Expression in E. coli and purification of Thermus thermophilus translation initiation factors IF1 and IF3.
The initiation of protein translation in bacteria requires in addition to mRNA, fMet-tRNA, and ribosomal subunits three protein factors, the initiation factor 1 (IF1), initiation factor 2 (IF2), and initiation factor 3 (IF3). The genes coding for IF1 and IF3 from Thermus thermophilus have been identified and cloned into pET expression vector and were expressed as soluble proteins in Escherichia coli. IF1 was purified by a DEAE-cellulose chromatography, followed by heat denaturation, chromatography on Hydroxylapatit, and gel permeation chromatography using Sephacryl 200HR. For the purification of IF3, a heat denaturation step is followed by anion-exchange chromatography on Q-Sepharose FF and gel permeation chromatography on Sephacryl 200HR. Using these procedures we obtained chromatographically pure and biologically active preparations of both T. thermophilus IF1 and IF3.
Laboratorium für Biochemie, AG Sprinzl, Universität Bayreuth, Universitaetsstrasse 30, 95440 Bayreuth, Germany. alexandra.nickles1@uni-bayreuth.de
Alexandra
A
Wolfrum
Stephan
S
Brock
Thi
T
Mac
Norbert
N
Grillenbeck
D000595:Amino Acid Sequence; D002845:Chromatography; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009876:Operon; D010448:Peptide Initiation Factors / Q000737:chemistry / Q000378:metabolism*; D039861:Prokaryotic Initiation Factor-1 / Q000737:chemistry / Q000378:metabolism*; D039665:Prokaryotic Initiation Factor-3 / Q000737:chemistry / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D012358:RNA, Transfer, Met / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D016963:Thermus thermophilus / Q000378:metabolism*; D013997:Time Factors
D010448:Peptide Initiation Factors; D039861:Prokaryotic Initiation Factor-1; D039665:Prokaryotic Initiation Factor-3; D012333:RNA, Messenger; D012358:RNA, Transfer, Met; C089238:fMet-tRNA(fMet)