Purification of the Caenorhabditis elegans transposase Tc1A refolded during gel filtration chromatography.
Full-length recombinant transposase Tc1A from Caenorhabditis elegans (343 amino acids) expressed in Escherichia coli BL21 in inclusion bodies has been purified in a high yield in a soluble form. The procedure includes denaturation of the inclusion bodies followed by refolding of the Tc1A protein by gel filtration. This last step is absolutely crucial to give a high yield of soluble and active protein since it allows the physical separation of the aggregates from intermediates that give rise to correctly refolded protein. This step is very sensitive to the concentration of protein. Good yields of refolded protein are obtained by refolding 2 to 12 mg of denatured protein. The other purification steps involve the initial use of gel filtration under denaturing conditions and a final step of ion-exchange chromatography. Biological activity of the purified protein was confirmed in an in vitro transposon excision assay and its DNA-binding capacity by UV crosslinking. This new Tc1A purification procedure gives a yield of 12-16 mg/liter E. coli culture, in a form suitable for crystallization studies.
Department of Molecular Biology, The Netherlands Cancer Institute, Plesmanlaan 121, Amsterdam, 1066 CX, The Netherlands. garcia@nki.nl
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D000818:Animals; D017173:Caenorhabditis elegans / Q000201:enzymology* / Q000235:genetics; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003432:Cross-Linking Reagents; D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D004795:Enzyme Stability; D002479:Inclusion Bodies / Q000378:metabolism; D010957:Plasmids; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D012995:Solubility; D019895:Transposases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins; C081454:Tc1 transposase; D019895:Transposases