Expression of mRNA cap-binding protein and structure analysis of its cap recognition mechanism

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Cap-binding protein (named CBP or IF-4E), the content of which is very small amount in living cell, plays an important role in initiating the protein synthesys on ribosome. In order to elucidate the biochemical and structural nature of this protein and to make clear the selective recognition of mRNA cap structure, we carried out this research project. The main results obtained so far are as follows:(1) We succeeded in the chemical synthsis of gene of human cap-binding protein (hCBP)(total base pairs of 660).(2) We established the inclusion of this gene into pBR322 vector and the large amount of expression of hCBP as the inclusion body with human growth hormone.(3) We established the separation of hCBP from the confused protein and the HPLC purification method.(4) The functional amino acid residues which probably functions in the selective recognition for the mRNA cap structure characterized by 7-methylguanine base (m7G) were surveyed by a series of model interaction studies.(5) Based on the above insight, the functional amino acids of hCBP were transformed to the non-functional amino acids by the site-directed mutagenesis. The methods for the gene mutations and the isolation and purification of mutant hCBP proteins were established.(6) A series of mRNA cap analogues were chemically synthesized.(7) The interaction modes of these cap analogues with the native and mutant hCBP proteins were investigated by UV and fluorescence spectroscopies. As a result, it was made clear that the His-33, His-37, Trp-102 and Glu-105 residues play important role in interaction with mRNA cap structure. Further, the same results were obtained from the binding experiments of the affinity of m7G-containing column with native and mutant hCBPs.(8) The X-ray crystal and NMR solution analyses are now in progress.