Preliminary X-ray characterization of the ribonuclease P (C5 protein) from Escherichia coli: expression, crystallization and cryoconditions.
The gene for Escherichia coli ribonuclease P (RNase P) protein (also known as C5 protein) and its mutant C5-C113A have been expressed as GST fusion proteins in E. coli at a high level. After cleavage of the fusion protein, highly purified functional C5 protein is obtained that can be crystallized with 2.5-2.6 M (NH(4))(2)HPO(4)/(NH(4))H(2)PO(4) pH 7.0 at room temperature. These crystals are suitable for X-ray analysis, belong to the space group P3(1)21 or P3(2)21 (unit-cell parameters a = b = 66.67, c = 142.09 A) and diffract to 2.9 A at 100 K using sorbitol and glycerol as cryoprotectants. For three molecules in the asymmetric unit a V(M) of 2.17 A(3) Da(-1) was calculated.
Choe
Hui-Woog
HW
Department of Chemistry, College of Natural Science, Chonbuk National University, 561-756 Chonju, South Korea. hwchoe@moak.chonbuk.ac.kr
Jeong
Dae-Gwin
DG
Park
Jung Hee
JH
Schlesinger
Ramona
R
Labahn
Jörg
J
Hofmann
Klaus Peter
KP
Büldt
Georg
G
D019943:Amino Acid Substitution; D003451:Cryoprotective Agents / Q000737:chemistry; D003460:Crystallization; D018360:Crystallography, X-Ray; D004722:Endoribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D005982:Glutathione Transferase / Q000235:genetics; D005990:Glycerol / Q000737:chemistry; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D043262:Ribonuclease P; D012261:Ribonucleoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013012:Sorbitol / Q000737:chemistry
D003451:Cryoprotective Agents; D029968:Escherichia coli Proteins; D016337:RNA, Catalytic; D011993:Recombinant Fusion Proteins; D012261:Ribonucleoproteins; D013012:Sorbitol; D005982:Glutathione Transferase; D004722:Endoribonucleases; D043262:Ribonuclease P; C470865:ribonuclease P, E coli; D005990:Glycerol