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US 7,252,932 B1Granted
Methods for the detection, analysis and isolation of nascent proteins
AssigneeAmberGen, Inc.
InventorsKenneth J. Rothschild, Sadanand Gite, Jerzy Olejnik, Sergey Mamaev, Mark Lim
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Abstract
This invention relates to non-radioactive markers that facilitate the detection and analysis of nascent proteins translated within cellular or cell-free translation systems. Nascent proteins containing these markers can be rapidly and efficiently detected, isolated and analyzed without the handling and disposal problems associated with radioactive reagents. Preferred markers are dipyrrometheneboron difluoride (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) dyes.
Claims48
A gel-free method, comprising:
- a) providing i) a misaminoacylated initiator tRNA molecule which only recognizes the first AUG codon that serves to initiate protein synthesis, said misaminoacylated initiator tRNA molecule comprising a first marker, and ii) a nucleic acid template encoding a protein, said protein comprising an affinity marker and a C-terminal marker;
- b) introducing said misaminoacylated initiator tRNA to a translation system comprising said template under conditions such that a nascent protein is generated, said protein comprising said first marker, said affinity marker, and said C-terminal marker;
- and
- c) testing said nascent protein under gel-free conditions that permit detection of a truncated protein.
1 more element
A gel-free method, comprising:
- a) providing i) a misaminoacylated initiator tRNA molecule which only recognizes the first AUG codon that serves to initiate protein synthesis, said misaminoacylated initiator tRNA molecule comprising a first marker, and ii) a nucleic acid template encoding a protein, said protein comprising an affinity marker and a C-terminal marker;
- b) introducing said misaminoacylated initiator tRNA to a translation system comprising said template under conditions such that a nascent protein is generated, said protein comprising said first marker at the N-terminus of said protein, said C-terminal marker, and said affinity marker adjacent to said first marker;
- and
- c) testing said nascent protein under gel-free conditions that permit detection of a truncated protein.
1 more element
A gel-free method, comprising:
- a) providing i) a misaminoacylated tRNA molecule which only recognizes the first codon designed to serve to initiate protein synthesis, said misaminoacylated initiator tRNA molecule comprising a first marker, and ii) a nucleic acid template encoding a protein, said protein comprising an affinity marker and a C-terminal marker;
- b) introducing said misaminoacylated initiator tRNA to a translation system comprising said template under conditions such that a nascent protein is generated, said protein comprising said first marker, said affinity marker and said C-terminal marker;
- and
- c) testing said nascent protein under gel-free conditions that permit detection of a truncated protein.
1 more element
A gel-free method, comprising:
- a) providing i) a misaminoacylated tRNA molecule which only recognizes the first codon designed to serve to initiate protein synthesis, said misaminoacylated initiator tRNA molecule comprising a first marker, and ii) a nucleic acid template encoding a protein, said protein comprising an affinity marker and a C-terminal marker;
- b) introducing said misaminoacylated initiator tRNA to a translation system comprising said template under conditions such that a nascent protein is generated, said protein comprising said first marker at the N-terminus of said protein, a C-terminal marker, and said affinity marker adjacent to said first marker;
- and
- c) testing said nascent protein under gel-free conditions that permit detection of a truncated protein.
1 more element
Dependencies are inferred from the claim text.