Difference between revisions of "Part:BBa K1188002"

Revision as of 15:31, 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.

Experimentally results show that calcium concentrations of 10 mM or above results in effective precipitation while conserving the function of the purified protein, in the the case of GFP. Calcium concentrations below ~1 mM do not produce a useful amount of precipitate.

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