1991 Volume 109 Issue 6 Pages 882-889
An artificial gene coding for the human cap binding protein (hCBP: human IF-4E) was chemically synthesized and expressed in Escherichia coli under the control of a trp promoter. The DNA duplex of 662 by was designed and constructed from 44 oligodeoxynu-cleotide fragments of typically 30 nucleotides in length. Although the hCBP gene was not directly expressed in E. coli HB101, we succeeded in its high-level expression as a fusion protein connected with a portion of human growth hormone through a tetradecapeptide (Asp-Asp-Pro-Pro-Thr-Val-Glu-Leu-Gln-Gly-Leu-Val-Pro-Arg) that contains the recognition sequence for a site-specific protease α-thrombin. Upon induction with 3-indoleacrylic acid, the fusion protein accumulated with a yield of about 20% of the total proteins of the host cell. Upon the treatment of the fusion protein with α-thrombin, which recognizes the sequence “Val-Pro-Arg, ” specific proteolysis at the fused junction occurred efficiently. In this system, nonspecific digestion by α-thrombin was not marked. About 15 mg of recombinant hCBP was obtained from a 1-liter culture. Association constants between the recombinant hCBP and mRNA cap structure analogues were determined by fluorescence spectros-copy. The values obtained for the m7GpppA, m7GTP, and m7GMP were almost the same as those reported for the IF-4E isolated from human erythrocyte cells. The results show that the binding constants for nonmethylated analogues GpppA, GTP, and GMP, a 2-amino deletion analogue m7IMP, a nonphosphorylated analogue 7-methylguanosine, and a ribose-deleted analogue 7-methyl-9-ethylguanine, were smaller than those for methylated cap structure analogues by about one order of magnitude. Moreover, m7AVP (an analogue containing the 7-methylguanine base and phosphate group) showed nearly the same association constant as the cap structure. Based on these results, a possible model for mRNA cap structure recognition by eIF-4E has been proposed.
