US2009257950A1PendingUtilityA1

Membrane Scaffold Proteins

Assignee: UNIV ILLINOISPriority: Nov 20, 2000Filed: Oct 10, 2007Published: Oct 15, 2009

Est. expiryNov 20, 2020(expired)· nominal 20-yr term from priority

Inventors:Stephen G. Sligar Timothy H. Bayburt Mary A. Schuler Natanya R. Civjan Yelena V. Grinkova Ilia G. Denisov Stephen James Grimme

A61P 31/12C07K 14/775A61P 35/00A61P 43/00C07K 14/47

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References

Claims

Abstract

The membrane scaffold proteins (MSP) of the present invention assemble with hydrophobic or partially hydrophobic proteins to form soluble nanoscale particles which preserve native structure and function; they are improved over liposomes and detergent micelles, both in terms of stability and preservation of biological activity and native conformation. In the presence of phospholipid, MSPs form nanoscopic phospholipid bilayer disks, with the MSP stabilizing the particle at the perimeter of the bilayer domain. The particle bilayer structure allows manipulation of incorporated proteins in solution or on solid supports, including for use with such surface-sensitive techniques as scanning probe microscopy or surface plasmon resonance. The nanoscale particles, which are robust in terms of integrity and maintenance of biological activity of incorporated proteins, facilitate pharmaceutical and biological research, structure/function correlations, structure determinations, bioseparations, and drug discovery.

Claims

exact text as granted — not AI-modified

1 . A nanoscale particle comprising at least one phospholipid, a membrane scaffold protein and an additional hydrophobic or partially hydrophobic molecule. 
     
     
         2 . The nanoscale particle of  claim 1 , wherein the additional hydrophobic or partially hydrophobic molecule is a therapeutic molecule, and wherein the membrane scaffold protein is an artificial membrane scaffold protein. 
     
     
         3 . The nanoscale particle of  claim 2 , wherein the at least one phospholipid is phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl inositol, dipalmitoyl-phosphatidylcholine, dimyristoyl phosphatidyl choline, 1-palmitoyl-2-oleoyl-phosphatidyl choline, 1-palmitoyl-2-oleoyl-phosphatidyl serine, 1-palmitoyl-2-oleoyl-phosphatidyl ethanolamine, dihexanoyl phosphatidyl choline, dipalmitoyl phosphatidyl ethanolamine, dipalmitoyl phosphatidyl inositol, dimyristoyl phosphatidyl ethanolamine, dimyristoyl phosphatidyl inositol, dihexanoyl phosphatidyl ethanolamine, dihexanoyl phosphatidyl inositol, 1-palmitoyl-2-oleoyl-phosphatidyl ethanolamine and 1-palmitoyl-2-oleoyl-phosphatidyl inositol. 
     
     
         4 . The nanoscale particle of  claim 2 , wherein the therapeutic molecule is an antimicrobial agent, an antineoplastic agent, an angiogenic factor, a thrombolytic agent, a calcium channel blocker, an antiatherogenic agent, an antihypertensive agent, an inotropic agent, an anti-inflammatory agent, an antiarrythmia agent, an antiviral agent, an antifungal agent, an anticoagulant, an anti-restenosis agent, a therapeutic protein, a photodynamic agent, a therapeutic peptide, a therapeutic nucleic acid molecule, a therapeutic carbohydrate or at least one vitamin. 
     
     
         5 . The nanoscale particle of  claim 4 , wherein said antimicrobial agent is Amphotericin B or ketoconazole. 
     
     
         6 . The nanoscale particle of  claim 2 , wherein said membrane scaffold protein comprises an amino acid sequence as set forth in SEQ ID NO: 6, SEQ ID NO:9, SEQ ID NO:17, amino acids 13-414 of SEQ ID NO:17, SEQ ID NO:19, amino acids 13-422 of SEQ ID NO:19, SEQ ID NO:23, amino acids 13-168 of SEQ ID NO:23, SEQ ID NO:29, amino acids 13-168 of SEQ ID NO:29, SEQ ID NO:43, amino acids 13-201 of SEQ ID NO:43, SEQ ID NO:44, amino acids 13-201 of SEQ ID NO:44, SEQ ID NO:45, amino acids 13-392 of SEQ ID NO:45, SEQ ID NO:73, amino acids 13-234 of SEQ ID NO:73, SEQ ID NO:74, amino acids 13-256 of SEQ ID NO:74, SEQ ID NO:75, amino acids 13-278 of SEQ ID NO:75, SEQ ID NO:76, amino acids 24-223 of SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, amino acids 24-212 of SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, amino acids 24-201 of SEQ ID NO:80, SEQ ID NO:81, amino acids 13-168 of SEQ ID NO:81, SEQ ID NO:82, amino acids 13-168 of SEQ ID NO:82, SEQ ID NO:83, amino acids 13-190 of SEQ ID NO:83, SEQ ID NO:84, amino acids 13-201 of SEQ ID NO:84, SEQ ID NO:85, amino acids 13-190 of SEQ ID NO:85, SEQ ID NO:86, amino acids 24-381 of SEQ ID NO:86, SEQ ID NO:91, amino acids 24-201 of SEQ ID NO:91, SEQ ID NO:92, amino acids 24-190 of SEQ ID NO:92, SEQ ID NO:93, amino acids 24-179 of SEQ ID NO:93, SEQ ID NO:94, amino acids 24-289 of SEQ ID NO:94, SEQ ID NO:95, amino acids 24-289 of SEQ ID NO:94, SEQ ID NO:95, amino acids 24-278 of SEQ ID NO:95, SEQ ID NO:96, amino acids 24-423 of SEQ ID NO:96, SEQ ID NO:97, amino acids 24-199 of SEQ ID NO:97, SEQ ID NO:98, amino acids 24-401 of SEQ ID NO:98, SEQ ID NO:99, amino acids 24-392 of SEQ ID NO:99, SEQ ID NO:111, amino acids 24-397 of SEQ ID NO:111, SEQ ID NO:113, amino acids 24-383 of SEQ ID NO:113, SEQ ID NO:115, amino acids 24-379 of SEQ ID NO:115, SEQ ID NO:117, amino acids 24-381 of SEQ ID NO:117, SEQ ID NO:119, amino acids 13-1094 of SEQ ID NO:119, SEQ ID NO:129, amino acids 25-214 of SEQ ID NO:129, SEQ ID NO:131, amino acids 25-212 of SEQ ID NO:131, SEQ ID NO:133, amino acids 25-212 of SEQ ID NO:133, SEQ ID NO:135 and amino acids 13-212 of SEQ ID 135. 
     
     
         7 . The nanoscale particle of  claim 2 , further comprising a targeting agent which specifically binds to a surface of a cell to which the therapeutic agent is to be delivered. 
     
     
         8 . The nanoscale particle of  claim 7 , wherein said targeting agent is a lectin, single chain antibody or an antigen-binding fragment of an antibody. 
     
     
         9 . The nanoscale particle of  claim 8 , wherein the targeting agent is covalently linked to said membrane scaffold protein. 
     
     
         10 . The nanoscale particle of  claim 8 , wherein a targeting agent is noncovalently bound to said particle. 
     
     
         11 . The nanoscale particle of  claim 1 , wherein the additional hydrophobic or partially hydrophobic molecule is a protein. 
     
     
         12 . The nanoscale particle of  claim 11 , wherein the protein is a membrane receptor protein. 
     
     
         13 . The nanoscale particle of  claim 12 , wherein the membrane receptor protein is a G protein coupled receptor. 
     
     
         14 . The nanoscale particle of  claim 13 , wherein the G protein coupled receptor is a 5-hydroxytryptamine receptor, a beta adrenergic receptor, a chemokine receptor, a lysophosphatidic acid receptor or a dopamine receptor. 
     
     
         15 . The nanoscale particle of  claim 14 , wherein the chemokine receptor is a CXCR4 receptor or CCR5 receptor. 
     
     
         16 . The nanoscale particle of  claim 11 , wherein the protein is a transhydrogenase or an aspartate receptor protein. 
     
     
         17 . The nanoscale particle of  claim 11 , wherein the protein is a cytochrome P450 protein. 
     
     
         18 . The nanoscale particle of  claim 11 , further comprising an electron transfer system, cytochrome P450 reductase or cytochrome b5. 
     
     
         19 . The nanoscale particle of  claim 2 , further comprising cholesterol or a cholesterol ester. 
     
     
         20 . The nanoscale particle of  claim 11 , wherein said membrane scaffold protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO:9, SEQ ID NO:17, amino acids 13-414 of SEQ ID NO:17, SEQ ID NO:19, amino acids 13-422 of SEQ ID NO:19, SEQ ID NO:23, amino acids 13-168 of SEQ ID NO:23, SEQ ID NO:29, amino acids 13-168 of SEQ ID NO:29, SEQ ID NO:43, amino acids 13-201 of SEQ ID NO:43, SEQ ID NO:44, amino acids 13-201 of SEQ ID NO:44, SEQ ID NO:45, amino acids 13-392 of SEQ ID NO:45, SEQ ID NO:73, amino acids 13-234 of SEQ ID NO:73, SEQ ID NO:74, amino acids 13-256 of SEQ ID NO:74, SEQ ID NO:75, amino acids 13-278 of SEQ ID NO:75, SEQ ID NO:76, amino acids 24-223 of SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, amino acids 24-212 of SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, amino acids 24-201 of SEQ ID NO:80, SEQ ID NO:81, amino acids 13-168 of SEQ ID NO:81, SEQ ID NO:82, amino acids 13-168 of SEQ ID NO:82, SEQ ID NO:83, amino acids 13-190 of SEQ ID NO:83, SEQ ID NO:84, amino acids 13-201 of SEQ ID NO:84, SEQ ID NO:85, amino acids 13-190 of SEQ ID NO:85, SEQ ID NO:86, amino acids 24-381 of SEQ ID NO:86, SEQ ID NO:91, amino acids 24-201 of SEQ ID NO:91, SEQ ID NO:92, amino acids 24-190 of SEQ ID NO:92, SEQ ID NO:93, amino acids 24-179 of SEQ ID NO:93, SEQ ID NO:94, amino acids 24-289 of SEQ ID NO:94, SEQ ID NO:95, amino acids 24-289 of SEQ ID NO:94, SEQ ID NO:95, amino acids 24-278 of SEQ ID NO:95, SEQ ID NO:96, amino acids 24-423 of SEQ ID NO:96, SEQ ID NO:97, amino acids 24-199 of SEQ ID NO:97, SEQ ID NO:98, amino acids 24-401 of SEQ ID NO:98, SEQ ID NO:99, amino acids 24-392 of SEQ ID NO:99, SEQ ID NO:111, amino acids 24-397 of SEQ ID NO:11, SEQ ID NO:113, amino acids 24-383 of SEQ ID NO:113, SEQ ID NO:115, amino acids 24-379 of SEQ ID NO:115, SEQ ID NO:117, amino acids 24-381 of SEQ ID NO:117, SEQ ID NO:119, amino acids 13-1094 of SEQ ID NO:119, SEQ ID NO:129, amino acids 25-214 of SEQ ID NO:129, SEQ ID NO:131, amino acids 25-212 of SEQ ID NO:131, SEQ ID NO:133, amino acids 25-212 of SEQ ID NO:133, SEQ ID NO:135 and amino acids 13-212 of SEQ ID NO:135. 
     
     
         21 . The nanoscale particle of  claim 11 , said membrane scaffold protein comprising an amino acid sequence of SEQ ID NO: 6, SEQ ID NO:9, SEQ ID NO:17, amino acids 13-414 of SEQ ID NO:17, SEQ ID NO:19, amino acids 13-422 of SEQ ID NO:19, SEQ ID NO:23, amino acids 13-168 of SEQ ID NO:23, SEQ ID NO:29, amino acids 13-168 of SEQ ID NO:29, SEQ ID NO:43, amino acids 13-201 of SEQ ID NO:43, SEQ ID NO:44, amino acids 13-201 of SEQ ID NO:44, SEQ ID NO:45, amino acids 13-392 of SEQ ID NO:45, SEQ ID NO:73, amino acids 13-234 of SEQ ID NO:73, SEQ ID NO:74, amino acids 13-256 of SEQ ID NO:74, SEQ ID NO:75, amino acids 13-278 of SEQ ID NO:75, SEQ ID NO:76, amino acids 24-223 of SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, amino acids 24-212 of SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, amino acids 24-201 of SEQ ID NO:80, SEQ ID NO:81, amino acids 13-168 of SEQ ID NO:81, SEQ ID NO:82, amino acids 13-168 of SEQ ID NO:82, SEQ ID NO:83, amino acids 13-190 of SEQ ID NO:83, SEQ ID NO:84, amino acids 13-201 of SEQ ID NO:84, SEQ ID NO:85, amino acids 13-190 of SEQ ID NO:85, SEQ ID NO:86, amino acids 24-381 of SEQ ID NO:86, SEQ ID NO:91, amino acids 24-201 of SEQ ID NO:91, SEQ ID NO:92, amino acids 24-190 of SEQ ID NO:92, SEQ ID NO:93, amino acids 24-179 of SEQ ID NO:93, SEQ ID NO:94, amino acids 24-289 of SEQ ID NO:94, SEQ ID NO:95, amino acids 24-289 of SEQ ID NO:94, SEQ ID NO:95, amino acids 24-278 of SEQ ID NO:95, SEQ ID NO:96, amino acids 24-423 of SEQ ID NO:96, SEQ ID NO:97, amino acids 24-199 of SEQ ID NO:97, SEQ ID NO:98, amino acids 24-401 of SEQ ID NO:98, SEQ ID NO:99, amino acids 24-392 of SEQ ID NO:99, SEQ ID NO:111, amino acids 24-397 of SEQ ID NO:111, SEQ ID NO:113, amino acids 24-383 of SEQ ID NO:113, SEQ ID NO:115, amino acids 24-379 of SEQ ID NO:115, SEQ ID NO:117, amino acids 24-381 of SEQ ID NO:117, SEQ ID NO:119, amino acids 13-1094 of SEQ ID NO:119, SEQ ID NO:129, amino acids 25-214 of SEQ ID NO:129, SEQ ID NO:131, amino acids 25-212 of SEQ ID NO:131, SEQ ID NO:133, amino acids 25-212 of SEQ ID NO:133, SEQ ID NO:135 and amino acids 13-212 of SEQ ID NO:135, wherein said sequence is modified by from one to five conservative amino acid, an insertion of from one to 5 amino acids or a deletion of from one to five amino acids. 
     
     
         22 . The nanoscale particle of  claim 1 , wherein the hydrophobic or partially hydrophobic molecule is a lipophilic dye or a fluorescent dye. 
     
     
         23 . The nanoscale particle of  claim 1 , wherein the hydrophobic or partially hydrophobic molecule is an imaging agent. 
     
     
         24 . The nanoscale particle of  claim 23 , wherein the imaging agent comprises gadolinium, technetium, tellurium, iridium or iodine. 
     
     
         25 . The method of  claim 1 , wherein the molar ratio of MSP:solubilizing agent:membrane lipid is from 1:25:50 to 1:2000:1000. 
     
     
         26 . The method of  claim 25 , wherein the molar ratio of MSP:solubilizing agent:membrane lipid is 1:75:150.

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