US 12,202,859 B2
Hepatitis c virus E1/E2 heterodimers and methods of producing same
Michael Houghton, Danville, CA (US); Darren Hockman, Edmonton (CA); John L. Law, Edmonton (CA); Chao Chen, Edmonton (CA); and Michael Logan, Edmonton (CA)
Assigned to The Governors of the University of Alberta, Edmonton (CA)
Filed by The Governors of the University of Alberta, Edmonton (CA)
Filed on Oct. 25, 2021, as Appl. No. 17/509,808.
Application 17/509,808 is a continuation of application No. 15/763,370, granted, now 11,186,615, previously published as PCT/IB2016/056000, filed on Oct. 7, 2016.
Claims priority of provisional application 62/239,157, filed on Oct. 8, 2015.
Claims priority of provisional application 62/337,212, filed on May 16, 2016.
Prior Publication US 2022/0041661 A1, Feb. 10, 2022
Int. Cl. C07K 14/005 (2006.01); A61K 39/00 (2006.01); A61K 39/12 (2006.01); A61K 39/29 (2006.01); A61P 31/14 (2006.01); C07K 1/22 (2006.01); C07K 19/00 (2006.01); C12N 7/00 (2006.01); C12N 15/62 (2006.01); C12P 21/02 (2006.01)
CPC C07K 14/005 (2013.01) [A61K 39/12 (2013.01); A61K 39/29 (2013.01); A61P 31/14 (2018.01); C07K 1/22 (2013.01); C07K 19/00 (2013.01); C12N 7/00 (2013.01); C12N 15/62 (2013.01); C12P 21/02 (2013.01); A61K 2039/54 (2013.01); A61K 2039/55505 (2013.01); A61K 2039/55572 (2013.01); A61K 2039/575 (2013.01); C07K 2319/30 (2013.01); C07K 2319/50 (2013.01); C12N 2770/24222 (2013.01); C12N 2770/24234 (2013.01); C12N 2770/28122 (2013.01)] 15 Claims
 
1. A method of purifying a hepatitis C virus (HCV) E1/E2 heterodimer from a lysate, the method comprising:
a) contacting a lysate of a genetically modified host cell with an affinity tag-binding polypeptide immobilized on an insoluble support, wherein the genetically modified host cell is genetically modified with a nucleic acid comprising a nucleotide sequence encoding a polyprotein comprising, in order from N-terminus to C-terminus:
a) an HCV E1 polypeptide; and
b) an HCV E2-affinity tag fusion polypeptide comprising, in order from N-terminus to C-terminus: i) an affinity tag polypeptide; ii) a proteolytically cleavable linker; and iii) an HCV E2 polypeptide
i) a first nucleotide sequence encoding an HCV E1 polypeptide; and
ii) a second nucleotide sequence encoding an HCV E2 polypeptide,
wherein the polyprotein is processed in the host cell to produce the affinity tagged HCV E1/E2 heterodimer and the lysate comprises the affinity tagged HCV E1/E2 heterodimer,
wherein the affinity tagged HCV E1/E2 heterodimer present in the lysate binds to the immobilized affinity tag-binding polypeptide, generating an immobilized affinity tagged HCV E1/E2 heterodimer;
b) contacting the immobilized HCV E1/E2 heterodimer with a protease that cleaves the proteolytically cleavable linker present in the immobilized affinity tagged HCV E1/E2 heterodimer, thereby releasing the HCV E1/E2 heterodimer from the affinity tag; and
c) collecting the HCV E1/E2 heterodimer.