Superexpression and fast purification of E coli initiation factor IF2.
For the production of large quantities of E coli initiation factor IF2 we have constructed an improved overexpression system. The gene infB was cloned into the thermo-inducible runaway plasmid pCP40 [1] and subsequently transformed into the E coli strain C600[pcI857]. In this system the expression of infB is under the control of the strong promoter lambda PL and the cells carry the plasmid pcI857, which contains a thermosensible lambda cI repressor. Overexpression of IF2, which is approximately 30 times higher than the expression in wild-type-cells, is induced at 42 degrees C and continues for 2 h at 37 degrees C. From these cells pure and active IF2 was obtained using a novel 3-step FPLC-procedure consisting of ion-exchange liquid chromatography on Q-sepharose HP, MonoQ and MonoS. In approximately 8 h, 5 mg of pure and active IF2 can be obtained from 10 g overproducing cells. This corresponds to 5 mg of IF2 per litre of medium. The purification was monitored by Western immunoblotting and the activity of the purified factor was tested by measuring the stimulation of binding of the initiator fMet-tRNA(Met)f to 70S ribosomes in the presence of GTP and poly(A,U,G) as messenger RNA. Compared with previous methods our purification procedure avoids the use of materials such as DEAE-cellulose and phosphocellulose which have relatively poor flow rates. In addition to the higher flow capacity of Q-sepharose HP, this new matrix can be loaded with an S30 supernatant.(ABSTRACT TRUNCATED AT 250 WORDS)
Mortensen
K K
KK
Department of Chemistry, Aarhus University, Denmark.
Nyengaard
N R
NR
Hershey
J W
JW
Laalami
S
S
Sperling-Petersen
H U
HU
D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D010448:Peptide Initiation Factors / Q000235:genetics* / Q000302:isolation & purification*; D010957:Plasmids; D039881:Prokaryotic Initiation Factor-2
D001426:Bacterial Proteins; D010448:Peptide Initiation Factors; D039881:Prokaryotic Initiation Factor-2