Direct expression of a synthetic gene in Escherichia coli: purification and physicochemical properties of human initiation factor 4E.
An artificial synthetic gene coding for human eIF-4E was cloned into an expression vector and direct expression was attempted in Escherichia coli [BL21(DE3) strain] under the control of T7 promoter. The active gene product which was induced in high yield (ca. 4 mg/100 ml) by isopropyl-beta-D-thiogalactopyranoside was purified to homogeneity by a two-step chromatographic procedure with a good yield (ca. 74%), and was confirmed to be recombinant human eIF-4E by amino acid composition and sequence analyses, isoelectric focusing, and absorption spectral measurements. The identity of three-dimensional structures between the recombinant and native human eIF-4Es was confirmed by CD and fluorescence measurements.
Department of Physical Chemistry, Osaka University of Pharmaceutical Sciences.
S
S
Morino
M
M
Yasui
M
M
Doi
M
M
Inoue
T
T
Ishida
H
H
Ueda
S
S
Uesugi
D000595:Amino Acid Sequence; D001483:Base Sequence; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003001:Cloning, Molecular; D004926:Escherichia coli; D039561:Eukaryotic Initiation Factor-4E; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D005813:Genes, Synthetic; D006801:Humans; D008969:Molecular Sequence Data; D010448:Peptide Initiation Factors / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification
D039561:Eukaryotic Initiation Factor-4E; D010448:Peptide Initiation Factors; D011994:Recombinant Proteins