Membrane scaffold proteins and tethered membrane proteins
View Patent ↗Membrane proteins are difficult to express in recombinant form, purify, and characterize, at least in part due to their hydrophobic or partially hydrophobic properties. Membrane scaffold proteins (MSP) assemble with target membrane or other hydrophobic or partially hydrophobic proteins or membrane fragments to form soluble nanoscale particles which preserve their native structure and function; they are improved over liposomes and detergent micelles. 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 facilitate pharmaceutical and biological research, structure/function correlation, structure determination, bioseparation, and drug discovery.
1. A nanoscale particle comprising a non-naturally occurring amphipathic membrane scaffold protein and at least one tethered membrane protein, and further comprising a phospholipid or a mixture of phospholipids, wherein said nanoscale particle has a diameter between 5 nm and 500 nm, wherein said artificial membrane scaffold protein, in an aqueous environment, self-assembles in the presence of phospholipids or in the presence of a mixture of phospholipids, into a nanoscale particle, wherein said membrane scaffold protein is amphipathic, 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 to 414 of SEQ ID NO:17, SEQ ID NO:19, amino acids 13 to 422 of SEQ ID NO:19, SEQ ID NO:23, amino acids 13 to 168 of SEQ ID NO:23, SEQ ID NO:29, amino acids 13 to 169 of SEQ ID NO:29, SEQ ID NO:43, amino acids 13 to 201 of SEQ ID NO:43, SEQ ID NO:44, amino acids 13 to 201 of SEQ ID NO:44, SEQ ID NO:45, and amino acids 13 to 392 of SEQ ID NO:45.
2. The nanoscale particle of claim 1 , wherein said tethered membrane protein is a NADPH-cytochrome reductase.
3. The nanoscale particle of claim 1 , wherein said tethered membrane protein is a cytochrome b5.
4. The nanoscale particle of claim 1 comprising a non-naturally occurring amphipathic membrane scaffold protein and at least one tethered membrane protein, and further comprising a phospholipid or a mixture of phospholipids, wherein said nanoscale particle has a diameter between 5 nm and 500 nm, wherein said artificial membrane scaffold protein, in an aqueous environment, self-assembles in the presence of phospholipids or in the presence of a mixture of phospholipids, into a nanoscale particle, wherein said membrane scaffold protein is amphipathic, wherein said membrane scaffold protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:17, amino acids 13 to 414 of SEQ ID NO:17, SEQ ID NO:19, amino acids 13 to 422 of SEQ ID NO:19, SEQ ID NO:23, aminoacids 13 to 168 of SEQ ID NO:23, SEQ ID NO:29, amino acids 13 to 169 of SEQ ID NO:29, SEQ ID NO:43, amino acids 13 to 201 of SEQ ID NO:43, SEQ ID NO:44, amino acids 13 to 201 of SEQ ID NO:44, SEQ ID NO:45, and amino acids 13 to 392 of SEQ ID NO:45.
5. The nanoscale particle of claim 4 , wherein said tethered membrane protein is a NADPH-cytochrome reductase.
6. The nanoscale particle of claim 4 , wherein said tethered membrane protein is a cytochrome b5.
7. The nanoscale particle of claim 4 , wherein said particle is from 5 to 100 nm in diameter.