Journal club

Constructing the optimal promotor

A human factor IX cDNA clone isolated from a liver cDNA library constructed in phage Agtl 1 vector was shown to express factor IX protein in Escherichia coli. A factor IX immunospecific protein of 46.8 kDa was expressed, but was not a β-galactosidase-factor IX fusion protein. Expression was seen when the factor IX cDNA was cloned into two different vector systems, λgtl 1 and pUC9, in both orientations with respect to the vector lacZ promoter. The expression of factor IX was not under control of the lacZ promoter of either vector system. In addition, when the factor IX cDNA fragment was subcloned in both orientations into a promoterless cloning vector (pCPP3), the factor IX cDNA fragment demonstrated promoter activity when inserted in only one orientation resulting in expression of chloramphenicol acetyl transferase in E. coli and Bacillus subtilis. A DNA computer search of the N-terminal sequences of the factor IX gene revealed prokaryotic-like promoter and ribosome-binding site (RBS) sequences with strong homology to the E. coli consensus sequences. The predicted sites homologous with prokaryotic promoter and RBS consensus sequences are followed by an in-frame methionine which could correspond to the translation start codon of the expressed factor IX. This report provides the first evidence that a eukaryotic gene encodes the information necessary for both transcription and translation to control gene expression in a prokaryotic host.