	US 12,442,050 B2
	Cleavable nucleic acid linkers for protein quantification rationing
Brian Chen, Brossard (CA)
Filed by Brian Chen, Brossard (CA)
Filed on Jun. 3, 2020, as Appl. No. 16/891,205.
Application 16/891,205 is a continuation of application No. 15/534,025, granted, now 10,752,965, previously published as PCT/CA2015/051281, filed on Dec. 7, 2015.
Claims priority of provisional application 62/088,823, filed on Dec. 8, 2014.
Prior Publication US 2021/0002731 A1, Jan. 7, 2021
Int. Cl. C12N 15/11 (2006.01); C12N 15/63 (2006.01); C12Q 1/6897 (2018.01); G01N 33/50 (2006.01); G01N 33/58 (2006.01)

CPC C12Q 1/6897 (2013.01) [C12N 15/11 (2013.01); C12N 15/63 (2013.01); G01N 33/5008 (2013.01); G01N 33/5023 (2013.01); G01N 33/582 (2013.01)]

20 Claims

1. A method for quantifying a protein of interest in a host or a host cell, said method comprising:

a) expressing a vector in the host or the host cell, said vector comprising a first nucleic acid molecule encoding a cleavable peptide, a second nucleic acid molecule encoding a reporter protein, and a third nucleic acid molecule encoding a protein of interest, the first nucleic acid molecule comprising a protein quantitation reporter linker molecule:

i) encoding the cleavable peptide having the amino acid sequence of SEQ ID NO: 23:

	(SEQ ID NO: 23)
	GSGX₁X₂X₃X₄X₅X₆X₇X₈X₉LX₁₀X₁₁X₁₂GDVEX₁₃NPGP

in which X₁is V or absent; X₂is K or absent; X₃is Q or absent; X₄is T, C, A or absent; X₅is L, T or E; X₆is N or G; X₇is F, Y or R; X₈is D, A, G or S; X₉is L or S; X₁₀is K or L; X₁₁is L, T or Q; X₁₂is A or C and X₁₃at position 21 is S or E; and

with the proviso that said cleavable peptide is not as set forth in amino acid sequence of SEQ ID NO: 1, 2, or 17, and

ii) being the nucleic acid sequence of SEQ ID NO: 25:

(SEQ ID NO: 25)
N₁N₂N₃ N₄N₅N₆ N₇N₈N₉ N₁₀N₁₁N₁₂ N₁₃N₁₄N₁₅ N₁₆N₁₇N₁₈ N₁₉N₂₀N₂₁

N₂₂N₂₃N₂₄ N₂₅N₂₆N₂₇ N₂₈N₂₉N₃₀ N₃₁N₃₂N₃₃ N₃₄N₃₅N₃₆ N₃₇N₃₈N₃₉

N₄₀N₄₁N₄₂ N₄₃N₄₄N₄₅ N₄₆N₄₇N₄₈ N₄₉N₅₀N₅₁ N₅₂N₅₃N₅₄ N₅₅N₅₆N₅₇

N₅₈N₅₉N₆₀ N₆₁N₆₂N₆₃ AAY CCN₆₄ GGA CCN₆₅

in which N₁to N₆₃are any nucleic acid capable of forming codons encoding GSGX₁X₂X₃X₄X₅X₆X₇X₈X₉LX₁₀X₁₁X₁₂GDVEX₁₃of SEQ ID NO: 23, N₆₄is T or U and N₆₅is T or U; and

at least 50% of the codons encoding X₁X₂X₃X₄X₅X₆X₇X₈X₉LX₁₀X₁₁X₁₂GDVEX₁₃of SEQ ID NO: 23 are de-optimized in relation to the host cell,

wherein the second nucleic acid molecule and the third nucleic acid molecule are operatively linked to the first nucleic acid molecule and wherein the first nucleic acid molecule is located between the second nucleic acid molecule and the third nucleic acid molecule, so that the that the first nucleic acid molecule, the second nucleic acid molecule and the third nucleic acid molecule are transcribed as a single nucleic acid transcript from the vector, to generate the single nucleic acid transcript encoding a poly-protein, said poly-protein comprising the protein of interest, the cleavable peptide and the reporter protein, wherein the cleavable peptide can be cleaved during translation of the nucleic acid transcript to generate a cleaved reporter protein; and

b) measuring a signal associated with the cleaved reporter protein to quantify the protein of interest in the host or the host cell.