Difference between revisions of "Part:BBa K1188002"
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RTX (repeats-in-toxin) is a polypeptide motif consisting of a repeating sequence of amino acids. It undergoes a conformation change in the presence of calcium which causes it to precipitate from solution. This is the shorter (8-mer) version of this part, and is in Freiburg format. This allows RTX to be fused to itself to create a longer RTX chain, or to other proteins which can then be precipitated from a solution. This is useful for the purpose of protein purification, or alternatively to remove an unwanted protein from a solution. | RTX (repeats-in-toxin) is a polypeptide motif consisting of a repeating sequence of amino acids. It undergoes a conformation change in the presence of calcium which causes it to precipitate from solution. This is the shorter (8-mer) version of this part, and is in Freiburg format. This allows RTX to be fused to itself to create a longer RTX chain, or to other proteins which can then be precipitated from a solution. This is useful for the purpose of protein purification, or alternatively to remove an unwanted protein from a solution. | ||
| − | Experimental results show that calcium concentrations of 10 mM or above result in effective precipitation | + | Experimental results show that calcium concentrations of 10 mM or above result in effective precipitation and is capable of a high level of purification of a fused protein (in this case, GFP) without disrupting protein function (green fluorescence is present). Additionally, calcium concentrations below ~1 mM do not produce a useful amount of precipitate for the purpose of protein purification. |
[[File:RTXPrecipitation.PNG|200px]] | [[File:RTXPrecipitation.PNG|200px]] | ||
Revision as of 15:35, 27 September 2013
RTX - Calcium Binding Protein
RTX (repeats-in-toxin) is a polypeptide motif consisting of a repeating sequence of amino acids. It undergoes a conformation change in the presence of calcium which causes it to precipitate from solution. This is the shorter (8-mer) version of this part, and is in Freiburg format. This allows RTX to be fused to itself to create a longer RTX chain, or to other proteins which can then be precipitated from a solution. This is useful for the purpose of protein purification, or alternatively to remove an unwanted protein from a solution.
Experimental results show that calcium concentrations of 10 mM or above result in effective precipitation and is capable of a high level of purification of a fused protein (in this case, GFP) without disrupting protein function (green fluorescence is present). Additionally, calcium concentrations below ~1 mM do not produce a useful amount of precipitate for the purpose of protein purification.
SDS-PAGE - From Left to Right:
Lane 1: Clarified cell lysate
Lane 2: 0mM supernatant after addition of 0mM CaCl2 and 2min spin at 13.2k RPM
Lane 3: 0mM supernatant after wash and resuspension in 50mM trisHCl with 0mM EDTA
Lane 4: 0.1mM supernatant after addition of 0.1mM CaCl2 and 2min spin at 13.2k RPM
Lane 5: 0.1mM supernatant after wash and resuspension in 50mM trisHCl with 0.1mM EDTA
Lane 6: 1mM supernatant after addition of 1mM CaCl2 and 2min spin at 13.2k RPM
Lane 7: 1mM supernatant after wash and resuspension in 50mM trisHCl with 1mM EDTA
Lane 8: 10mM supernatant after addition of 10mM CaCl2 and 2min spin at 13.2k RPM
Lane 9: 10mM supernatant after wash and resuspension in 50mM trisHCl with 10mM EDTA
Lane 10: 100mM supernatant after addition of 100mM CaCl2 and 2min spin at 13.2k RPM
Lane 11: 100mM supernatant after wash and resuspension in 50mM trisHCl with 100mM EDTA
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 4
Illegal AgeI site found at 223 - 1000COMPATIBLE WITH RFC[1000]