A sixty base pair DNA duplex containing the nucleotide sequence of the bacteriophage T5 early (T5P25) promoter has been constructed using a combination of chemical synthesis and enzymatic methods. Subsequent to cloning into pBR322, the promoter has been demonstrated to be biologically active being capable of directing the efficient expression of genes under its control. This serves as a prototype for an approach to the study of the in vivo structure-function relationships and efficiency of promoters.
A chemically synthesized gene for human interferon-y has been cloned into a prokaryotic expression vector under the regulation of a synthetic constitutive transcriptional-translational control unit that contains a strong bacteriophage T5 early promoter and a strong ribosome-binding site. Cells harboring the recombinant plasmid express high levels (4 x 109 units per liter of culture) of antiviral activity specific for interferon-y. Analysis of total cell lysates on NaDodSO4/polyacrylamide gels revealed a 17,200-dalton protein, expected for the nonglycosylated form of human interferon-y, that constitutes >15% of total cell protein.Human interferon-y (HuIFN--y) is an interesting protein that exhibits a number of different biological activities. In addition to its antiviral activity, HuIFN-y has been shown to exhibit potent immunomodulation and cell proliferation-inhibition properties (reviewed in ref. 1). Recently, the nucleotide sequence of its gene has been determined (2, 3), and its expression in Escherichia coli has been achieved by using natural control signals from either the trp operon (2) or the lac UV5 operon (4). However, the reported yields were relatively low compared to those for IFN-a (5-7) and IFN-,B (8, 9).Among the large numbers of DNA fragments containing bacterial promoters that have been used for in vitro binding studies, the bacteriophage T5 early promoters (T5P25 and T5P26) have been found to far exceed other promoter fragments in the rate of complex formation with E. coli RNA polymerase (10, 11). Recently, we have reported the chemical synthesis and insertion of the T5P25 promoter in front of either the tetracycline-resistance (TcR) gene or the chloramphenicol acetyltransferase (CAT) gene, and have demonstrated that the synthetic T5P25 promoter is highly efficient in vivo (12).We have also shown that the introduction of a synthetic ribosome-binding site (RBS) in front of the coding sequence for simian virus 40 small tumor antigen, and its subsequent insertion at the Pst I site within the pBR322 ampicillin-resistance (ApR) gene, resulted in the synthesis of authentic simian virus 40 small tumor antigen in E. coli (13). Using this model system, we were able to compare the efficiency of different synthetic RBS sequences in vivo (14).In this communication, we describe the construction of a plasmid expression vector containing both the synthetic T5P25 promoter and a strong synthetic RBS; this vector (pJPlR3) is used for the efficient expression of a synthetic gene for human IFN-y.
MATERIALS AND METHODSConstruction of the IFN-y Gene. The deoxyoligonucleotides (Fig. 1) comprising the entire IFN-y sequence including initiation and termination signals were chemically synthesized using a modified solid-phase phosphite method. -Details of the synthesis and construction of the gene will be published elsewhere.Construction of the pJPjR3 Expression Vector. The pJPj plasmid DNA, a derivative of pBR322 in which the TcI promoter between the EcoRI and HindIII sites has been replaced by the strong synth...
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