrier
within or around a cell). In particular, target proteins include
proteins that contain large regions or structural domains that are
hydrophobic (the regions that are embedded in or bound to the
membrane); those proteins can be extremely difficult to work with
in aqueous systems, since when removed from their normal lipid
bilayer environment those proteins tend to aggregate and become
insoluble. Accordingly, target proteins are protein that typically
can assume an active form wherein the target protein exhibits one
or more functions or activities, and an inactive form wherein the
target protein doe not exhibit those functions/activities.
Exemplary target proteins include but are not limited to membrane
proteins, i.e. proteins that can be attached to, or associated with
the membrane of a cell or an organelle, such as integral membrane
proteins (i.e. proteins (or assembly of proteins) that are
permanently attached to the biological membrane.), or peripheral
membrane proteins (i.e. proteins that adhere only temporarily to
the biological membrane with which they are associated). Integral
membrane proteins can be separated from the biological membranes
only using detergents, nonpolar solvents, or sometimes denaturing
agents. Peripheral membrane proteins are proteins that attach to
integral membrane proteins, or penetrate the peripheral regions of
the lipid bilayer with an attachment that is reversible.
[0066] The term "membrane forming lipid" or "amphipatic lipid" as
used herein indicates a lipid possessing both hydrophilic and
hydrophobic properties that in an aqueous environment assemble in a
lipid bilayer structure that consists of two opposing layers of
amphipathic molecules know as polar lipids. Each polar lipid has a
hydrophilic moiety, i.e., a polar group such as, a derivatized
phosphate or a saccharide group, and a hydrophobic moiety, i.e., a
long hydrocarbon chain. Exemplary polar lipids include
phospholipids, sphingolipids, glycolipids, ether lipids, sterols
and alkylphosphocholins. Amphipatic lipids include but are not
limited to membrane lipids, i.e. amphipatic lipids that are
constituents of a biological membrane, such as phospholipids like
dimyrisoylphosphatidylcholine (DMPC) or Dioleoylphosphoethanolamine
(DOPE) or dioleoylphosphatidylcholine (DOPC).
[0067] The membrane forming lipid and the protein components of the
NLP are generally able to self-assemble in a biological (largely
aqueous) environment according to the thermodynamics associated
with water exclusion (increasing entropy) during hydrophobic
association. In the methods and systems herein provided, the
amphipatic lipid and the protein components of the NLP are allowed
to assembly in a cell free expression system.
[0068] In the methods and systems herein disclosed, the production
of the target protein in a NLP is monitored by way of an indicator
protein. The term "indicator protein" as used herein refers to a
protein that is capable of assuming an active form wherein the
indicator protein is capable of exhibiting a detectable activity
and a non active form wherein the indicator protein is not capable
of exhibiting the detectable activity. In the methods and systems
herein disclosed, the indicator protein is selected so that the
detectable activity exhibited by the indicator protein of choice is
predictive and can be associated with the active form of the target
protein. In particular, in some embodiments the indicator protein
is a target protein itself and/or is structurally related to the
target protein of interest so that the production of the indicator
protein in an active form can be associated and considered
predictive of the production of the target protein in an active
form.
[0069] In some embodiments, the indicator protein and the target
protein are contacted with the membrane forming lipid and the
scaffold protein for a time and under conditions that allow
self-assembly of the indicator protein, target protein, scaffold
protein and membrane forming lipid in an NLP.
[0070] In some embodiments, the indicator protein and the target
protein expressed in a cell free expression system in presence of
the membrane forming lipid and the scaffold protein for a time and
under condition that allow the expression of the indicator protein
and the target protein and self-assembly of the indicator protein,
target protein, scaffold protein and membrane forming lipid in an
NLP.
[0071] In some embodiments, the indicator protein, target protein
and scaffold protein are expressed in a cell-free expression system
in presence of the membrane forming lipid that allow the expression
of the indicator protein, the target protein and the scaffold
protein and self-assembly of the indicator protein, target protein,
scaffold protein and membrane forming lipid in an NLP
[0072] As used herein, "the wording cell free expression" "cell
free translation", "in vitro translation" or "IVT" refer to at
least one compound or reagent that, when combined with a
polynucleotide encoding a polypeptide of interest, allows in vitro
translation of said polypeptide/protein of interest.
[0073] The term "polynucleotide" as used herein indicates an
organic polymer composed of two or more monomers including
nucleotides, nucleosides or analogs thereof. The term "nucleotide"
refers to any of several compounds that consist of a ribose
(ribonucleotide) or deoxyribose (deoxyribonucleotides) sugar joined
to a purine or pyrimidine base and to a phosphate group, and that
are the basic structural units of nucleic acids. The term
"nucleoside" refers to a compound (as guanosine or adenosine) that
consists of a purine or pyrimidine base combined with deoxyribose
or ribose and is found especially in nucleic acids. The term
"nucleotide analog" or "nucleoside analog" refers respectively to a
nucleotide or nucleoside in which one or more individual atoms have
been replaced with a different atom or a with a different
functional group.
[0074] Accordingly, the term polynucleotide includes nucleic acids
of any length DNA RNA analogs and fragments thereof. A
polynucleotide of three or more nucleotides is also called
nucleotidic oligomers or oligonucleotide.
[0075] In some embodiments, the polynucleotide is an engineered
polynucleotide designed such that the resulting protein may be
expressed as a full-length protein. In some embodiments the
polynucleotide is an engineered polynucleotide designed to encode a
protein fragment. Protein fragments include one or more portions of
the protein, e.g. protein domains or subdomains. In some
embodiments the polynucleotide is an engineered polynucleotide
designed to encode a mutated proteins. In particular, in some
embodiments the polynucleotide can also be designed such that the
resulting protein, protein fragment or mutated protein is expressed
as a fusion, or chimeric protein product (i.e. it is joined via a
peptide bond to a heterologous protein sequence of a different
protein), for example to facilitate purification or detection. A
chimeric product can be made by ligating the appropriate nucleic
acid sequences encoding the desired amino acid sequences to each
other using standard methods and expressing the chimeric product.
In particular, in some embodiments wherein the polynucleotide
encodes for a target protein, the polynucleotide can be engineered
so that target protein are labeled or tagged. Labeling or tagging
can be performed with methods that include, for example, FRET
pairs, NHS-labeling, fluorescent dyes, and biotin, as well as
coding for a "His-tag" to enable protein isolation and purification
via established Ni-affinity chromatography. In some embodiments
herein disclosed, the polynucleotide is a DNA molecule that can be
in a linear or circular form, and encodes the desired polypeptide
under the control of a promoter specific to an enzyme such as an
RNA polymerase, that is capable of transcribing the encoded portion
of the DNA.
[0076] In embodiments where the polynucleotide is DNA, the DNA may
be transcribed as part of the cell free reactions or system. In
those embodiments the DNA contains appropriate regulatory elements,
including but not limited to ribosome binding site, T7 promoter,
and T7 terminator, and the reagents or compounds include
appropriate elements for both transcription and translation
reactions. In other embodiments, the polynucleotide can be prepared
prior to addition to the cell free reactions/system, wherein the
polypeptide of interest is produced, and the reagents or compounds
include appropriate elements for and translation reactions
only.
[0077] Accordingly, as used herein, the term "cell free
expression", "cell free translation", "in vitro translation" or
"IVT" refer to methods and systems wherein the transcription and
translation reactions are carried out independently, and to systems
in which the transcription and translation reactions are carried
out simultaneously in a non-cellular compartment, e.g. glass vial.
In each of these methods and systems, the reagents or compounds
typically include a cell extract capable of supporting in vitro
transcription and/or translation as appropriate. In any case the
cell extracts must contain all the enzymes and factors to carry out
the intended reactions, and in addition, be supplemented with amino
acids, an energy regenerating component (e.g. ATP), and cofactors,
including factors and additives that support the solubilization of
the protein of interest.
[0078] These methods and systems are known in the art and can be
identified by the skilled person upon reading of the present
disclosure, and exist for eukaryotics, yeast, plants and
prokaryotic applications. Exemplary cell free expression systems
that can be used in connection with the methods and systems of the
present disclosure includes but are not limited to commercial kits
for various species such as extracts available from Invitrogen
Ambion, Qiagen and Roche Molecular Diagnostics, cellular extracts
made from E. coli or wheat germ or rabbit reticulocytes or prepared
following protocols, such as published laboratory protocols,
identifiable by a skilled person upon reading of the present
disclosure.
[0079] In some embodiments, the cell free system can operate in
batch mode or in a continuous mode. In the batch mode the reaction
products remain in the system and the starting materials are not
continuously introduced. Therefore, in batch mode, the system
produces a limited quantity of protein. In a continuous mode
instead, the reaction products are continuously removed from the
system, and the starting materials are continuously restored to
improve the yield of the protein products and therefore the system
produces a significantly greater amount of product.
[0080] In some embodiments, the cell free expression system is a
high-throughput expression system, where an array (i.e., at least
two) of polynucleotides (coding for the same or different
polypeptides) is processed simultaneously in multi-well reaction
plates, where each polynucleotide is in a well of the plate. The
reaction plate can typically have at least 2 wells, and typically
has 12-, 24-, 96-, 384-, or 1536-wells; other sizes may also be
used.
[0081] In some of those embodiments the array is carried out to
explore the function and potential relationships of proteins
encoded within any genome. In some of those embodiments the array
is carried out for parallel analysis of multiple binary
interactions between proteins and other molecules. In addition, in
some embodiments engineering and tagging techniques allows the
orientation of proteins of interest and expands the capabilities
and use of protein microarrays. Some of those embodiments wherein
cell-free expression is combined with array-based proteomics are
applicable in particular in protein biochemistry, molecular
diagnostics and therapeutics. In some embodiments array-based
methods and systems provide a high-throughput format with which to
investigate protein-protein, protein-DNA, and protein-small
molecule interactions on the NLP.
[0082] In some embodiments, the target protein and the indicator
protein are expressed in the cell free reaction system where a
preformed NLP is included. In those embodiments addition of
pre-formed NLPs to an actively expressing cell-free protein
synthesis reaction is performed for a time and under condition to
allow direct insertion of the target protein and indicator protein
as they are synthesized in the cell-free system, into the NLP.
[0083] In some embodiments, the target protein, indicator protein
and scaffold protein are co-expressed in a cell free system wherein
both kinds of proteins can be expressed in a single reaction in a
system that can include the appropriate additives directed to
facilitate reactant solubilization. In those embodiments, the
co-expressed target protein, indicator protein and scaffold protein
are then allowed to assembly of membrane proteins into NLP
nanostructures; possibly within the same reaction mixture. Some of
those embodiments allow providing NLPs overcoming the requirement
for the purification and reassembly of the NLP complex. Some of
those embodiments also provides a single-step process for the
production of soluble membrane proteins that eliminates the need
for cell growth, cell lysis, and subsequent purification,
refolding. Some of those embodiments allow avoiding use of
detergents while allowing single-step addition of lipids and other
molecules important to protein function.
[0084] In particular, co-expression of both scaffold protein and
target membrane protein in presence of phospholipids and
surfactant/detergents can be performed in a single reaction
mixture, wherein a "one-pot" reaction generates, in situ, both
scaffold protein and target membrane protein; NLP self-assembly
will ensue using phospholipid already in the reaction mixture. Some
of those embodiments are exemplified by the co-expression of a
truncated apolipoprotein (Ll-ApoA1) and the bacteriorhodopsin gene,
which results in the functionally active seven transmembrane helix
bacteriorhodopsin protein (bR) upon addition of retinal cofactor as
illustrated in Examples 1-22 and in FIGS. 1-32.
[0085] In some embodiments, each of the target protein, indicator
protein and/or the scaffold protein expressed in the IVT system is
comprised of more than one protein, thus resulting in NLP including
two or more target proteins and/or two or more scaffold proteins.
In particular, in some of those embodiments cell-free co-expression
of membrane proteins in NLP complexes enable production in a same
NLP of multiple classes of membrane associated proteins previously
not conveniently obtainable.
[0086] In some embodiments the target proteins are membrane
proteins such as protein coupled receptors (GPCRs), which include,
for example acetylcholine receptors (AChRs) and rhodopsin. GPCRs
conform to a shared common structure that is believed to traverse
the cell surface membrane seven times forming a helical structure
encompassing a ligand binding site. Of the three cytoplasmic
dominions the C-loop has a C-terminal tail that recognized and
activates specific hetero-trimeric GTP binding proteins (G
proteins) in an agonist dependent manner.
[0087] Further exemplary target proteins include Ion channels (IC)
and small multidrug resistance transporter (SMR), and additional
membrane proteins that mediate essential cellular processes
including signal transduction, transport, recognition,
bioenergetics, and cell-cell communication. This would include
G-coupled receptors, Toll receptors and various kinases that
important for the aforementioned processes. Additional examples
include targeting whole family of proteins within any species such
as membrane proteins from Thiobacillus denitrificans that contain
unusual membrane associated [NiFe]hydrogenase complex, a group of
highly expressed membrane bound c-type cytochromes and a group of
highly upregulate membrane proteins of unknown functions that
contribute to the bioenergetics of an organism.
[0088] Exemplary proteins suitable to be used as target proteins in
the methods and systems herein disclosed are indicated in Table
1.
TABLE-US-00001 TABLE 1 Exemplary target proteins CB FL CF Target
Endogenous ligand Family Fluorescent ligand Availability 1 X X X
V2R Vasopressin GPCR FAM-Vasopressin commercial 2 X X X CRF
Corticotropin RF I (CRF) GPCR FAM/Rhod-CRF commercial 3 X X X ETB
Endothelin GPCR FAM/Rhod-Endothelin commercial 4 X X X MC5R
Melanocortin GPCR BODIPY-TMR NDP-.alpha.-MSH commercial 5 V X X
NTR1 Neurotensin GPCR FAM-neurotensin commercial 6 X S 5HT1A
Serotonin GPCR synthesize 7 X S D1 Dopamine GPCR synthesize 8 X S
H2 Histamine GPCR synthesize 9 X X M1 Acetylcholine/Muscarine GPCR
FITC-pirenzapine commercial 10 X X hERG Voltage IC IVGN-0107
synthesized 11 X .alpha.1AR Epinephrine GPCR BODIPY FL prazosin
commercial 12 X .beta.1AR Epinephnne GPCR BT-CGP12177 commercial 13
V X OP1R Opiods GPCR FL-naltrexone/naloxone commercial 14 X
.beta.2AR Epinephrine GPCR synthesize 15 V X M2
Acetylcholine/Muscarine GPCR synthesize CB = Cell Based Assay
available (X = CBA exists, V = vector exists for assay in
development) FL = Fluorescent Ligand available (X = Yes, S =
requires synthesis) CF = Expressed in Cell-Free system
[0089] In general, target proteins that can advantageously be
included in NLP using methods and systems herein disclosed,
comprise all the proteins and in particular membrane protein, whose
over-expression results in cell toxicity (in vivo), protein
aggregation, mis-folding, and low yield and that are instead
expressed in a cell free system that includes appropriate
additives.
[0090] In some embodiments, the additives used in the cell free
reaction systems include any substance that improves the
solubilization of the protein of interest and/or of any other
protein components that are present in the reaction mixtures, any
substance that may augment protein production and any substance
that improves protein functions. Those additives include but are
not limited to cofactors (e.g. retinal, heme) other proteins that
facilitate modification (e.g. glycosylases, phosphatases,
chaperonins) lipids, redox factors, detergents and protease
inhibitors, and in particular, phospholipids such as
dimyristoylphosphatidyl choline (DMPC) and the like, and
surfactants/detergents such as cholate, triton X-100 and the likes.
Exemplary detergents that can be used for protein solubilization in
the methods and systems herein disclosed, include
Heptanoyl-N-methyl-glucamide, Octanoyl-N-methyl-glucamide,
Nonanoyl-N-methyl-glucamide, n-Nonyl-b-D-gluco-pyranoside,
N-Octyl-b-D-glucopyranoside, Octyl-b-D-thiogluco-pyranoside,
N,N-Dimethyldodecylamine-N-oxide and Glycerol.
[0091] Suitable indicators include Bacteriorhodopsin (bR) from H.
salinarium, which is also an exemplary target protein having 7
transmembrane spanning regions that can be produced, purified and
regenerated by exogenously adding all-trans retinal. bR was used in
a series of experiments exemplified in the Examples section as a
model target protein to be included in NLPs according to methods
and systems herein disclosed. bR is in particular suitable as an
indicator for monitoring the production in an NLP of a GPCR, a heme
containing protein or other chromospheres containing membrane
protein. Additional exemplary indicator proteins that can be used
in the methods and systems herein disclosed and in particular for
in situ monitoring of cell-free membrane protein synthesis with
concomitant NLP formation, include GFP, GFP-fused to a membrane
protein, cytochromes and dye labeled proteins such as sensory
rhodopsin, proteorhodopsin, and phytochromes. Like bR, these
proteins can be produced by the cell-free transcription/translation
technology in the presence of detergents, lipids and phospholipids,
as well as other reaction additives such as chromophores and
coupled to NLP formation. The ability of the protein to turn color
through the binding of the chromophore provides a direct
calorimetric measure assay for a properly folded and active
protein. This allows the inference to assay conditions that are
favorable to proper conditions for folding and obtaining functional
activity of any target protein such as GPCRs. Additional target
proteins that can be monitored using the indicator proteins herein
described can be identified by a skilled person upon reading of the
present disclosure and will not be further discuss in details.
[0092] In some embodiments, the detectable activity of the
indicator protein in its active form is the ability of the
indicator protein to directly or indirectly bind a label or labeled
molecule wherein binding of the indicator protein to the labeled
molecule is associated with the emission of a labeling signal.
[0093] The terms "label" and "labeled molecule" as used herein
refer to a molecule capable of detection, including but not limited
to radioactive isotopes, fluorophores, chemiluminescent dyes,
chromophores, enzymes, enzymes substrates, enzyme cofactors, enzyme
inhibitors, dyes, metal ions, nanoparticles, metal sols, ligands
(such as biotin, avidin, streptavidin or haptens) and the like. The
term "fluorophore" refers to a substance or a portion thereof which
is capable of exhibiting fluorescence in a detectable image. As a
consequence the wording and "labeling signal" as used herein
indicates the signal emitted from the label that allows detection
of the label, including but not limited to radioactivity,
fluorescence, chemolumiescence, production of a compound in outcome
of an enzymatic reaction and the likes.
[0094] In some embodiments, detecting the indicator protein
detectable activity can be performed by providing a labeled
molecule that specifically binds to the indicator protein, the
labeled molecule providing a labeling signal. The labeled molecule
is in particular contacted with the nanolipoprotein particle for a
time and under condition to allow binding of the labeled molecule
with the indicator protein in the nanolipoprotein particle. The
labeling signal is then detected from the labeled molecule bound to
the indicator protein in the nanolipoprotein particle.
[0095] In some embodiments, the labeled molecule comprises a
molecule that specifically binds to the indicator protein (or to
another polypeptide or protein to be detected) and a label attached
to the molecule, with the label providing the labeling signal.
[0096] In some embodiments, (e.g. when the label or labeled
molecule is a chromophore), the signal can be a visual signal (e.g.
calorimetric) detectable by naked eye and/or with the aid of
appropriate equipment.
[0097] In particular, indicators able to provide a detectable
visual signal, such as calorimetric indicators e.g.
bacteriorhodopsin will only incorporate the chromophore when
properly folded. UV-visible spectroscopy is an example of a
detection method that could be used to confirm proper folding of
the indicator protein. Empirical observation of color development
is the primary detection method. Visualization can take place
before, during and after a cell-free reaction is complete. The
measurement/visualization of color can also be used to follow
purification/isolation steps. Visualization is primarily by eye or
UV-visible spectroscopy, and is compatible/possible using other
technologies such as IR, and radiolabeling and additional
technologies identifiable by the skilled person upon reading of the
present disclosure.
[0098] Additional indicators able to provide a detectable visual
signal include proteins able to bind a specific antibody labeled
with a fluorophore only when properly folded. In those embodiments
the antibody can be labeled for detection, which can occur by
visualization of the antibody bound to the indicator protein or by
other methods identifiable by a skilled person upon reading of the
present disclosure.
[0099] In the methods and systems herein disclosed, detection can
be performed without visualization. For example, in some
embodiments, (e.g. wherein binding of the indicator protein to the
label results in the production of a compound in outcome of an
enzymatic reaction), the labeling signal can be provided by the
measured amounts of compound produced in outcome of the enzymatic
reaction or other detecting techniques identifiable by a skilled
person upon reading of the present disclosure.
[0100] In some embodiments, the methods and systems herein
disclosed are used to produce proteins for structural studies,
including for example NMR and X-ray crystallography. In particular,
these cell free methods can be applied to integral membrane
proteins in a high-throughput manner, as a variety of conditions
can be rapidly tested to identify optimal expression
parameters.
[0101] In some embodiments, the methods and systems herein
disclosed are used to produce NLPs suitable as drug delivery
vehicles, wherein the particles are formed by taking advantage of
the ability of amphipathic apolipoproteins to solubilize certain
phospholipid vesicle substrates, transforming them into a
relatively homogeneous population of disk-shaped bilayers whose
perimeter is circumscribed by apolipoprotein molecules.
[0102] In some embodiments, NLPs are provided by the methods and
systems herein disclosed by using different scaffold proteins,
which allow to tailor the average size of the particles, e.g. from
10 to 60 nm (+/-3%), and in particular 10 to 30 nm (+/-3%), at an
average height of 5.0 nm. The nanoscale bilayers so obtained can be
used to investigate and control assembly of oligomeric integral
membrane proteins critical to macromolecular recognition and
cellular signaling. Those embodiments can be performed using any
apolipoprotein-like molecules as potential structures for
solubilizing the membrane proteins via NLP formation. Examples
include, but are not limited to ApoE4, ApoA1, MSP1 (ApoA1
truncations), synthetic peptides and insect lipophorins.
[0103] In some embodiments, the methods and systems herein
disclosed are performed at predefined lipid protein ratio, assembly
conditions and/or with the use of preselected protein component and
amphipatic lipid so to increase the yield, control the size of the
resulting NLP and/or provide an NLP of pre-determined dimensions so
to include a predetermined target protein.
[0104] In particular, in some embodiments the scaffold protein is
selected to define the size of NLPs. Lipophorin III lipoproteins
make assemble into larger NLPs with diameters 10-30 nm range,
apolipoprotein A1 NLPs range in size from 10-25 nm, truncated
.DELTA.(1-49)Apolipoprotein .DELTA.115-35 nm. Adjustment of protein
to lipid ratios increasing lipid will also increase the size of the
NLP.
[0105] In some embodiments the amphipatic lipid is tested to
provide the most stable and native-like environment. For example a
target protein that is naturally found in the inner mitochondrial
membrane would contain lipids specific to that region of the cell.
In particular the protein of the inner mitochondrial membrane
requires a membrane compose of 20% cardiolipin for proper function.
A protein that requires more flexibility in it function may require
lipids with a higher degree of unsaturation creating a bilayer with
more fluidity. While incorporating a target protein the stability
of the protein may be improve by using a detergent that has been
proven to allow the protein to retain native activity as
measured/monitored by our indicator protein.
[0106] In some embodiments the amphipatic lipid is selected to
resemble the native lipid composition in which the membrane protein
is known to function.
[0107] In some embodiments the lipid to scaffold protein ratio: is
selected to optimize and maximize the yield leading to NLP
formation.
[0108] In some embodiments, the assembly parameters are selected to
allow the constituents reach maximum NLP formation reflective of a
thermodynamic endpoint.
[0109] In some embodiments, the methods and systems herein
disclosed can be used to monitor the efficiency of selected
cell-free reagents in producing a predetermined target protein.
[0110] In some embodiments, the methods and systems herein
disclosed can be used to monitor the efficiency of refolding of a
denatured target protein.
[0111] In some embodiments, the indicator protein can be used be
used as a marker for various gel and column separations methods and
system, which are identifiable by a skilled person upon reading of
the present disclosure.
[0112] In some embodiments, the indicator protein can be used as a
marker for protein microarrays, in various applications
identifiable by a skilled person upon reading of the present
disclosure.
[0113] In some embodiments, the indicator protein can be used as
sizing calibrators for microfluidics, in applications identifiable
by a skilled person upon reading of the present disclosure.
[0114] The systems herein disclosed can be provided in the form of
kits of parts. For example the protein can be provided as embedded
in the NLP to follow process controls. The indicator protein can be
included as a protein alone or in the presence of lipids/detergents
for transition in to nano-particles. The indicator protein can be
included as a plasmid or PCR DNA product for
transcription/translation. The indicator protein may be included as
encoded RNA for translation.
[0115] In a kit of parts, a polynucleotide, amphipatic lipid,
target protein, indicator protein and/or scaffold protein are
comprised in the kit independently possibly included in a
composition together with suitable vehicle carrier or auxiliary
agents. For example a polynucleotide--can be included in one or
more compositions alone and/or included in a suitable vector. Also
each polynucleotide can be included in a composition together with
a suitable vehicle carrier or auxiliary agent. Furthermore, the
indicator protein can be included in various forms suitable for
appropriate incorporation into the NPL. For example, in embodiments
wherein the indicator protein is bR, the cofactor all-trans-retinal
would be included in a kit that also contained the encoded genetic
information for the production of bacteriorhodopsin as the
calorimetric indicator.
[0116] In some embodiments, the labeling molecule can also be
included in the kit herein disclosed, including but not limited to
labeled polynucleotides, labeled antibodies, other labels
identifiable by the skilled person upon reading of the present
disclosure.
[0117] . Additional components can also be included and comprise
microfluidic chip, reference standards, and additional components
identifiable by a skilled person upon reading of the present
disclosure.
[0118] In the kit of parts herein disclosed, the components of the
kit can be provided, with suitable instructions and other necessary
reagents, in order to perform the methods here disclosed. In some
embodiments, the kit can contain the compositions in separate
containers. Instructions, for example written or audio
instructions, on paper or electronic support such as tapes or
CD-ROMs, for carrying out the assay, can also be included in the
kit. The kit can also contain, depending on the particular method
used, other packaged reagents and materials (i.e. wash buffers and
the like).
[0119] Further details concerning the identification of the
suitable carrier agent or auxiliary agent of the compositions, and
generally manufacturing and packaging of the kit, can be identified
by the person skilled in the art upon reading of the present
disclosure.
EXAMPLES
[0120] The methods and system herein disclosed are further
illustrated in the following examples, which are provided by way of
illustration and are not intended to be limiting.
[0121] In particular, in the following examples, the methods and
systems herein disclosed are exemplified by a calorimetric assay
that indicates production, correct folding, and incorporation of
bacteriorhodopsin (bR)-- a 7-transmembrane (TM) protein and
prototypical of the 7-TM class of membrane receptors--into a
soluble nanolipoprotein particle. In particular, in the following
examples NLP are membrane bilayer mimetics resulting from
self-assembly of an apolipoprotein, (including related fragments,
peptides and derivative thereof) and phospholipid.
[0122] The results of the experiments illustrated in the following
examples show that synthesis of bR membrane proteins in cell-free
reactions was functional by UW/V is and that the bR proteins
associate directly with the NLPs. The bR protein is only purple
when retinal ligand is present allowing proper folding. In
particular, as illustrated in some of the examples below, when bR
and apolipoprotein are co-expressed in a cell-free system, assembly
into bR-NLP constructs follows; if retinal is present in the
reaction mixture, a clear pink-purple colored solution results
indicating formation of bR-NLPs.
[0123] In some experiments herein exemplified the approach did
provide both structural and functional results within minutes. This
assay approach is demonstrated' for example, by the expression of
bacterioOpsin gene in the presence and absence of different
additives, which results in properly folded and functionally active
seven transmembrane helices bacteriorhodopsin protein (bR)
contained within an apo-AI apolipoprotein NLP construct.
Example 1
Cell-Free Production of NLPs bR Protein
[0124] The experimental strategy for cell-free membrane protein-NLP
self-assembly was based on the ability of membrane proteins to
insert into lipid bilayers during cell-free synthesis, the
apolipoprotein ability to sequester lipid bilayer patches, and the
demonstrated ability of NLPs to than solubilize membrane proteins.
Individual plasmid DNAs encoding the membrane protein and the
apolipoprotein are added to the cell-free reaction with the
addition of phospholipids and cofactors to produce membrane protein
associated discoidal nanolipoprotein particles (NLPs) in a single
reaction. In particular, as shown in FIG. 1 constituents (DNA,
lipid vesicles, cofactors and cell-free lysates) are added together
in a single reaction vial. The cell-free lysates take advantage of
the T7 coupled transcription and translation system to produce a
mixed population of self-assembled NLPs with and without associated
integral membrane protein.
[0125] In a first series of experiments, two plasmids were used to
generate the integral membrane protein and the lipoprotein NLP
support, one encoding membrane protein bacterioOpsin (bOp) and the
second encoding a .DELTA.1-49 apolipoprotein A-1 fragment
(.DELTA.49A1). The plasmids were co-expressed, in the presence of
all-trans-retinal and the phospholipid
dimyristoyl-phosphatidylcholine (DMPC), resulting in functional
bacteriorhodopsin (bR) protein solubilized in a discoidal
bR-NLP.
[0126] In particular, the truncated form of Apo A1 (.DELTA.1-49) or
.DELTA.49A1 was cloned using the following primers: forward,
5'-atgctaaagctccttgacaactgg-3' (SEQ ID NO: 1) and reverse,
5'-ttactgggtgttgagcttcttagtg-3' (SEQ ID NO: 2). This construct is
six amino acids shorter than our truncated form of Apo A1
(.DELTA.1-49) or .DELTA.49A1, and was expected to perform similarly
in NLP assembly and characterization.
[0127] The resulting PCR product was cloned into the vector
pIVEX2.4d using NdeI and SmaI restriction sites. This vector also
contains a His-tag for nickel affinity purification. The
bacterioOpsin sequence (bOp), which encodes the bacteriorhodopsin
protein, was amplified from a plasmid p72bop (Sonar et al., 1993;
obtained from Kenneth Rothschild) using the following primers:
5'-ggggcatatgcaagctcaaat-3' (SEQ ID NO: 3) and
5'-ggggatccaaaaaaaacgggcc-3' (SEQ ID NO: 4). The gene represents a
synthetic form of bOp that was designed for E. coli-based
expression (1). The resulting PCR product was cloned directionally
into the HIS-tagged pIVEX 2.4b vector using the NdeI and BamHI
restriction enzyme sites. All constructs were verified by DNA
sequencing.
[0128] Cell-free reactions were then performed. In particular,
preparative reactions are carried out using the Invitrogen's
Expressway Maxi kit or Roche's RTS 500 ProteoMaster Kit. Basically,
lyophilized reaction components (Lysate, Reaction Mix, Amino Acid
Mix, and Methionine) are dissolved in Reconstitution Buffer and
combined as specified by the manufacturer. For co-expression a
total of 5 .mu.g of each plasmid DNA (bOp and .DELTA.49A1) was
added to the lysate mixture with added DMPC vesicles and retinal
cofactor (see below). The reactions were incubated at 30.degree. C.
or 37.degree. C. for 4-24 h. For membrane protein survey studies
co-expression of 0.2 ug .DELTA.49A1 DNA and 1 ug of each membrane
protein DNA was added to the cell-free mixture where [.sup.35S]Met
(135 mCi/mmol final) (Perkin Elmer, Waltham, Mass.) was added in
place of methionine. The soluble fraction was obtained by
centrifuging the reactions at 14000.times.g for 5 min.
Autoradiograms were generated by overnight exposures to proteins
separated by SDS-PAGE (data not shown). Percent solubility was
determined using ImageJ software (U.S, National Institutes of
Health) to quantize autoradiogram bands for the soluble fractions
in the presence and absence of apolipoprotein .DELTA.49A1.
[0129] In some reactions a retinal cofactor was added. To this
purpose, an all trans-retinal (Sigma) solution was prepared with
100% ethanol at a stock concentration of 0.586 or 10 mM. The stock
solution was diluted to achieve a final working concentration of
30-50 .mu.M in cell-free reactions.
[0130] The lipid component of the NLPs was also prepared. Small
unilamellar vesicles of DMPC (liposomes) were prepared by probe
sonicating a 68 mg/mL aqueous solution of DMPC until optical
clarity is achieved; typically 15 min on ice is sufficient. A 2
min. centrifugation step at 13700 RCF was used to remove any metal
contamination from the probe tip. The individual lipid component
was added to the cell-free reaction at a concentration of 2
mg/mL.
[0131] Soluble fractions were purified. In particular, NLP
complexes were then purified through Affinity purification. In
particular, immobilized metal affinity chromatography was used to
isolate the proteins of interests (truncated 49A1 and bOp) from the
cell-free reaction mixture based on affinity of the N-terminal
poly-His tag. The soluble fraction was separated from precipitated
protein by centrifugation for 5 min at 18K RCF at 4.degree. C. The
soluble fraction was mixed with Ni-NTA Superflow resin (Qiagen)
according to the manufacturer's protocol using native purification
conditions with the following modifications; 5 mM imidazole in PBS
buffer was used for washing and 400 mM imidazole PBS buffer was
used for elution of the His-tagged proteins. All elutions were
combined, concentrated and buffer exchanged into TBS using a 100K
MWCO molecular weight sieve filters (Vivascience) in a volume of
200 .mu.L.
[0132] The samples were also characterized by SDS-PAGE, Native
PAGE, UV-visible spectroscopy, and atomic force microscopy (AFM) as
illustrated in the following examples. A survey study of other
membrane proteins co-expressed with the truncated apolipoprotein
also significantly increased solubility all of the membrane
proteins surveyed
Example 2
Characterization of NLPs Produced By Cell-Free System
Solubilization of the bR-NLP Complex
[0133] The experimental design outlined in Example 1 of cell-free
co-expression for refolding and incorporation into NLPs was also
demonstrated using bR from Halobacterium salinarium, and truncated
apolipoprotein A-1 (A 1-49) or .DELTA.49A1. The bR protein is a
seven transmembrane (TM) helical protein and serves as a structural
model protein for rhodopsin and other 7-TM proteins such as GPCR
family members.
[0134] Simultaneous cell-free protein expression of both bR and
.DELTA.49A1 in the presence of DMPC in a single reaction produces a
functional bR-NLP complex (FIG. 2 and FIG. 3).
[0135] In particular, as illustrated in FIG. 2, bOp & DMPC
(sample 1) shows bR is insoluble in the absence of co-expression of
.DELTA.49A1; bOp, .DELTA.49A1 co-expressed in the presence of DMPC
(sample 2) shows bR remains in the soluble fraction with
co-expressed .DELTA.49A1; .DELTA.49A1 & DMPC (sample 3) shows
production of "empty"-NLPs; the control (sample 4) shows cell-free
reaction (No DNA) in the presence of DMPC only. All were expressed
in the presence of 30-50 .mu.M all-trans-retinal and 2 mg/mL DMPC.
Purple color development observed in sample 1 and 2 indicates
incorporation of retinal into the bOp transcript representing
proper folding of bR.
[0136] Single-step co-expression, assembly and purification of the
soluble bR-NLP complex was completed within 4 hours giving
analogous yields and functions comparable to previous published
findings (2). This extremely rapid approach was also applicable to
a wide variety of other transmembrane proteins (FIG. 4).
[0137] Although bR coloration was observed in the presence of DMPC
without .DELTA.49A1, very little of the material was soluble
compared to when the .DELTA.49A1 was co-expressed in the reaction
mixture (FIG. 2 and FIG. 5), as indicated in the soluble (S) and
pelleted (P) lanes with and without .DELTA.49A1 co-expression). Two
methods for refolding of cell-free expressed bR into lipid vesicles
have been previously reported by Sonar et al., and Kalmbach et al
(2, 3). However, these two approaches required multiple steps over
a lengthy period of time and were further encumbered by limited
membrane protein accessibility due to the nature of liposomes (2,
3). Similar results, cell-free synthesis of bR in the presence of
liposomes and cofactor alone, produced functional membrane protein
(purple color) that was insoluble (FIG. 2, and FIG. 5). In contrast
the co-expressed bR-NLP complexes were functional, stable and
soluble using our procedure.
[0138] Demonstration using bacteriorhodopsin (bR) and truncated
apolipoprotein A1 (.DELTA.49A1) produced bR-NLP complexes that were
shown to be soluble, discoidal in shape and light active FIGS. 2,
4, 8, 10, and 11. Distinct purple coloration, an indication of
properly folded functional bR protein, was observed when all-trans
retinal and phospholipid were included in the reaction mixtures
(see FIGS. 2, 3, 4, and 21). Solubility survey results indicate
this rapid approach may also be applicable to a wide variety of
other transmembrane proteins (FIG. 9).
Example 3
Characterization of NLPs produced by cell-free system
SDS Page,
Native Page SEC and AFM
[0139] bR-NLP complex heterogeneity was also observed by both
native gel electrophoresis and SEC.
[0140] In particular, NLPs produced as described in Example 2 were
first analyzed by SEC, to detect the separation of NLPs from larger
lipid-rich material. Size exclusion chromatography identified a
size shift in the bR-NLP complex compared to empty NLPs or
liposomes. The bR-NLP complexes eluted primarily before empty NLPs
and after liposomes (FIG. 5). A size range of approximately 470-680
kDa was observed for bR-NLP complexes, which was 160-370 kDa larger
than the empty self-assembled NLPs (FIG. 6).
[0141] The NLPs were also analyzed by SDS Page. In particular, a 1
.mu.L aliquot of the total (T) cell-free reaction, soluble (S)
fraction and resuspended pellet (P) were diluted with 1.times.LDS
Sample buffer with reducing agents (Invitrogen), heat denatured and
loaded on to a 4-12% gradient pre-made Bis-Tris gel (Invitrogen)
along with the molecular weight standard SeeBlue plus2
(Invitrogen). The running buffer was 1.times. MES-SDS (Invitrogen).
Samples were electrophoresed for 38 minutes at 200V. Gels were
stained with coomassie brilliant blue.
[0142] The particles were also analyzed by native PAGE. Equal
amounts of NLP samples (0.5-1.0 .mu.g) were diluted with 2.times.
native gel sample buffer (Invitrogen) and loaded onto 4-20%
gradient pre-made Tris-glycine gels (Invitrogen). Samples were
electrophoresed for 2 hrs. at a constant 125 V. After
electrophoresis, gels were incubated with SYPRO Ruby protein gel
stain (Bio-Rad) for 2 hours and then de-stained using 10% MeOH, 7%
Acetic acid. Following a brief wash with ddH.sub.2O, gels were
imaged using the green laser (532 nm) of a Typhoon 9410 (GE
Healthcare) with a 610 nm bandpass 30 filter. Molecular weights
were determined by comparing migration vs. log molecular weight of
standard proteins found in the NativeMark standard
(Invitrogen).
[0143] This heterogeneity may have been due to multiple factors
such as number of lipids per NLP, bR oligomerization within the
NLPs and/or generation of NLPs with varying diameters. Particle
diameters measured by atomic force microscopy AFM (data not shown)
supports the latter.
[0144] To this extent NLPs were imaged using and Asylum MFP-3D-CF
atomic force microscope. Images were captured in tapping mode with
minimal contact force and scan rates of 1 Hz. Asylum software was
used for cross-sectional analysis to measure NLP height and
diameter. For experimental analysis, the heights and diameters were
measured on 182 NLPs produced by cell-free expression in the
absence of bR and 430 total NLPs (empty-NLPs 185 and 255 bR-NLPs)
produced by cell-free co-expression. Two-tailed student T-tests
were run to compare both the height and diameter of the "empty"-NLP
population in the sample co-expressed with bR compared to the
sample with no bR expressed. A p-value of <0.01 was considered
significant. A student T-test compares two populations of data and
can determine if the difference between the two sets is statically
significant or insignificant.
[0145] Size and shape of the NLPs determined by AFM showed a height
of the "empty"-NLP to be 5.0+/-0.3 while the height of bR-NLPs was
6.4+/-0.3. UV-visible spectroscopy identified a 5 nm shift upon
light adaption indicating functionality
Example 4
Characterization of NLPs Including Membrane Protein Indicator
Demonstration of Membrane Protein Activity
[0146] Functional activity of the soluble, self-assembled,
co-expressed bR-NLP complex was determined by light-dark adaptation
(FIG. 8).
[0147] The light-dark adaptation yielded a 5 nm shift with a dark
absorption maximum of 549 nm and light absorption maxima of 554 nm.
These results indicated that the majority of active bR was in a
monomeric form (4). This is in agreement with other studies that
used pre-purified apolipoprotein scaffolds to solubilize native
forms of bR (5). The major advantage of our approach is that it
allows to obtain folded light active bR-NLP assemblies in less than
four hours that have been self-assembled in a single step, thereby
eliminating the need for isolation of membrane protein, protein
purification, dialysis and refolding protocols prior to the
formation of NLP-membrane protein complexes.
Example 5
Characterization of NLPs
Co-Expression Survey of Membrane Proteins
[0148] In order to determine if the co-expression method increased
solubility a series of membrane proteins with varying numbers of
transmembrane domains were expressed using cell-free methods.
Cell-free expression of the membrane protein alone and
lipid-assisted expression of the membrane protein were used for
comparison. The reactions included .sup.35S-methionine in order to
quantify the protein. Autoradiograms (not shown) were generated
from total and soluble fractions separated by SDS-PAGE.
Densitometry using ImageJ software (National Institutes of Health)
was used to analyze the autoradiograms. The percent solubilized
membrane protein compared to the total membrane protein was plotted
in FIG. 9.
[0149] In particular in FIG. 9 a comparison was made between (MP
alone--Grey) the membrane protein expressed alone, (Lipid
Assisted-striped) expression of the membrane protein in the
presence of DMPC vesicles, and (Co-expressed--Black) membrane
protein co-expressed with apolipoprotein (.DELTA.49ApoA1) in the
presence of DMPC vesicles. The data was generated from
autoradiograms by the incorporation of .sup.35S-Methionine in to
the cell-free reaction (data not shown) that were quantified using
ImageJ software (U.S. National Institutes of Health).of
autoradiograms generated from SDS-PAGE.
[0150] In all cases co-expression with the truncated apolipoprotein
.DELTA.49A1 was greater that the expression of the membrane protein
alone or lipid-assisted membrane protein expression (FIG. 9). Also
in all cases the solubility of the membrane protein increased with
co-expression of .DELTA.49ApoA1 with added (FIG. 9).
Example 6
Compared Self-Assembly of Cell-Free Produced and Conventionally
Produced NLPs
[0151] NLPs produced by cell-free methods and NLPs assembled by
conventional means (6) were both examined by AFM to assess NLP size
and shape and to demonstrate the association between bR with NLPs.
For comparison to cell-free produced bR-NLP complexes, both bR-NLPs
and "empty"-NLPs were also prepared using previously described
methods (6, 7).
[0152] Conventional assembly of NLPs is described herein in
Examples 13 to 22. Briefly, the truncated form of Apo A1
(.DELTA.1-55) called MSP1T2 or .DELTA.55A1 was purchased from
Nanodisc Inc. For "empty"-NLPs .DELTA.55A1 was combined with DMPC
liposomes in a ratio of 1:4 by mass in TBS buffer. The mixture was
then incubated at room temp for 2 hours. The NLPs were then
purified by size exclusion chromatography. Assembly of bR-NLPs:
.DELTA.55A1 was mixed with DMPC in a ratio of 1:4 by mass in TBS
buffer. Sodium cholate solution was then added to a final
concentration of 20 mM. Purple membrane bacteriorhodopsin was then
added in a 0.67 mass ratio to the .DELTA.55A1 apolipoprotein.
Incubation proceeded as described above, followed by dialysis in
TBS for detergent removal. The NLPs were then purified by size
exclusion chromatography.
[0153] In particular, the size exclusion chromatography was
performed as follows. The NLPs made with and without incorporated
membrane protein were purified from `free protein` and `free lipid`
by HPLC (Shimadzu) using a Superdex 200 10/300 GL column (GE
Healthcare), with TBS at a flow rate of 0.5 ml/min. The column was
calibrated with four protein standards HMW Gel filtration
calibration kit (GE Healthcare), of known molecular weight and
Stokes diameter that span the separation range of the column and
the NLP samples. The void volume was established with blue dextran.
The NLP fraction is concentrated about 10-fold to approximately 1.0
mg/ml using molecular weight sieve filters (Vivascience) having
molecular weight cutoffs of 50 kDa. Protein concentration was
determined using the ADV01 protein concentration kit
(Cytoskeleton), which is based on Coomassie dye binding.
[0154] These NLPs and bR-NLP complexes were made with a similarly
truncated form of Apolipoprotein A1 (.DELTA.1-55) or .DELTA.55A1
(MSP1T2, Nanodisc Inc.), purple membrane bR and DMPC liposomes (6,
7). Both the co-expressed and conventionally assembled bR-NLPs
showed similar increases in particle height relative to an "empty"
NLP indicating likely association of bR protein within the NLPs
(FIG. 10).
[0155] Empty NLPs produced either by conventional methods or
cell-free displayed heights of approximately 5.0.+-.0.3 nm (s.d.)
as determined by AFM (FIGS. 10B and 10C respectively). The NLPs
produced by either conventional assembly of .DELTA.49A1 and bR
(FIG. 10B) or co-expression of .DELTA.49A1 and bR (FIG. 10C)
appeared as two distinct discoidal populations when examined by AFM
cross-sectional height analysis. The first population is
approximately 5.1.+-.0.3 nm (s.d.) in height, analogous to
"empty"-NLPs (FIG. 10C). The second population, which was not
observed in control experiments lacking bR, was approximately
6.4.+-.0.3 nm (s.d.) in height (FIG. 10C). The increased height
observed in the presence of bR is located in the center region of
the NLP (bright green dot, pseudo color) is consistent with the bR
being contained within the NLP lipid bilayer (FIG. 10A).
Additionally, the increased height particles produced in the
presence of bR also had an associated increase mean diameter
(27.8.+-.5.8 nm (s.d.)) relative to the "empty" .DELTA.49A1-NLPs
(22.0.+-.5.1 nm) (Table 2).
[0156] Using solely the increase in height as a basis for
distinguishing bR-NLPs from "empty" .DELTA.49A1-NLPs, an overall
yield of protein incorporation of 58% was determined (Table 2).
Two-tailed student T-tests indicated that there was no
statistically significant difference between the diameter and
height of the "empty" .DELTA.49A1-NLPs produced by cell-free
methods in the presence (n=185; 2a) and absence (n=182; 1) of bR
(Table 2) with p-values of 0.94 and 0.04 respectively. However, a
statistically significant increase in diameter and height was
observed between the bR-NLPs (n=255; 2b) and "empty"
.DELTA.49A1-NLPs (n=185, 2a) with p-values of 1.8 E.sup.-24 and 3.9
E.sup.-155 respectively (Table 2). Those results are illustrated in
Table 2 that includes a summary of analysis of cell-free expressed
NLPs with and without co-expressed bR
TABLE-US-00002 TABLE 2 Relative % Height Increase % Sample (nm) +/-
s.d. Diameter NLP 1 "empty" .DELTA.49A1 NLPs 5.0 +/- 0.3 1 100 2a
Co-expressed "empty"-.DELTA.49A1 5.1 +/- 0.3 1 42 NLPs 2b
Co-expressed .DELTA.49A1/bR-NLPs 6.4 +/- 0.3 * 1.3 * 58 *
Statistically significant, see text for specific values.
[0157] AFM was also used to visualize the first SEC fraction, where
high molecular weight lipid complexes were observed consistent with
results reported in Chromy et al (6) in which was described
lipid-based macro-molecular formations unable to enter a
native-PAGE and had the appearance of liposom-like material (FIG.
7). The majority of this material was distinctly different in size,
ranging 35-60 nm in diameter and 6.5-20 nm in height indicating the
majority of the material was large lipid complexes such as
liposomes or membrane patches (FIG. 7).
Example 7
Cell-Free Expression and Purification of Apolipoproteins
[0158] The methods described below outline cell-free expression and
purification of apolipoproteins. In particular, it is described the
cell-free production of a selected N-terminal truncation of human
apolipoprotein E4 which does not require post-translational
modification.
[0159] The following materials and instruments were used:
Apolipoprotein (ApoA1, MSP, Apo E4, lipophorin III, or truncations
.DELTA.49ApoA1 and ApoE4 22k) clones of interest from the
LLNL-IMAGE Consortium cDNA collection or as a gift from
collaborating labs, subcloned in to an expression vector such as,
pET32a thioredoxin (Novagen) (8, 9), pIVEX-2.4b (Roche), or pEXP4
(Invitrogen); Spectrophotometer UV-visible A.sub.260/A.sub.280
quantification or PicoGreen dsDNA Quantification Kit
(Invitrogen/Molecular Probes); Cell-Free Expression System:
Expressway.TM. Maxi Cell-Free E. coli Expression System
(Invitrogen) or RTS 500 ProteoMaster E. coli HY Kit (Roche);
Thermomixer, Eppendorf Thermomixer R (for Roche lysates) or
Incubator shaker for example New Brunswick C24 (for Invitrogen
lysates); Disposable fritted columns 3 mL capacity (Bio-Rad);
Ni-NTA Superflow resin (Qiagen); Ni-NTA buffers (modified Qiagen
recipes) Binding buffer: 50 mM NaH.sub.2PO.sub.4, 300 mM NaCl; pH
8.0; Wash Buffer: 50 mM NaH.sub.2PO.sub.4; 300 mM NaCl; 10 mM
Imidazole; pH 8.0; Elution Buffer: 50 mM NaH.sub.2PO.sub.4; 300 mM
NaCl; 400 mM Imidazole; pH 8.0; Gel electrophoresis equipment;
NuPAGE 4-12% Bis-Tris SDS-PAGE gel with 1.times.MES-SDS running
buffer (Invitrogen), Protein Quantification Kit and standards, such
as Bio-Rad Protein Assay (Bio-Rad) Vivaspin6, ultrafiltration
Devices, 10 k MWCO (Sartorius Biotech); Centrifuge such as
Eppendorf 5804R (Needs to fit 15 mL Falcon tubes); Thrombin
(Novagen); DMPC; 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine
(Avanti Polar Lipids); Probe or bath sonicator;
.beta.-mercaptoethanol; TBS Buffer: 10 mM Tris-HCl; 0.15 M NaCl;
0.25 mM EDTA; 0.005% NaN.sub.3 (sodium azide) adjust to pH 7.4; and
FPLC Instrument (Shimadzu SCL-10A), size exclusion column (Superdex
200 10/300 GL (GE Healthcare Life Sciences).
[0160] In particular, Lipophorin III DNA clones (M. sexta and B.
mori) were obtained from the lab of Robert Ryan at Children's
Hospital Oakland Research Institute (CHORI). Truncated
Apolipoprotein E4 22kDa N-terminal thioredoxin fusion plasmid was
obtained from Karl Weisgraber at the University of California, San
Francisco. The 193 amino acid protein sequence of the 22 kD
Apolipoprotein E4 construct is as follows, with the two initial
amino acids, Gly-Ser, are left over from the thrombin cleavage site
in pET32a. Midi or Maxi prepped plasmid DNA was prepared according
to the Qiagen protocol.
TABLE-US-00003 (SEQ ID NO: 5)
GSKVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQ
EELLSSQVTQELRALMDETMKELKAYKSELEEFQLTPVAEETRARLSKEL
QAAQARLGADMEDVRGRLVQYRGEVQAMLGQSTEELRVRLASHLRKLRKR
LLRDADDLQKRLAVYQAGAREGAERGLSAIRERLGPLVEQGRVR
[0161] The cDNAs for apolipoprotein were selected and cloned into
expression vector of interest such as pIVEX-2.4b (Roche Applied
Science), GFP folder or pETBlue-2 (Novagen.), pET32a (thioredoxin
fusion vector). The plasmids were propagated by transforming into
Top10 or DH5.alpha. chemically competent cells (Invitrogen) and
isolate DNA using HiSpeed Plasmid Maxi or Midi Kits (Qiagen). The
N-terminal truncated apolipoprotein E4-22kD (ApoE422k) thioredoxin
(trx) fusion protein construct in pET32a (ApoE422k-trx) is
illustrated here (FIG. 13).
[0162] The Midi or Maxi prepped plasmid DNA concentrations were
determined by PicoGreen dsDNA Quantification Kit (Molecular Probes)
or by UV-visible spectroscopy A.sub.260/A.sub.280. The cell-Free
protein production reactions were performed using either the
Expressway.TM. Maxi Cell-Free E. coli Expression System
(Invitrogen) or RTS 500 ProteoMaster E. coli HY Kit (Roche) using
.about.15 .mu.g of midi or maxi prepped DNA in a 1 mL reaction
size.
[0163] The reactions were incubated at 30.degree. C. shaking at 990
rpm in a thermomixer (Roche RTS ProteoMaster or Eppendorf
Thermomixer R)-(Roche Lysates) or 37.degree. C. shaking at 225 rpm
in a shaker incubator (New Brunswick)-(Invitrogen Lysates). All
reactions were run overnight (although 4 hours is sufficient). A
5-10 .mu.l sample was collected for further analysis.
[0164] The His-tagged apolipoprotein (ApoE422k-trx) was purified by
using Ni-NTA native affinity chromatography, and 1 mL of the Ni-NTA
slurry, equivalent to 500 .mu.L column bed volume (Qiagen) was
equilibrated with binding buffer and resuspend the resin to form a
50% slurry again. The equilibrated slurry was added to the
cell-free post-reaction mixture and mix at 4.degree. C. for 1-2
hours. The mixture was added to a 3-mL fritted plastic column and
collected the flow through for SDS-PAGE analysis.
[0165] The column was washed with eight column volumes (500 .mu.L)
of native wash buffer. Fractions are collected for SDS-PAGE
analysis.
[0166] The bound apolipoprotein was eluted with six column volumes
of native elution buffer.
[0167] All collected fractions were analyzed by denatured gel
electrophoresis using a NuPAGE 4-12% Bis-Tris SDS-PAGE gel with
1.times. MES-SDS running buffer for 38 minutes at 200V
(Invitrogen). The load buffer is LDS Sample Buffer (Invitrogen).
Volumes to load for SDS-Page gels were as follows: 1 .mu.L of total
reaction and non-bound flow through, 5 .mu.L wash fractions 1-2, 20
.mu.L of remaining washes and all elutions. Gels were stained with
Coomassie brilliant blue.
[0168] Elution fraction of interest determined by gel
electrophoresis were combined and concentrated and buffer exchanged
into TBS using an ultrafiltration device vivaspin6. In particular,
concentration from 6 mL to 100 .mu.L was easily achieved in
.about.15 min at 5000 RCF in an Eppendorf 5804R centrifuge with a
fixed angle rotor check each 3-5 min. Buffer exchange into TBS pH
7.4 required at least 3 dilutions and re-concentration steps.
Alternatively eluted protein could were dialyzed (spectrapor 1 MWCO
3500) against TBS buffer overnight and concentrated by immersion of
the dialysis membrane in PEG 8000 (polyethylene glycol).
[0169] The final protein concentration was determined by Bradford
total protein concentration following the manufacturer's
protocol.
[0170] Small unilamellar vesicles of DMPC were then prepared by
probe sonicating 20 mg DMPC lipid into 1 mL TBS at 6 amps for
approximately 15 minutes or until optical clarity is achieved.
Typically fifteen minutes is sufficient to achieve optical clarity.
An appropriate container choice was a thick walled 3 mL glass
conical vial. In particular, lipid solution were vortexed lightly
before sonication to help get to lipid into the buffer. Lipid
should be stored at -20.degree. C. when not in use, and protected
from water absorption. When sonicating lipid overheating of the
lipids was avoided by either sonicating in a beaker of ice or
cooling the sample every few minutes. The solution was practically
water clear at the end of the sonication. If the probe hits the
side of the glass vessel metal will be sloughed off into the
solution and the solution will become grayish. The metal can be
removed by a short centrifugation at 13,700 RCF for two minutes
after transferring to a 1.5 ml Eppendorf tube. Remove the
supernatant and use. Any white pellet indicated DMPC that is not in
vesicle form. Alternatively, sonicate in bath sonicator to optical
clarity and skip the centrifugation step.
[0171] The sample was transferred to an 1.5 mL tube. Any
contaminant metal was removed from the probe by centrifugation at
13700 RCF for 2 minutes in a 1.5 mL tube.
[0172] Thioredoxin fusion protein tags were removed by incubating
2-4 mg of the produced protein with 100 .mu.g/mL of the sonicated
DMPC overnight at 24.degree. C. Thrombin was added at 1:500 w/w
ratio (thrombin:apolipoprotein) and incubated at 37.degree. C. for
one hour. The reaction was halted by the addition of
.beta.-mercaptoethanol to a final concentration of 1%. 5 .mu.g of
the product were analyzed by SDS-PAGE as described above. The
results are shown in FIG. 13.
[0173] Contaminant thioredoxin (trx), thrombin and
.beta.-mercaptoethanol were then removed from the apolipoprotein,
ApoE422k by size exclusion chromatography using a FPLC Instrument
(Akta, GE Healthcare and Life Sciences or Shimadzu SCL-10A), and
size exclusion column (Superdex 200 10/300 GL) with a TBS buffer at
a flow rate of 0.5 mL/min. The fractions of interest were
determined by gel electrophoresis combine and concentrate as
above.
Example 8
Nanolipoprotein Particle (NLP) Formation and Purification
[0174] The methods described below outlines nanolipoprotein
Particle (NLP) formation and purification. The following materials
and equipments were used: DMPC:
1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (Avanti Polar Lipids);
Purified apolipoprotein protein or truncation (ApoE422k construct);
TBS Buffer: 10 mM Tris-HCl; 0.15 M NaCl; 0.25 mM EDTA; 0.005% NaN3
(sodium azide); adjust to pH 7.4. 30.degree. C. and 20.degree. C.
water baths; Probe or bath sonicator; Spin filter, 0.45 .mu.m;
Concentrator 50 kD MWCO, Vivaspin 2 (Sartorius Inc.) (Other
concentrator brands that are angled are also acceptable such as
Agilent, because the nanolipoprotein particle produced will be
larger than 200 kD, a 100 kD filter may be useful); FPLC Instrument
(Shimadzu SCL-10A), size exclusion column (Superdex 200 10/300 GL
(GE Healthcare Life Sciences).
[0175] Nanolipoprotein particles (NLPs) form in a self assembly
process in the correct mass ratio of apolipoprotein to lipid. This
ratio needs to be optimized for each different apolipoprotein. The
ratio described below is for ApoE422k (6). Other ratios can be
found in the literature (7, 10, 11).
[0176] The water bath incubators were started with temperatures at
30.degree. C. and 20.degree. C. 34 mg of DMPC were probe sonicated
into 1 mL of TBS at 6 amps for approximately 15 minutes or until
optical clarity is achieved. DMPC solution was centrifuged at 13700
RCF for 2.5 min to remove residual metal from probe sonicator. The
supernatant was transferred into a new tube. Apo E422K was combined
with DMPC in a ratio of 1:4 by mass in TBS buffer in a 1.5 mL
Eppendorf tube. Typically batches are of the 250 .mu.L size.
[0177] Transition temperature procedure was performed as follows:
the tube was immersed in water bath for 10 minutes each 30.degree.
C. (above DMPC transition temp.) followed by 20.degree. C. (below
DMPC transition temp.). The procedure was repeated three times then
the tube was incubated at 23.8.degree. C. overnight.
[0178] Filter preparation was performed through a 0.45 .mu.m spin
filter at 13700 RCF for 1 min. Purify NLPs using size exclusion
chromatography. A Shimadzu SCL-10A FPLC was used that was equipped
with a Superdex 200 10/300 GL column with TBS buffer, a 200 .mu.L
sample injection volume, and a flow rate of 0.5 mL/min. Collect 0.5
mL fractions see FIG. 14.
[0179] Fractions were concentrated using a Vivaspin 2
ultrafiltration device with a 50k MWCO as described in Example
7.
Example 9
Biotinylation of Membrane Protein
[0180] The methods described below outline the biotinylation of
membrane bound proteins, and in particular of Bacteriorhodopsin
(bR). The following materials and equipments were used: EZ-Link
Sulfo-NHS-LC-Biotin (Pierce); Bacteriorhodopsin (Sigma); Bath
sonicator; Ultracentrifuge (Beckman-Coulter Optima TLX, TLA-120.2
fixed angle rotor); 1.times. BupH PBS buffer (Pierce): 0.1 M
NaH2PO4, 0.15 M NaCl; pH 7.0. Bacteriorhodopsin can also be
produced in a cell-free manner and purified in the denatured state.
A re-folding procedure is then employed to incorporate the retinal
according to the methods of Rothschild et al (2, 12).
[0181] Biotinylation of the membrane protein (MP) provides a tool
for investigating the incorporation of the MP with the NLP.
Biotinylation using of bacteriorhodopsin supplied in membrane
sheets from Sigma selectively labels only the solvent exposed
lysine residues when using EZ-Link Sulfo-NHS-LC-Biotin (Pierce)
which is impermeable to membranes. Bacteriorhodopsin in membrane
sheets is easily separated from the aqueous phase by
centrifugation. For other membrane proteins that may be solubilized
in detergent micelles removal of excess biotin solution will need
to be accomplished using a desalting column or other means.
Membrane proteins including bR may be expressed in a cell free
manner and biotinylated (2, 13-16).
[0182] In particular, bacteriorhodopsin (bR) purchased from Sigma
and stored as a lyophilized powder at 4.degree. C. was resuspended
in BupH PBS buffer in the original bottle. Amine containing buffers
such as TBS, were avoided due to the interaction with the
biotinylation reagent. The sample was bath sonicated eight times
for 1 min. each chilling the bottle on ice for one minute in
between each burst. UV-visible spectra were recorded to confirm the
concentration of bR in solution using the molar extinction
coefficient at 568 nm of 63,000 M-1-cm-1.
[0183] A freshly made 10 mM solution of EZ-Link Sulfo-NHS-LC-Biotin
(Pierce) was prepared according to the manufactures recommendation
in ddH2O.
[0184] The biotin solution was added to the bacteriorhodopsin
solution in a 20-fold molar excess, and incubated on ice for two
hours.
[0185] The excess biotin was removed by centrifugation of the
solution in an ultracentrifuge at an RCF of 89,000 (although 50,000
should be sufficient) for 20 minutes at 4.degree. C. The
supernatant was removed and the bR pellet resuspended in TBS
buffer. This process was repeated two times total. In particular,
Bacteriorhodopsin in membrane sheets was extremely sticky, and did
pellet well at the RCF listed. 85-90% recovery of bR was achieved
with careful resuspension and washing of tips and tubes.
Resuspension should be in the TBS buffer used for assembly (or
other buffer of interest that will be used for assembly).
[0186] UV-visible spectra were collected as described above to
calculate the concentration of the solution and the percent
recovery typically around 85-90% with careful resuspension.
Example 10
Membrane Protein Incorporation into Nanolipoprotein Particles
(MP-NLPs)
[0187] The methods described below outline incorporation of a
membrane protein into nanolipoprotein particles (NLPs). The
following materials and equipments were used: DMPC
[1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine] (Avanti Polar
Lipids); Purified apolipoprotein or truncation (ApoE4 22kD
construct); TBS Buffer: 10 mM Tris-HCl; 0.15 M NaCl; 0.25 mM EDTA;
0.005% NaN.sub.3 (sodium azide), adjusted to pH 7.4; Sodium Cholate
(Sigma) 500 mM solution in TBS; Biotinylated Bacteriorhodopsin (bR)
(Sigma) from Example 21; 30.degree. C. and 20.degree. C. and
23.8.degree. C. water baths; Probe Sonicator; Dialysis cups 10,000
MWCO (Pierce) or D-Tube Dialyzers, mini (Novagen); Spin filter,
0.45 .mu.m; FPLC Instrument (Shimadzu SCL-10A); size exclusion
column (Superdex 200 10/300 GL (GE Healthcare Life Sciences);
Concentrator 50 kD MWCO, Vivaspin 2 (Sartorius Inc.).
[0188] The water bath incubators were started at temperatures at
30.degree. C. and 20.degree. C. 34 mg of DMPC were probe sonicated
into 1 mL of TBS at 6 amps for approximately 15 minutes or until
optical clarity was achieved. Alternatively, the DPMC can be
sonicated in bath sonicator to optical clarity (see Example 7).
[0189] The solution was centrifuged at 13K for 2 minutes to remove
residual metal sloughed off from probe sonicator. 250 .mu.L were
batched in a 1.5 mL Eppendorf tube. Combine Apo E422K with DMPC in
a ratio of 1:4 by mass in TBS buffer. Sodium cholate solution was
then added to a final concentration of 20 mM. The biotinylated
bacteriorhodopsin membrane protein was then added in a 0.67 mass
ratio to the Apo E422k apolipoprotein.
[0190] The transition temperature procedure was performed as
follows: the tube was immersed in water bath for 10 minutes each
30.degree. C. (above DMPC transition temp.) followed by 20.degree.
C. (below DMPC transition temp.). The procedure was repeated three
times and the tube was then incubated at 23.8.degree. C.
overnight.
[0191] The cholate detergent was removed and MP-NLPs (bR-NLPs) were
allowed to self-assembly; the sample was loaded into a pre-soaked
D-Tube Dialyzers, mini (Novagen). The sample was then dialyzed
against 3 changes each of IL TBS buffer over a 2-3 day period at
room temperature. In particular, dialysis at 4.degree. C. was used
for unstable membrane proteins. Detergent use was compatible with
the membrane protein of interest. Adsorbent beads (Bio beads,
Bio-Rad) were also used to remove the detergent. If dialysis cups
were used (Pierce) the sample was split into three pre-soaked
dialysis cups. Care was taken not to create bubbles or droplets on
the sides of the cups.
[0192] The sample was then concentrated using an ultrafiltration
device, Vivaspin 2 (Sartorius) MWCO 50K to 200 .mu.L.
[0193] The supernatant was transferred into new tube. Size
exclusion chromatography was performed using a Shimadzu SCL-10A
FPLC, equipped with a Superdex 200 10/300 GL column (GE Healthcare
Life Sciences). The buffer was TBS with a 200 .mu.L sample
injection volume, a 0.5 mL/min flow rate and 0.5 mL-1.0 mL fraction
size.
[0194] The fractions of interest were concentrated using an
ultrafiltration device, Vivaspin 2 (Sartorius) MWCO 50K for NLP
peaks.
Example 11
Validating Nlp Formation by Native Gel Electrophoresis and
Confirmation of Membrane Protein Association and Functionality with
NLPs by Microarray, UV Visible Spectroscopy, AFM and EM
[0195] The methods described below outlines a procedure to validate
protein association by microarray and UV visible spectroscopy. The
following materials and equipments were used: 4-20% Tris-Glycine
polyacrylamide gel, 1.times. Tris-Glycine native running buffer,
2.times. Native Sample buffer, Native Mark molecular weight marker
(Invitrogen); Sypro Ruby Stain (Bio-Rad) light sensitive, Aqueous
destain solution: 10% Methanol; 7% acetic acid; Fluoroimager with
appropriate filter for SyproRuby stain; Biotinylated positive
control protein such as biotinylated-bR; Bovine serum albumin 1
mg/ml solution; PBS-Tween buffer: 1.06 mM KH2PO4; 2.97 mM Na2HPO4;
NaCl 1551.72 mM, 0.05% tween-20 (v/v) pH 7.4; 1.times.PBS buffer,
(Gibco): 1.06 mM KH2PO4; 2.97 mM Na2HPO4; NaCl 1551.72 mM, pH 7.4;
Cyanine-5-Strepavidin (Rockland) solution (5 .mu.g/mL); Barcoded
.gamma.-Aminopropylsilane coated glass slides (GAPS-II; Corning);
Robotic arrayer; Hybridization Chamber (Grace Bio-Labs); Blocking
buffer: 1 mg/mL BSA in 1.times.PBS, Wash Buffer: 1.times.PBS;
Laser-based confocal scanner (ScanArray 5000 XL; Perkin-Elmer);
UV-visible plate reader (Bio-TEK Synergy HT); 96 well flat bottom
UV plate (Corning Costar UV Plate).
Validation Through Native Polyacrylamide Gel Electrophoresis
[0196] Native polyacrylamide gel electrophoresis is used to
validate the association of proteins of interest (apolipoprotein
and/or membrane protein) with NLP fractions eluted from the size
exclusion column. Protein identification is confirmed with mass
spectrometry.
[0197] Native-PAGE gels, 4-20% Tris-glycine, were run with 0.75
.mu.g total loaded protein estimated by A.sub.280 absorbance. 10
.mu.L of molecular weight standards, Native mark (Invitrogen)
diluted 20.times. in native sample buffer were loaded on the gels.
The gels were run at 125V for approximately 2 hours.
[0198] The gels were stained with .about.150 mL of SyproRuby
protein stain (Bio-Rad) following the microwave staining method: 30
sec. microwave, 30 sec. mixing on shaker table, 30 sec microwave, 5
min. shake, 30 sec. microwave, finally 23 min. on shaker table at
room temperature. The gels were destained for 1.5 hours on a shaker
table at room temperature.
[0199] The gels were imaged using a Typhoon Imager with appropriate
filters selected for the SyproRuby fluorescence.
[0200] The results are illustrated in FIG. 15.
Confirmation of Membrane Protein Association and Functionality with
NLPs by Microarray
[0201] Microarray spotting technology was used to attach NLPs to an
amino-silane coated glass slide in an array format for streptavidin
binding studies (17-19). Biotinylated bacteriorhodopsin (bR) was
used to validate the incorporation of bR into nanolipoprotein
particle fractions eluted from size exclusion chromatography.
Cyanine-5-Strepavidin was used for fluorescence detection of
biotinylated bacteriorhodopsin.
[0202] Microarray single print head was used to deposit
approximately 1 mL of diluted protein solution on the slide. It was
determined that robotic spotting is best when the humidity is
greater than 30%. Proteins were spotted in 4.times.4 squares with
16 replicates of each sample, generating .about.300 .mu.m diameter
spots with a spot-to-spot distance of 350 .mu.m.
[0203] Protein microarrays were spotted on GAPSII amino silane
glass slides (Corning) with bacteriorhodopsin bR
(non-biotinylated), biotinylated-bR, biotinylated-bR-NLPs, using a
robotic arrayer. Non-biotinylated bR was used as a negative
control, and biotinylated-bR was used as a positive control.
[0204] Bacteriorhodopsin (bR) concentrations of 10 mM, as
determined by UV-visible spectroscopy as described above were used
for all samples.
[0205] Proteins were cross-linked to the glass slides by exposure
to UV light for five minutes. Unused slides were stored at
4.degree. C. without UV cross-linking.
[0206] The hybridization chamber was applied with a volume capacity
of 950 .mu.L to the slide carefully as to not disrupt the array.
Carefully add reagents below without injecting bubbles.
[0207] The slides were blocked with BSA (1 mg/mL) for 30 minutes.
The slides were washed with 1.times.PBS for 15 minutes.
Cyanine-5-streptavidin (5 .mu.g/mL) was bound for 15 minutes. The
slides were washed in 1.times.PBS then nanopure water each for 15
min. The slides were dried by centrifugation or air dry.
[0208] Protein microarrays of bR, biotinylated-bR and bR-NLPs were
imaged with a laser-based confocal scanner (ScanArray 5000 XL;
Perkin Elmer) using the VheNe 594 nm laser for detection of any
bound Cyanine-5-streptavidin.
[0209] Images were collected and analyzed using the mean pixel
intensities with Scan Array software (Perkin Elmer) (data analysis
not shown).
[0210] The results are illustrated in FIG. 16.
Confirmation of Membrane Protein Association and Functionality with
NLPs by UV-Visible Spectroscopy, AFM and EM
[0211] UV-visible spectroscopy of light and dark adapted
bacteriorhodopsin can be used to determine the functionality of the
protein and relates information regarding the conformation of the
protein (4).
[0212] UV-visible spectra were collected in 96-well plate reader
using 100 .mu.L of sample in a UV detectable flat bottom plate.
Dark adapted spectra were collected after keeping the sample
wrapped in foil overnight taking care not to expose the sample
prior to spectral collection. Light adapted spectra were collected
after exposure to a full spectrum bright lamp for 15 min. The
results are illustrated in FIG. 17. A 5-10 nm visible shift between
light and dark adapted spectra indicates a functional protein
(4).
[0213] Further in-depth physical characterization of these
particles was used to demonstrate functional protein
insertion/association. Combined with the biochemical evidence
methods such as Atomic force microscopy (AFM) and Electron
microscopy (EM) addresses whether the end product of
self-assembly/association was successful by determining physical
parameters to identify insertion and localization of membrane
proteins. Atomic force microscopy (AFM) (FIG. 18), and Electron
microscopy (FIG. 19) although not fully described here, but are
used to image the prepared discs and determine diameter and height
measurements as well as sample heterogeneity.
Example 12
Membrane Protein Synthesis and Purification in a Single Step Using
Cell-Free Synthesis in Conjunction with Pre-Formed Nanolipoprotein
Particles (NLPs)
[0214] The methods described below allow membrane protein synthesis
and determination of solubility in a single step using cell-free
synthesis in conjunction with pre-formed nanolipoprotein particles
(NLPs).
[0215] Cell-free expression of membrane proteins has usually
employed either of two possible methods; one: expression and
purification in a denatured state followed by refolding in the
presence detergents and/or lipids as well as any cofactors such as
all trans-retinal for bacteriorhodopsin or two: expression in the
presence of detergents or lipids (2, 13-15). Solubilization of the
membrane protein with detergent is generally followed by a dialysis
step to return the membrane protein to a lipid bilayer vesicle. The
method described here utilizes preformed NLPs as an additive to
increase the membrane protein production, solubility and
stabilization by incorporation into a NLP lipid bilayer
(Co-translation). The procedure uses commercially available
cell-free extracts with the addition of membrane protein plasmid
DNA (pEXP4 expression vector (Invitrogen)), and pre-formed NLPs to
synthesize folded functional membrane protein in one step.
[0216] Cloned membrane protein cDNAs of interest were into the
expression plasmid pEXP4 (Invitrogen) and were propagated by
transforming into Top10 or DH5.alpha. chemically competent cells
(Invitrogen). Isolate plasmid DNA using a HiSpeed Plasmid Maxi or
Midi Kits (Qiagen).
[0217] Cell-Free expression reactions were carried out using the
Expressway.TM. Maxi Cell-Free E. coli Expression System
(Invitrogen) protocols with the addition of 15 .mu.g of membrane
protein DNA, for a 1 mL reaction, 300 .mu.g of purified NLPs (ApoE4
22k assembled with DMPC see above section). For scintillation
counting the manufacturer protocol for the incorporation of
35S-Methionine was followed. Reactions were scalable to other
volumes following the same ratios. Control experiments were carried
out without the addition of NLPs using the same lysate batch.
[0218] The reactions were incubated at 37.degree. C. shaking at 225
rpm in a shaker incubator (New Brunswick). The reactions were
continued for 1.5-2 hours.
[0219] A 5 .mu.L aliquot of the total (T) reaction was retained for
SDS-PAGE and autoradiograms (not shown), the reaction was then
centrifuged for 5 min. at 4.degree. C., and 18000 RCF. The
supernatant was collected and a 5 .mu.L aliquot of the soluble (S)
fraction placed into a 12.times.75 mm glass tube.
[0220] 100 .mu.l of 1N NaOH was added and the resulting mixture was
incubated at room temperature for 5 minutes. 2 ml of cold 10% TCA
(trichloroacetic acid) were further added to the 12.times.75 mm
tube. Place at 4.degree. C. for 10 minutes.
[0221] The precipitate was collected via vacuum filtration through
a Whatman GF/C glass fiber filter (or equivalent). The filter was
pre-wetted with a small amount of 10% TCA prior to adding the
sample.
[0222] The tube was rinsed twice with 1 ml of 10% TCA and then
rinsed once with 3-5 ml of 95% ethanol. Each of the rinses was
passed through the GF/C filter.
[0223] The filter was placed in a scintillation vial, aqueous
scintillation cocktail was added, and counted in a scintillation
counter. The cpm did reflect the amount of radiolabel that was
incorporated.
[0224] FIG. 20 shows protein yield for the soluble (S) fraction
based on scintillation counting of incorporated 35S-Methionine in
the presence and absence of added NLPs.
[0225] In particular, in FIG. 20, a comparison was made between the
membrane protein expressed alone (Black bars) or in presence of
pre-formed ApoE4 22k NLPs (Co-Translation) (Grey bars).
[0226] In all cases the expression in the presence of NLPs
increased membrane protein solubility. Solubility is determined by
removing a 5 ul of the reaction supernatant after a 10 minute
centrifugation at 14000 rpm and determining yield by TCA
precipitation and scintillation counting as described in section
3.6.
[0227] A survey of several membrane proteins with various numbers
of transmembrane (TM) segments are expressed using this method.
Solubility of the membrane protein is clearly increased in the
presence of pre-formed NLPs indicating association with the
NLP.
Example 13
Cell-Free Production of NLPs
[0228] In a further series of experiments performed following the
approach outlined in Example 1 and illustrated in FIG. 1,
preparative reactions were carried out using the Invitrogen's
Expressway Maxi kit and/or for comparison the RTS High Yield Kits
as outlined below.
[0229] Basically, lyophilized reaction components (Lysate, Reaction
Mix, Amino Acid Mix, Methionine) were dissolved in Reconstitution
Buffer and combined as specified by the manufacturer. Then, 1-5
.mu.g of each plasmid DNA were added and the reactions are
incubated at 30 DC-37 DC for 14-24 h. Small-scale reactions, can
make use of PCR products. This is especially convenient for
conducting screening experiments in volumes as low as 2 .mu.L. PCR
products are quantified using a fluorescence-based TpicoGreen assay
Then 0.1 .mu.g of linear template DNA is added to initiate the
reaction, which is incubated at 30 DC for 4 h.
[0230] For expression screening, reactions were performed in 12-25
.mu.L volumes and the resulting products were analyzed by
immunoblotting or using a 96-sample format dot blot or array using
previously described techniques adapted to NLP-GPCRs.
[0231] The DNA constructs to produce the scaffold proteins E422K,
E22K, and apoLp-III from B. mori were provided. The ApoAI and MSPI
(truncated form of ApoAI) were also cloned (see Table 3 of Example
15 below).
[0232] In general, the bacterial overexpression of these scaffold
proteins was started by transferring 20 ml of a bacterial overnight
culture into I LM9 minimal medium supplemented with 50 .mu.g/l
ampicillin. The expression was induced with 2 M
isopropyl-thio-galactopyranoside (IPTG) at an OD600 nm of 0.55.
Four hours later the bacteria were centrifuged (10 min, 4500 rpm,
Beckman JA 10), the supernatant filtrated (0.8 urn) and
subsequently concentrated to a volume of 2 ml by ultrafiltration
through a 10 kDa membrane (Amicon). The concentrate was heated for
5 min at 100.degree. C., centrifuged (15 min, 13,000 rpm,
Eppendorf5415 C), and the supernatant was exchanged against 20 mM
BisTris (pH 6.5) by 3-kDa ultrafiltration (Centriprep, Amicon). The
prepared sample as applied onto a DEAE-Sepharose CL-6B anion
exchange column (bed volume 20 ml, Sigma) connected with a
Gradifrac-system (Pharmacia). Flow rate was I ml/min, and I.-ml
fractions were collected. The late fractions of the flow-through
containing apoLp-III were pooled, exchanged against physiological
saline (172 mM KCI, 68 M NaCI, 5 mM NaHCO3, pH 6.1) and applied in
a volume of 2 ml onto a gel filtration column (HiLoad 16/60,
Superdex 75, Pharmacia) operated with an FPLC system (Pharmacia).
Protein purity was checked by sodium dodecylsulfate-polyacrylamide
gel electrophoresis (SDS-PAGE).
[0233] Lipid (20 mg) was weighed out and combined in a glass, round
bottom tube. Chloroform (200 .mu.l) was added to dissolve lipid.
Chloroform is evaporated in a stream of nitrogen gas, rotating
rapidly to distribute the lipid evenly. Samples are placed under
vacuum for 30 min. to assure removal of solvent. The individual
components are placed into an assembly solution with the
appropriate ratios of lipid (800 .mu.g), scaffold protein (200
.mu.g), detergent (21 mM), creating a mass ratio of 4:I for lipid
to protein and maintaining the cholate above the critical micellar
concentration. The self-assembly process is started with 3 repeated
sets of transition temperature incubations, bracketing the
transition temperature of DMPC (23.80 C), by incubating at
30.degree. C. for 10 minutes, then at 20.degree. C. for 10 minutes
with light hand mixing between incubations.
[0234] Following these transitions, the samples are incubated at
23.8.degree. C. overnight. Following assembly, samples with cholate
are dialyzed against 1OOO.times. volume of TBS buffer using 3
changes in 24 hrs. The NLPs are purified from `free protein` and
`free lipid` by a VP HPLC (Shimadzu) with a Superdex 200 HR 10/30
column (GE healthcare), using TBS at a flow rate of 0.5 ml/min. The
column was calibrated with four protein standards of known
molecular weight and stokes diameter that span the separation range
of the column and the NLP samples.
[0235] The void volume was established with blue dextran. The NLP
fraction is concentrated about 10-fold to .about.approximately 0.1
mg/ml using molecular weight sieve filters (Vivascience) having
molecular weight cutoffs of 50 kDa. Protein concentration was
determined using the ADVOI protein concentration kit
(Cytoskeleton), which is based on Coomassie dye binding.
[0236] To performed Native PAGE validation, equal amounts of NLP
samples (0.5-2 .mu.g) were diluted with 2.times. native gel sample
buffer (Invitrogen) and loaded onto 4-20% gradient pre-made
Tris-glycine HCI gels (Invitrogen). Samples were electrophoresed
for 250 V/hrs at a constant 125 V. After electrophoresis, gels are
incubated with Sypro Ruby for 2 hours and then de-stained using 10%
MeOH, 7% Acetic acid. Following a brief wash with ddH20, gels are
imaged using the green laser (532 nm) of a Typhoon 9410 (GE
Healthcare) with a 610 nm bandpass 30 filter.
[0237] Molecular weights were determined by comparing migration vs.
log molecular weight of standard proteins found in the NativeMark
standard (Invitrogen). The Stokes diameter of the NLPs is
calculated from the known Stokes diameter of the same proteins in
the standard sample.
Example 14
Characterization of NLPs Produced by Cell-Free Systems
Solubilization of the bR-NLP Complex
[0238] The experimental approach described in Example 13 was
applied to obtain cell free expression for single step production
and refolding of the membrane protein bacteriorhodopsin (bR) from
Halobacterium alinarium, which serves as model protein for
G-protein coupled receptors (GPCRs), an important membrane protein
family.
[0239] Cell-free expression of bR in the presence of NLPs is shown
in FIGS. 21 and 22. Preliminary results show that cell-free
synthesis of bR in the presence of NLPs yields nanoparticles
containing bR, i.e. NLP-bR.
[0240] Additionally, these NLP-bR constructs are functional as
assessed by UV/Vis spectrometry (data not shown). The bR protein is
only purple when co-factors are present to allow proper protein
folding. The figure also demonstrates that the bR protein is
incorporated within the NLP following size exclusion chromatography
to purify and isolate the complex (FIG. 21, Panel B). The bR
containing NLPs were also assayed using a Western microarray format
as preliminary development of rapid fluorescent screening
techniques (FIG. 21, Panel C) and by Atomic Force Microscopy, which
showed an increase in particle height (FIG. 22). Interestingly,
co-expression in RTS or Expressway) cell-free reactions of an
apolipoprotein (scaffold protein) with two other membrane protein
targets showed significantly enhanced soluble expression levels
(FIG. 22). These results, suggesting similar incorporation of
membrane protein into NLPs, support the claim of a single step
reaction process yielding soluble NLP/membrane protein constructs.
These results therefore establish cell-free co-protein expression
as a viable expression strategy for membrane proteins in general,
which comprise a significant fraction of any genome and are
notoriously difficult to isolate and characterize.
Example 15
apoE422K and apoLp-III Protein Production
[0241] Apolipoproteins apo E422K and apoLp-111 where selected from
the ones illustrated in Table 3 below.
TABLE-US-00004 TABLE 3 Apolipoprotein Mol. Wt. Lipid Cholate Key
REFs ApoAI 28.1 kDa DMPC No Jonas et al., 1980.sup.8 MSP1T2
(.DELTA.1-55 ApoAI) 24.8 kDa DMPC No Sligar et al., 2005.sup.9;
Denisov et al., 2005.sup.10 ApoE422K 22.3 kDa DMPC No Lu et al,
2000.sup.11 ApoLp-III 18 kDa DMPC No Weintzek, M. et al.
1994.sup.12; Weers and Ryan, 2003.sup.13 ApoAI 28.1 kDa DMPC Yes
Jonas et al., 1980.sup.8 MSP1T2 (.DELTA.1-55 ApoAI) 24.8 kDa DMPC
Yes Shaw et al., 2004.sup.14, Bayburt et al., 2006.sup.15 ApoE422K
22.3 kDa DMPC Yes This work ApoLp-III 18 kDa DMPC Yes Garda HA et
al., 2002.sup.16 Cy3-apoE422K 22.3 kDa DMPC Yes &No This work
Cy3-apoE422K 22.3 kDa DMPC + 1% Yes &No This work NBD-DMPC
[0242] The expression clone to produce apoE422K, the N-terminal 22
kDa fragment of apolipoprotein E4 (apoE4), as a 6His and thyrodoxin
tagged construct was kindly provided by Dr. Karl Weisgraber.
ApoE422K was over-expressed and in E. coli as previously reported.
Pelletted E. coli cells expressing apoE422K were re-suspended in
lysis buffer (50 mM sodium phosphate, 300 mM sodium chloride, 10 mM
imidazole, pH 8.0) and lysed with an Emulsiflex-05 homogenizer
(Avestin Inc., Ottawa, Canada) at 4.degree. C. Following
centrifugation, the clarified supernatant was first partially
purified by nickel affinity chromatography using a 5 ml His Trap FF
crude nickel column (GE Healthcare) on an Akta FPLC (GE Healthcare)
then further purified with a 320 ml Superdex 75 HiLoad 26/60 column
(GE Healthcare) using TBS running buffer (10 mM Tris, pH 7.4, 0.15
M sodium chloride, 0.25 mM EDTA, 0.005% sodium azide) giving one
predominant peak.
[0243] The collected material was cleaved with bovine
.alpha.-Thrombin (Haematologic Technologies) 1/500 enzyme/protein
for 1 hour at 37.degree. C. Resulting products were separated by
SEC on a 320 ml Superdex 75 HiLoad 26/60 FPLC column with one
column volume of TBS. Protein fractions were analyzed by SDS-PAGE
gels stained with Sypro Ruby (BioRad), gels were imaged with a
Typhooon 9410 (GE Healthcare). Relative purity of the proteins was
determined to be greater than 95% by densitometry and overall
yields are on the order of 6 mg/L bacterial culture.
[0244] The B. mori apoLp-111 expression clone was a kind gift from
Dr. Rob Ryan. ApoLp-III was over-expressed in E. coli as described.
The protein was expressed with a PEL leader sequence, targeting the
protein to the periplasm, where the leader sequence is cleaved and
protein secreted in to the media. Expression was induced for four
hours, bacteria were pelleted and the supernatant was collected,
filtered (0.8 .mu.m), and subsequently concentrated to a volume of
.about.20 ml using a Vivaflow 200 (Sartorius) with a 5-kDa MW
cutoff PES membrane. The concentrated protein was exchanged against
20 mM Tris pH 8.0 over a HiPrep 26/10 desalting column on an Akta
FPLC (GE Healthcare). The protein was then purified to homogeneity
by HPLC (Shimadzu) using a ProPac WAX-10 column (Dionex) and eluted
as follows: 0-100% gradient between 20 mM Tris pH 8.0 and 20 mM
Tris pH 8.0 with 0.5M NaCl. Fractions showing highest protein
content by A.sub.280 were pooled.
[0245] Protein purity was checked by sodium
dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and
mass spectroscopy analysis. The protein was >90% pure by gel
electrophoresis, MS analysis gave expected molecular ion peaks;
overall yield was 40%.
[0246] Fluorescently labeled apoE422K was obtained by using a Cy3
labeling kit and following the manufacturer's instructions (GE
Healthcare). Dye:protein ratio was determined by comparing the
absorbance of the protein at 280 nm and the absorbance of the CyDye
at 532 nm. The ratio provided a 1:1 correlation suggesting that a
single Cy3 molecule is present on each apoE422K protein.
Example 16
Nanolipoprotein Particle (NLP) Formation Through Self-Assembly of
Lipids and Apolipoproteins
[0247] In order to better understand the self-assembly process and
the range of attributes of NLPs extensive comparison of particles
from a number of self-assembly conditions was performed, using four
different apoliporoteins, and a battery of characterization
techniques, was applied. In particular NLPs from each of the four
apolipoproteins apoA-I, .DELTA.-apoA-I fragment, apoE4 fragment,
and apolipophorin III (apoLp-III) assembled and characterized in
combination with DMPC, with and without cholate, with and without
fluorescent labels on the apolipoprotein and DMPC molecules
[0248] Phospholipids (DMPC and NBD-DMPC) were purchased from Avanti
Polar Lipids. Inc (Alabaster, Ala.). Full-length apoA-I was
purchased from Fitzgerald, Inc. (Concord, Mass.), apoA-I, .DELTA.
1-55 (MSP1T2) and Nanodisc.TM. particles were purchased from
Nanodisc, Inc., (Urbana, Ill.). The latter particles were made from
DMPC and apoA-I, .DELTA.1-55 protein fragment (MSP1T2); this
fragment has a modified NH 2-terminus containing a His tag and a
tobacco etch virus (TEV) cleavage site.
[0249] FIG. 23 schematically shows the assembly process while Table
4 details the NLP preparations undertaken in this example.
Individual reactants are combined, mixed and subjected to a series
of temperature transitions before overnight incubation. NLPs are
separated from the reaction mixture by chromatography, concentrated
and characterized.
[0250] DMPC (20 mg) is weighed out, added to a glass, round bottom
tube followed by chloroform (200 .mu.l) to dissolve lipid.
Chloroform is evaporated in a stream of nitrogen with constant
rotation to distribute the lipid evenly along the tube wall and
placed under vacuum overnight. DMPC is either re-suspended in TBS
with probe sonication or with TBS/cholate and gentle vortexing; the
final concentration of cholate (20 mM) is above its critical
micellar concentration (CMC). Apolipoproteins (200-250 .mu.g) are
added to the TBS/DMPC solution+/-cholate at a mass ratio of 4:1 for
apoE422K and 3:1 of apoLp-III. The particle formation process is
started with 3 repeated sets of transition temperature incubations,
above and below the transition temperature of DMPC (23.8.degree.
C.), i.e. 10 minutes at 30.degree. C., then 10 minutes at
20.degree. C., with light hand mixing between incubations. After 3
heating and cooling transitions, the samples are incubated at
23.8.degree. C. overnight.
[0251] Following assembly, samples containing cholate are dialyzed
against 1000.times. volume of TBS buffer using 3 changes in 24 hrs.
The NLPs are purified from `free protein` and `free lipid` by
size-exclusion chromatography (VP HPLC, Shimadzu) using a Superdex
200 HR 10/30 column (GE Healthcare), in TBS at a flow rate of 0.5
ml/min. The column was calibrated with four protein standards of
known molecular weight and Stokes diameter that span the separation
range of the column and the NLP samples. The void volume was
established with Blue dextran. The NLP fractions are concentrated
to approximately 0.1 mg/ml using molecular weight sieve filters
(Vivascience) with molecular weight cutoffs of 50 kDa. Protein
concentration was determined using the ADVOI protein concentration
kit (Cytoskeleton, Inc.).
Example 17
NLPs Purification from Free Lipid and Free Protein Starting
Reactants
[0252] Comparison of Size Exclusion Chromatography (SEC) traces
from NLP assemblies illustrated in FIG. 24 provides insight on
particle molecular size and homogeneity. In particular the
chromatogram of the ApoE422K-, Lipophorin III-, ApoAI-, and
MSP1T2-derived NLPs shows one to three peaks at A280 nm. The NLP
peak eluted as the predominant peak in the chromatogram, well
separated from lipid-rich and lipid-poor fractions and was isolated
for further analysis. The larger E422K and apoLpIII peaks are
eluted at about 21 min, while the smaller ApoAI derived NLPs elute
at about 26 min. Molecular size information from SEC is shown in
Table 4 below.
[0253] ApoE422K and apoLp-III derived NLPs eluted a few minutes
after the void volume, whereas the two apoA1-based NLPs eluted 3-4
minutes after the others. These data suggest larger particles are
formed from apoE422K and lipophorin apolipoproteins versus
particles derived from apoA-I proteins. The SEC profiles of
apoE422K and lipophorin-NLPs were quite similar eluting in nearly
the same position showing a diameter of .about.14-15 nm. Each had a
small `free lipid` peak and a larger `free protein` peak
surrounding the single predominant NLP peak; this elution pattern
for E422K is similar to previous results (20). Interestingly,
altering the lipid:protein ratio for apoLp-111 assembled NLPs
enhanced the NLP peak, while diminishing the free component peaks
consistent with previous, work (21). When cholate was used to
solubilize lipid films deposited by chloroform evaporation, the
`free lipid` peak is diminished or completely disappears suggesting
that altering lipid:protein ratio affects apparent yield of
lipophorin-based NLPs (data not shown).
Example 18
NLPs' Size and Heterogeneities Associated with Individual
Apolipoproteins
[0254] Equal amounts of NLP samples (0.5-2 .mu.g) are diluted with
2.times. native gel sample buffer (Invitrogen) and loaded onto
4-20% gradient pre-made Tris-HCl gels (Invitrogen). Samples are
electrophoresed for 250 Vhrs at a constant 125V. After
electrophoresis, gels are incubated with Sypro Ruby for 2 hours and
then destained using 10% MeOH, 7% Acetic acid. Following a brief
wash with ddH.sub.2O, gels are imaged using a Typhoon 9410 (GE
Healthcare) at 532 nm (green laser) with a 610 nm bandpass 30
filter. Molecular weights are determined by comparing migration vs.
log molecular weight of standard proteins found in the NativeMark
standard (Invitrogen). The Stokes diameter of the NLPs is
calculated from the known Stokes diameter of the same proteins in
the standard sample.
[0255] The results illustrated in FIG. 25 show predominant single
bands<700 kDa for E422K-NLPs (A) and apoLp-111 NLPs (B). The
apoAI-derived (Fitzgerald, Inc.) NLPs show five major species when
full-length ApoAI (C) or MSP1T2 (D) proteins (Nanodisc, Inc.) are
used for making NLPs. Panel E shows the commercially available NLP
sample from Nanodisc, Inc. Native gels of NLP fractions obtained
from cholate containing preparations were qualitatively similar to
those shown here. Each gel was stained with Sypro RUBY which has a
wider dynamic range than Coomassie stain and is more sensitive; it
is possible that, some protein species may not be detected.
[0256] Accordingly, native gel electrophoresis reveals (FIG. 25)
that apoE422K and apoLp-111 NLPs appear predominantly as single
bands. On the contrary NLP preparations using apoAI and MSP1T2
(.DELTA.1-55 apoAI) show multiple bands. Five bands on the gel
corresponding to putative NLPs were observed using purchased
MSP1T2; the three larger molecular weight bands constitute less
than 10% of the total protein amount. Overloading apoE422K NLPs on
native gels show minor larger molecular weight bands; AFM analysis
of the latter show NLP species of larger diameter. These larger
species likely do not affect size characterization shown in Table
4. Our MSP1T2-derived NLPs averaged 260 kDa, consistent with the
molecular weight obtained from purchased MSP1T2-based `nanodiscs`,
of 255 kDa. The Stokes diameter of all NLP assemblies was
determined by migration comparison to protein standards with known
Stokes diameters and shown in Table 4. The calculated Stokes
diameter of the apoAI-derived NLPs was approximately 11 nm, while
the apoE422K- and apoLp-III-derived NLPs showed around 13 nm
diameters. The apoLpIII-NLPs were slightly larger by native PAGE
when compared to apoE422K-NLPs; this observation is not consistent
with the SEC data that suggested apoE422K-derived particles were
larger. This discrepancy might be due to differences in protein
shape, charge and/or bound DMPC molecules. Size of NLPs determined
by SEC and native PAGE are based on calibration standards used for
soluble proteins. As such, these standards may not be appropriate
for calibrating lipid-containing NLPs by native PAGE.
Example 19
NLPs Characterization by Mass Spectrometry and Ion Mobility
Spectrometry (IMS)
[0257] In addition to using previously reported analytical methods
for examining NLP, ion mobility spectrometry (IMS) was also used, a
very sensitive and precise technique for measuring particle size
Mass determination was performed using Bruker APEX II 9.4 T FTICR
mass spectrometer through a homebuilt nanospray interface on an
Apollo (Bruker Daltonics, Billerica, Mass., USA) ESI source.
Protein solution concentrations were 1-10 .mu.M or 1 nM in 10 mM
ammonium acetate, pH 7.5. Solutions were desalted and concentrated
by centrifugal filtration using Microcon or Amicon Ultra-4 filters
(Millipore, Bedford, Mass.).
[0258] The aerodynamic diameter of NLPs was determined with a
Macroion Mobility Spectrometer (Model 3890, TSI Inc., Shoreview,
Minn.). The details of the instrumentation and a method for
measuring protein sizes have been described elsewhere (4,5).
Interestingly, this method has been used to measure the size
distribution for HDL, LDL and VLDL taken directly from serum (6).
Briefly, the instrument consists of an electrospray ionization
source with a charge-neutralizing chamber, a differential mobility
analyzer (DMA) and a condensation particle counter (CPC). Multiply
charged droplets generated by electrospray are charge-reduced by
interaction with air ions formed by .alpha.-radiation (.sup.210Po).
NLP samples are exchanged via dialysis (3.times. buffer exchange)
into a volatile buffer and then pumped into the electrospray source
at 100 mL/min. These conditions were chosen so primary electrospray
droplets contain, on average, less than one individual NLP in 25 mM
ammonium acetate. The droplets ultimately evaporate, leaving
individual NLPs in the gas phase carrying, predominantly, a single
charge (7). Charged NLPs pass through a scanning differential
mobility analyzer and are counted by a condensation particle
counter. The size distribution of a population of NLPs is
determined from the scanning parameters; mobility measurements are
used to infer NLP mean aerodynamic diameter.
[0259] FIG. 26 shows differential ion mobility spectra for four
representative NLP preparations. MSP1T2 scaffold without cholate,
apoLp-III scaffold with cholate, apoLp-III scaffold without
cholate, and apoE422K without cholate. The centroid and full width
at half maximum (FWHM) of the highest abundance peak within a trace
is used to represent the average mean aerodynamic diameter of the
particles within a sample. The lower abundance peaks at .about.17
and 19 nm in the apoLp-III and ApoE422K traces are respectively
likely due to slightly larger particles of lower abundance that are
not detected by native gel. The MSP1T2-NLPs appear significantly
smaller than the apoLp-III and apoE422K-NLPs while the addition of
cholate during formation of NLPs utilizing apoLp-III as the
scaffold has no significant effect on the ion mobility trace and
the average mean aerodynamic particle diameter. Ion mobility traces
of mean aerodynamic diameter size distributions for the other NLP
preparations shown in Table 4 were qualitatively and quantitatively
similar to those shown here.
[0260] Together with the ion mobility data summarized in Table 4
below, the spectra illustrated in FIG. 27 indicate that IMS can
resolve size differences in NLP arising from the use of differing
apolipoproteins. Moreover, these spectra also illustrate that, for
a given apolipoprotein, cholate addition and removal does not alter
particle size. The full-width half maximum of the predominant peak
in each spectrum is similar suggesting that, at least for the
predominant IMS peak, NLP size heterogeneity may not be strongly
dependent on choice of scaffold protein. Heterogeneity observed in
the native gel electrophoresis data for apoA1 and MSP1T2
preparations is not reflected in the ion mobility FWHM data. This
likely arises as different bands on a gel correspond to different
peak diameters within an IMS spectrum and consequently, the IMS
FWHM data are only assessing the heterogeneity within a single gel
band.
Example 20
NLPs Characterization by Transmission Electron Microscopy (TMS) and
Atomic Force Microscopy (AFM)
[0261] Samples were diluted using TBS to achieve a final
concentration of 0.02 mg/ml. Three .mu.l of each sample was
pipetted onto a carbon coated 400 mesh copper EM grid (Ted Pella).
After sitting for 1 minute, the sample was blotted with Whatman
filter paper. Three .mu.l of 2% Uranyl Acetate (Electron Microscopy
Sciences) was applied for one minute and then blotted. Grids were
dried for 30 minutes before use in the EM. Negative stain images
were recorded using a Philips CM300 FEG transmission electron
microscope operating at an accelerating voltage of 300 keV. Images
were recorded as 8-bit and 16-bit tiff at varying magnifications
onto a Gatan digital CCD and stored as jpegs and Gatan image format
files. Images were then analyzed using Gatan Digital Micrograph
software.
[0262] Micrographs in FIG. 27 show NLPs whose dimensions are
consistent with previously described observations (see Table 4
below). In particular, the micrographs of FIG. 27 show a negative
stain TEM and AFM of apoE422K-NLP preparations with and without
cholate. For TEM, each sample was stained with a 2% solution of
uranyl acetate, as described herein. Samples for AFM were measured
in solution, using non-contact mode. Both electron micrographs were
taken at 65,000.times. magnification, panel B shows sample prepared
with cholate. Panels C and D show 400.times.400 nm topographical
AFM images of apo4E22K-NLPs having similar shape and height; they
were prepared without cholate (C) or with cholate (D). The scale
bar represents 50 nm. A color bar scale identifies NLP height and
insets show zoomed in regions (50.times.50 nm), showing single
particles. A section line trace below each image show both the
typical heights and diameters in the slow scan direction for the
respective AFM images, Panel E (no cholate) and F (cholate). The
average height for NLPs with cholate is 4.8 nm+/-0.2 nm and the
heights of NLPs without cholate 4.8 nm+/-0.3 nm, which is
consistent with the theoretical size of the lipid bilayer. The data
suggest discoidal structures with a diameter of about 10-20 nm and
height consistent with the thickness of a bilayer.
[0263] Two of these assemblies made from apoE422K and DMPC are
shown, with (panel B) and without cholate (panel A). The lower
right-hand corner of each panel shows a region at higher
magnification to highlight the presence of discoidal structures.
Cholate has no effect on the size and structure of apoE422K-derived
NLPs. Like previous reports, images of stacked particles were
observed--described as "rouleaux"--but not in all samples. Others
have described these formations as artifacts of sample preparation
and concentration (22).
[0264] Atomically flat Muscovite mica disks were glued to metal
substrates to secure them to the scanner of a stand-alone MFP-3D
AFM (Asylum Research, Santa Barbara, Calif.). 2 uL of solution was
incubated for two minutes on the mica surface in imaging buffer (10
mM MgCl2, 10 mM Tris-HCL, and 0.1 M NaCl, adjusted to pH 8.0) and
then lightly rinsed. The AFM has a closed loop in the x, y, and z
axes. The topographical images were obtained with "Biolevers"
(Olympus, Tokyo, Japan) with a spring constant of 0.03 N/m. Images
were taken in alternate contact (AC) mode in liquid, with
amplitudes below 20 nm and an amplitude setpoint at 50% tapping
amplitude. Scan rates were below 1.5 Hz. Height, amplitude, and
phase images were recorded. Heights of features in images were
determined by histogram analysis of contiguous particles.
Experiments were carried out in a temperature controlled room at
23+/-1.degree. C.
[0265] All four apolipoprotein assemblies show a common discoidal
bilayer structure. In particular, it was observed that NLPs made
from different apolipoproteins examined by AFM showed discrete
bracketed structures even at high concentration, indicative of
individual particles. When DMPC without apolipoprotein is examined,
planar fusible features, .about.4-5 nm in thickness are observed
consistent with the presence of a lipid bilayer (data not shown).
Also, when apolipoprotein is examined alone, globular features on
the order of 2-3 nm are seen. NLPs have diameters ranging from 10
to 20 nm and heights of approximately 5 nm; these observations are
consistent with diameters measured by other techniques described
above. Particle size and structure is unaffected by cholate as
shown in FIG. 27 with apoE422K-derived NLPs. These AFM data
indicate that the particles are just less than 5 nm high with
diameters of .about.20 nm. This diameter size is larger than was
derived from TEM, but AFM is known to increase the size of x, y
resolution due to tip convolution effects. Combined, AFM and TEM
data suggests discoidal structures with height dimensions
consistent with a phospholipid bilayer and a diameter of about
10-20 nm; cholate addition during assembly does not appreciably
change the heights of the apoE422K and apoLp-III assemblies (see
Table 4).
Example 21
Monitoring NLPs Assembly Process by Fluorescent Labeling
[0266] FIG. 24 shows analyses of a labeled NLP using a Cy3-labeled
apoE422K mixed with 7-nitrobenz-2-oxa-1,3-diazol-4-yl labeled DMPC
(DMPC-NBD).
[0267] In the analysis illustrated in FIG. 28 fractions collected
from SEC were characterized by native PAGE (panel C), AFM (panel D,
right) and TEM (panel D, left). Importantly, it appears that
labeled NLP reactants do not affect NLP formation. The SEC trace
(panel A) shows similar lipid-rich, NLP, and lipid-poor peaks that
co-elute with those species in non-fluorescent samples. The SEC
fractions were analyzed for fluorescence (panel B) and the
fluorescent lipid (green pseudo color), fluorescent protein (red
pseudo color), and NLPs (yellow pseudo color) show up in the
expected vials based on the SEC trace. The results illustrated in
FIG. 28 indicate that fluorescently-labeled NLPs show similar
structure and size compared to unlabeled NLPs.
[0268] Table 4 shows the size characteristics of NLPs made using
fluorescently-labeled reactants do not appreciably change from the
unlabeled reactants. Moreover, the size and shape are maintained as
observed AFM and EM analyses show similar discoidal structures
(panel D). These data suggest that fluorescent dye attachment to
lipid and protein reagents can be used to track NLP assembly as
well as provide means to detect individual reactants within the
particle.
Example 22
NLPs Combined Characterization
[0269] Table 4 summarizes results from combined characterization
approaches and highlights particle size parameters of NLPs
assembled from each of four apolipoproteins in combination with a
single phospholipid, dimyristoylphosphatidylcholine (DMPC).
Apoliprotein can assume three different forms: fully extended,
doubled-back/"hairpin", and semi-extended/"double-hairpin" folds as
illustrated in FIG. 29.
[0270] Reaction of each protein with DMPC yields NLPs with unique
overall structural/shape characteristics. In general, particles
produced were found to be discoidal in shape with diameters ranging
from 10-20 nm dependent on the apolipoprotein or derivative used in
assembly; a height of .about.5 nm was determined for all NLP
preparations, consistent with a membrane bilayer formed by DMPC
(23).
[0271] The fundamental observations are that the apolipoprotein is
the primary determinant of NLP size and that a discoidal shape was
consistent among the four assemblies. These characterization
results, irrespective of the method or apolipoprotein used, show
remarkable consistency in measuring overall NLP size and shape for
any given apolipoprotein. Moreover, measured sizes and shapes did
not differ appreciably when formed in the presence of cholate and
when using fluorophore labeled reactants. The following sections
summarize results from each of the specific characterization
techniques.
TABLE-US-00005 TABLE 4 Native Gel SEC Apolipo- Mol. Wt. Stokes D
Mol. Wt. Stokes D Ion Mobility AFM (nm) TEM (nm) protein Lipid
Cholate (kDa) (nm) (kDa) (nm) AMAD .+-. FWHM Height Diameter
Nanodiscs 100% DMPC No 290 .+-. 10 10.8 .+-. 0.1 190 .+-. 15 9.3
.+-. 0.3 10.6 .+-. 1.4 6.1 .+-. 0.2 10.2 .+-. 3.1 (MSP1T2) apoAI
100% DMPC No 360 .+-. 10 11.4 .+-. 0.1 270 .+-. 30 12.6 .+-. 0.4
10.5 .+-. 1.1 4.3 .+-. 0.5 13.0 .+-. 1.4 MSP1T2 100% DMPC No 260
.+-. 30 10.1 .+-. 0.8 300 .+-. 120 12.8 .+-. 1.4 9.5 .+-. 0.9 4.8
.+-. 0.2 12.7 .+-. 3.0 (.DELTA.apoAI) apoE422K 100% DMPC No 505
.+-. 60 12.6 .+-. 0.3 560 .+-. 15 15.1 .+-. 0.1 13.2 .+-. 0.9 4.9
.+-. 0.2 17.6 .+-. 2.7 apoLp-III 100% DMPC No 620 .+-. 60 12.8 .+-.
0.4 480 .+-. 25 14.5 .+-. 0.2 13.1 .+-. 0.7 4.4 .+-. 0.3 17.6 .+-.
2.6 apoE422K 100% DMPC Yes 680 .+-. 40 13.2 .+-. 0.1 600 .+-. 15
15.3 .+-. 0.1 13.6 .+-. 1.0 4.9 .+-. 0.3 16.2 .+-. 2.4 apoLp-III
100% DMPC Yes 530 .+-. 10 12.6 .+-. 0.1 425 .+-. 12 14.1 .+-. 0.5
13.1 .+-. 0.7 4.0 .+-. 0.4 18.0 .+-. 2.6 Cy3-apoE422K 100% DMPC Yes
&No 510 .+-. 40 12.4 .+-. 0.4 660 .+-. 10 15.1 .+-. 0.1 14.0
.+-. 1.5 4.6 .+-. 0.3 20.8 .+-. 2.9 Cy3-apoE422K 100% DMPC Yes
&No 630 .+-. 70 13.0 .+-. 0.2 670 .+-. 15 15.1 .+-. 0.1 14.1
.+-. 1.5 5.1 .+-. 0.3 17.4 .+-. 2.7 (1% NBD)
[0272] Table 4 illustrates the results of the physical
characterization of NLPs by native gel electrophoresis, SEC, Ion
mobility spectrometry, AFM and negative stain TEM performed
according to Examples 1 to 7. Molecular weights and Stokes
diameters of the NLPs from native gels and SEC were determined
using known protein standards and are shown in kDa and nm,
respectively. The average mean aerodynamic diameter (AMAD)
corresponds to the centroid and full width at half maximum (FWHM)
of the most abundance peak within an ion mobility trace. The
centroid provides a robust measurement of the average mean
aerodynamic diameter of the particles within a sample while the
FWHM provides a comparative estimate of sample heterogeneity. AFM
derived measurements of height and TEM derived measurements of
diameter are reported as the mean+/-standard deviation of
individual measurements from typically 100 NLPs within a sample.
ApoAI and MSP1T2 assembled NLPs were noticeably smaller than E422K
and apoLp-III assembled NLPs, ranging in size from 10-13 nm in
diameter as compared to 12-20 nm in diameter for the E and
apoLp-III assemblies. Cholate addition during assembly did not
appear to appreciably change the size of any of the structures. The
addition of fluorescently labeled assembly components also had
little affect on the molecular size, but likely affects the
homogeneity of the assembled structures since fluorescent
components were unlikely to be uniformly distributed throughout the
NLP population. Characterization data using purchased empty
Nanodiscs.TM. are also shown for comparative purposes.
[0273] Additional experiments and related results concerning
monitoring of NLP assembly and related characterization are
illustrated in FIGS. 30 to 32.
[0274] The system exemplified in the preceding examples is useful
to monitor/indicate successful in situ cell-free protein synthesis
of membrane proteins, in general and their incorporation into NLPs.
Preceding examples although performed with indicator bR are also
indicative of the application of the methods and systems herein
disclosed with other such indicator proteins that could be used for
in situ monitoring of cell-free membrane protein synthesis with
concomitant NLP formation; these include, but are not limited to,
sesory rhodopsin, proteorhodopsin and phytochromes.
[0275] Like bR, these proteins can be produced by the cell-free
transcription/translation technology in the presence of detergents,
lipids and phospholipids as well as other reaction additives and
couples to NLP formation.
[0276] The examples set forth above are provided to give those of
ordinary skill in the art a complete disclosure and description of
how to make and use the embodiments of the devices, systems and
methods of the disclosure, and are not intended to limit the scope
of what the inventors regard as their disclosure. Modifications of
the above-described modes for carrying out the disclosure that are
obvious to persons of skill in the art are intended to be within
the scope of the following claims. All patents and publications
mentioned in the specification are indicative of the levels of
skill of those skilled in the art to which the disclosure pertains.
All references cited in this disclosure are incorporated by
reference to the same extent as if each reference had been
incorporated by reference in its entirety individually.
[0277] The entire disclosure of each document cited (including
patents, patent applications, journal articles, abstracts,
laboratory manuals, books, or other disclosures) in the Background,
Summary, Detailed Description, and Examples is hereby incorporated
herein by reference. Further, the hard copy of the sequence listing
submitted herewith and the corresponding computer readable form are
both incorporated herein by reference in their entireties.
[0278] It is to be understood that the disclosures are not limited
to particular compositions or biological systems, which can, of
course, vary. It is also to be understood that the terminology used
herein is for the purpose of describing particular embodiments
only, and is not intended to be limiting. As used in this
specification and the appended claims, the singular forms "a,"
"an," and "the" include plural referents unless the content clearly
dictates otherwise. The term "plurality" includes two or more
referents unless the content clearly dictates otherwise. Unless
defined otherwise, all technical and scientific terms used herein
have the same meaning as commonly understood by one of ordinary
skill in the art to which the disclosure pertains.
[0279] Although any methods and materials similar or equivalent to
those described herein can be used in the practice for testing of
the specific examples of appropriate materials and methods are
described herein.
[0280] A number of embodiments of the disclosure have been
described. Nevertheless, it will be understood that various
modifications may be made without departing from the spirit and
scope of the present disclosure. Accordingly, other embodiments are
within the scope of the following claims.
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Sequence CWU 1
1
5124DNAArtificial SequenceSynthetic Oligonucleotide 1atgctaaagc
tccttgacaa ctgg 24225DNAArtificial SequenceSynthetic
Oligonucleotide 2ttactgggtg ttgagcttct tagtg 25321DNAArtificial
SequenceSynthetic Oligonucleotide 3ggggcatatg caagctcaaa t
21422DNAArtificial SequenceSynthetic Oligonucleotide 4ggggatccaa
aaaaaacggg cc 225193PRTArtificial Sequence22 kD Apolipoprotein E4
construct 5Gly Ser Lys Val Glu Gln Ala Val Glu Thr Glu Pro Glu Pro
Glu Leu1 5 10 15Arg Gln Gln Thr Glu Trp Gln Ser Gly Gln Arg Trp Glu
Leu Ala Leu20 25 30Gly Arg Phe Trp Asp Tyr Leu Arg Trp Val Gln Thr
Leu Ser Glu Gln35 40 45Val Gln Glu Glu Leu Leu Ser Ser Gln Val Thr
Gln Glu Leu Arg Ala50 55 60Leu Met Asp Glu Thr Met Lys Glu Leu Lys
Ala Tyr Lys Ser Glu Leu65 70 75 80Glu Glu Gln Leu Thr Pro Val Ala
Glu Glu Thr Arg Ala Arg Leu Ser85 90 95Lys Glu Leu Gln Ala Ala Gln
Ala Arg Leu Gly Ala Asp Met Glu Asp100 105 110Val Arg Gly Arg Leu
Val Gln Tyr Arg Gly Glu Val Gln Ala Met Leu115 120 125Gly Gln Ser
Thr Glu Glu Leu Arg Val Arg Leu Ala Ser His Leu Arg130 135 140Lys
Leu Arg Lys Arg Leu Leu Arg Asp Ala Asp Asp Leu Gln Lys Arg145 150
155 160Leu Ala Val Tyr Gln Ala Gly Ala Arg Glu Gly Ala Glu Arg Gly
Leu165 170 175Ser Ala Ile Arg Glu Arg Leu Gly Pro Leu Val Glu Gln
Gly Arg Val180 185 190Arg
* * * * *