A preliminary solubility screen used to improve crystallization trials: crystallization and preliminary X-ray structure determination of Aeropyrum pernix flap endonuclease-1.
Crystallization of protein and protein complexes is a multi-parametric problem that involves the investigation of a vast number of physical and chemical conditions. The buffers, salts and additives used to prepare the protein will be present in every crystallization condition. It is imperative that these conditions be defined prior to crystal screening since they will have a ubiquitous involvement in the crystal-growth experiments. This study involves the crystallization and preliminary analysis of the flap endonuclease-1 (FEN-1) DNA-repair enzyme from the crenarchaeal organism Aeropyrum pernix (Ape). Ape FEN-1 protein in a standard chromatography buffer had only a modest solubility and minimal success in crystallization trials. Using an ion/pH solubility screen, it was possible to dramatically increase the maximum solubility of the protein. The solubility-optimized protein produced large diffraction-quality crystals under multiple conditions in which the non-optimized protein produced only precipitate. Only minor adjustments of the conditions were required to produce single diffraction-quality crystals. The native Ape FEN-1 crystals diffract to 1.4 A resolution and belong to space group P6(1), with unit-cell parameters a = b = 92.8, c = 80.9 A, alpha = beta = 90, gamma = 120 degrees.
Collins
Brandon K
BK
Department of Chemistry, The University of Toledo, Toledo, OH 43606, USA.
Tomanicek
Stephen J
SJ
Lyamicheva
Natasha
N
Kaiser
Michael W
MW
Mueser
Timothy C
TC
D041522:Aeropyrum / Q000201:enzymology*; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003460:Crystallization; D018360:Crystallography, X-Ray; D003627:Data Interpretation, Statistical; D004926:Escherichia coli / Q000378:metabolism; D045585:Flap Endonucleases / Q000737:chemistry* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D012995:Solubility
D011994:Recombinant Proteins; D045585:Flap Endonucleases