Integral membrane protein display on poxvirus extracellular enveloped virions
This disclosure provides compositions and methods for expressing and displaying isolated integral membrane proteins (IMPs) or fragments thereof in a native conformation for use in the screening, selecting, and identifying of antibodies or antibody-like molecules that bind to a target IMP of interest.
1. An isolated polynucleotide comprising:
(a) a first nucleic acid fragment that encodes an integral membrane protein (IMP) or fragment thereof, wherein the IMP or fragment thereof comprises at least one extra-membrane region, at least one transmembrane domain and at least one intra-membrane region, and wherein a portion of the first nucleic acid fragment encoding at least one intra-membrane region is situated at the 5′ or 3′ end of the first nucleic acid fragment; and
(b) a second nucleic acid fragment that encodes a vaccinia virus F13L protein comprising the amino acid sequence SEQ ID NO: 1 or functional fragment thereof, wherein the second nucleic acid fragment is fused in frame to a portion of the first nucleic acid fragment that encodes an intra-membrane region of the IMP;
wherein a poxvirus infected cell comprising the polynucleotide can express an IMP-F13L fusion protein as part of the outer envelope membrane of an extracellular enveloped virion (EEV).
2. The polynucleotide of claim 1 , wherein the IMP is a multi-pass membrane protein comprising at least two transmembrane domains.
3. The polynucleotide of claim 2 , wherein the IMP has an odd number of transmembrane domains, wherein the 5′ end of the first nucleic acid fragment encodes an extra-membrane region, wherein the 3′ end of the first nucleic acid fragment encodes an intra-membrane region, and wherein the 5′ end of the second polynucleotide is fused to the 3′ end of the first nucleic acid fragment.
4. The polynucleotide of claim 3 , wherein the IMP comprises a G-protein coupled receptor (GPCR).
5. The polynucleotide of claim 4 , wherein the IMP is the human frizzled-4 protein (FZD4), or a fragment thereof.
6. The polynucleotide of claim 4 , wherein the IMP is the CXC chemokine receptor CXCR4, or a fragment thereof.
7. The polynucleotide of claim 2 , wherein the IMP has an even number of transmembrane domains, and wherein both the 5′ and 3′ ends of the first nucleic acid fragment encode intra-membrane regions, and wherein the second nucleic acid fragment is fused to 3′ end of the first nucleic acid fragment.
8. The polynucleotide of claim 7 , wherein the IMP is human CD20 protein, or a fragment thereof.
9. The polynucleotide of claim 1 , which is operably associated with a poxvirus promoter.
10. The IMP-F13L fusion protein encoded by the polynucleotide of claim 1 .
11. A poxvirus genome comprising the polynucleotide of claim 1 .
12. The poxvirus genome of claim 11 , which is a vaccinia virus genome.
13. A recombinant vaccinia virus EEV comprising the vaccinia virus genome of claim 12 .
14. A method of producing the recombinant vaccinia virus EEV comprising:
(a) infecting a host cell permissive for vaccinia virus infectivity with a vaccinia virus comprising the poxvirus genome of claim 12 , and
(b) recovering EEV released from the host cell.
15. A method to display an integral membrane protein (IMP) or fragment thereof in a native conformation comprising:
(a) infecting host cells permissive for poxvirus infectivity with a recombinant poxvirus that expresses the IMP or fragment thereof as a fusion protein with poxvirus EEV-specific protein or membrane-associated fragment thereof, wherein EEV produced by the infected host cell comprise the IMP fusion protein as part of the EEV outer envelope membrane;
(b) recovering EEV released from the host cell;
wherein the IMP or fragment thereof displays on the surface of the EEV in a native conformation.
16. The method of claim 15 , wherein the EEV-specific protein is the vaccinia virus A33R protein, A34R protein, A56R protein, B5R protein, A36R protein, F13L protein, any membrane-associated fragment thereof, or any combination thereof.
17. The method of claim 16 , wherein the EEV-specific protein is F13L (SEQ ID NO: 1) or a functional fragment thereof.
18. The method of claim 17 , wherein the IMP is a multi-pass membrane protein comprising at least two transmembrane domains.
19. The method of claim 16 , wherein the membrane-associated EEV specific protein fragment comprises the stalk, transmembrane, and intra-membrane domains of the vaccinia virus A56R protein.
20. The method of claim 16 , wherein the membrane-associated EEV specific protein fragment comprises the transmembrane and intra-membrane domains of the vaccinia virus B5R protein.
21. The method of claim 20 , wherein the membrane-associated EEV specific protein fragment comprises the stalk, the transmembrane domain, and intra-membrane domain of the vaccinia virus B5R protein.
22. A method to select antibodies that bind to a multi-pass membrane protein comprising:
(a) attaching the recombinant vaccinia virus EEV of claim 13 to a solid support;
(b) providing an antibody display library, wherein the library comprises display packages displaying a plurality of antigen binding domains;
(c) contacting the display library with the EEV such that display packages displaying antigen binding domains that specifically binds to the IMP expressed on the EEV can bind thereto;
(d) removing unbound display packages; and
(e) recovering display packages that display an antigen binding domain specific for the IMP expressed on the EEV.
23. The method of claim 22 , wherein the EEV are attached to the solid surface via reaction with tosyl groups attached to the surface.
24. The method of claim 22 , wherein the EEV are biotinylated and attached to a streptavidin coated solid surface.