Integral membrane protein display on poxvirus extracellular enveloped virions
AssigneeVaccinex, Inc.
InventorsErnest S. Smith, Mark Paris, Maria G.M. Scrivens, Renee A. Kirk, Angelica A. Cornelison
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Abstract
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.
Claims28
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).
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.
A fusion protein comprising:
- (a) amino acids 20 to 892 of SEQ ID NO:
- 2;
- (b) SEQ ID NO:
- 3;
- (c) SEQ ID NO:
- 4;
- (d) amino acids 20 to 370 of SEQ ID NO:
- 6;
- (e) amino acids 20 to 855 of SEQ ID NO:
- 7;
- (f) amino acids 20 to 935 of SEQ ID NO:
- 8;
- (g) amino acids 20 to 243 of SEQ ID NO:
- 10;
- (h) amino acids 20 to 281 of SEQ ID NO:
- 11;
- (i) amino acids 20 to 506 of SEQ ID NO:
- 16, or
- (j) amino acids 20 to 235 of SEQ ID NO:
- 17;
- wherein the fusion protein, when expressed by a recombinant poxvirus, appears on the surface of a poxvirus extracellular enveloped virion (EEV) in a native conformation.
A recombinant poxvirus EEV comprising a heterologous IMP or fragment thereof fused to a poxvirus EEV-specific protein or membrane-associated fragment thereof, wherein the fusion protein is situated in the EEV outer envelope membrane, wherein the IMP or fragment thereof displays on the surface of the EEV in its native conformation.
Dependencies are inferred from the claim text.