科研之友
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摘要
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 bp was designed and constructed from 44 oligodeoxynucleotide 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 alpha-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 alpha-thrombin, which recognizes the sequence "Val-Pro-Arg," specific proteolysis at the fused junction occurred efficiently. In this system, nonspecific digestion by alpha-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 spectroscopy. 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.
- 出版日期1991-6
