Compositions and methods for synthesizing, purifying, and detecting biomolecules
Compositions and methods that can reduce or eliminate purification and detection difficulties related to SlyD polypeptides are disclosed. The compositions include cells that lack or contain a reduced amount of SlyD, cells that contain SlyD mutated to reduce or eliminate its ability to bind biarsenical reagents; anti-SlyD antibodies, and kits containing the same. The disclosed compositions can be used, e.g., to purify and detect recombinant polypeptides having polyhistidine or polycysteine tags.
1 . A cellular extract from an organism comprising a SlyD gene, substantially free of a SlyD polypeptide that binds to a bi-arsenical reagent.
2 . The cellular extract of claim 1 , wherein said cellular extract is competent for IVTT.
3 . The cellular extract of claim 1 , wherein the cellular extract is a bacterial extract.
4 . The cellular extract of claim 1 , wherein the cellular extract is an E. coli extract.
5 . The cellular extract of claim 1 , wherein the cellular extract is from an E. coli comprising a SlyD mutant gene.
6 . The cellular extract of claim 5 , wherein the SlyD mutant gene encodes a SlyD that is mutated in an amino acid sequence that binds a biarsenical molecule.
7 . The cellular extract of claim 5 , wherein the SlyD mutant gene encodes a truncated SlyD protein.
8 . The cellular extract of claim 1 , further comprising a nuclease inhibitor.
9 . The cellular extract of claim 8 , wherein the nuclease inhibitor is a Gam protein.
10 . The cellular extract of claim 1 , wherein the extract has reduced activity of at least one enzyme that catalyzes hydrolysis of high energy phosphate bonds or hydrolysis or formation of phosphodiester bonds.
11 . The cellular extract of claim 1 , further comprising at least one inhibitor of at least one enzyme that catalyzes hydrolysis of high energy phosphate bonds or hydrolysis or formation of phosphodiester bonds.
12 . The cellular extract of claim 1 , further comprising at least two energy sources providing chemical energy for synthesis.
13 . The cellular extract of claim 1 , further comprising a nucleic acid encoding a fusion protein encoding an exogenous protein fused to a tag.
14 . The cellular extract of claim 13 , wherein the tag binds a biarsenical reagent.
15 . A kit for the in vitro production of proteins, said kit comprising a cellular extract according to claim 1 .
16 . The kit of claim 14 , further comprising one or more packaged solutions selected from the group consisting of a buffer, a solution comprising magnesium, a solution comprising amino acids and a solution comprising ribonucleotide triphosphates.
17 . The kit of claim 16 , wherein the buffer is an in vitro transcription/translation buffer.
18 . A method for producing a protein comprising combining a nucleic acid that encodes said protein with the cellular extract of claim 1 .
19 . The method of claim 16 , wherein the nucleic acid encodes a fusion protein comprising a tag.
20 . The method of claim 19 , wherein the tag binds a biarsenical reagent.
21 . The method of claim 20 , further comprising contacting the protein with a biarsenical reagent.
22 . The method of claim 21 , further comprising detecting the biarsenical reagent.
23 . The method of claim 22 , wherein the detection is carried out in real-time.
24 . A method for producing a protein comprising combining a nucleic acid that encodes said protein with the cellular extract of claim 1 .
25 . A polypeptide comprising an amino acid sequence that binds that specifically binds to EDT 2 [4′,5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein-(1,2-ethanedithiol) 2 , selected from the group consisting of CCGGKGNGGCGC (SEQ ID NO. 2), CCGGHGHDHGHEHGGEGCCGGKGNGGCGC (SEQ ID NO. 3) and AAGGHGHDHGHEHGGEGCCGGKGNGGCGC (SEQ ID NO. 8).
26 . A fusion protein comprising an amino acid sequence that binds that specifically binds to EDT 2 [4′,5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein-(1,2-ethanedithiol) 2 , selected from the group consisting of CCGGKGNGGCGC (SEQ ID NO. 2), CCGGHGHDHGHEHGGEGCCGGKGNGGCGC (SEQ ID NO. 3), and AAGGHGHDHGHEHGGEGCCGGKGNGGCGC (SEQ ID NO. 8).
27 . A nucleic acid encoding an amino acid sequence that binds that specifically binds to EDT 2 [4′,5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein-(1,2-anedithiol) 2 , selected from the group consisting of CCGGKGNGGCGC (SEQ ID NO. 2), CCGGHGHDHGHEHGGEGCCGGKGNGGCGC (SEQ ID NO. 3) and GGHGHDHGHEHGGEGCCGGKGNGGCGC (SEQ ID NO. 8).