Expression of human interferon-α8 synthetic gene under P(BAD) promoter.

Recombinant human interferon-α8 (rhIFN-α8) was obtained by synthesizing a codon-optimized gene in a two-step polymerase chain reaction (PCR) and expressing it in Escherichia coli. The gene encoding human IFN-α8 shows a high content of rare codons. These were replaced based on E. coli codon usage and balancing TA-GC ratio contents of the entire gene. The two-step PCR was performed using long (45-60 nucleotides) overlapped primers and two Taq polymerases (pfu clone and GC-rich system) and resulted in a DNA band of 504 base pairs (bp) corresponding to the calculated size of the IFN-α8 coding sequence; the pfu clone failed to amplify the gene in the correct size without unspecific bands. The full gene was cloned into the pBAD-TOPO expression vector. After cloning, the gene was reoriented by NcoI restriction digestion and religation. The ligated pBAD-TOPO-IFN-α8 (pBAD-IFNα8) plasmid carried the IFN-α8 gene under transcriptional control of the L-arabinose-inducible P(BAD) promoter. IFN-α8 expression was optimized with respect to L-arabinose concentration, temperature, and time of induction in shake flask cultures to maximize the yield of soluble IFN-α8. The produced IFN-α8 was characterized by polyacrylamide gel electrophoresis and immunoassays. After purification on DEAE-Sepharose, the yield was 100 mg/liter. The antiviral and anticancer activities of the IFN-α8 were evaluated in comparison with IFN-α2a, and the results are discussed.

Mapping of a major surface-exposed site in herpes simplex virus protein Vmw65 to a region of direct interaction in a transcription complex assembly.

The cellular factor Oct-1 is selectively recruited, together with at least one other cellular protein (CFF), into a multicomponent transcription complex whose assembly is directed by the herpes simplex virus regulatory protein Vmw65 (VP16). The acidic carboxy terminus of Vmw65 is not involved in assembly of the complex but is absolutely required for subsequent transcriptional activation. Elucidation of the mechanism of action of Vmw65 is important for an understanding not only of combinatorial control of gene expression by POU- and homeodomain proteins but also of the interaction(s) between activation domains of regulatory proteins and components of the basal transcriptional apparatus. We used a combination of limited proteolysis with a number of site-specific proteases and immunological detection to demonstrate the presence of two main surface-exposed regions in Vmw65. We mapped these sites to within a few amino acids at positions 365-370 408/409. The site at 408/409 is indicative of a flexible exposed linker region between the acidic carboxy-terminal activation domain (residues 430-480) and an N-terminal domain involved in complex formation with the two cellular factors. The site around residues 365-370 is precisely within a region that results from this and other laboratories have shown to be critical for complex formation. Furthermore, we show that this site is selectively protected from proteolysis after complex assembly. Finally, using a series of overlapping peptide encompassing this region, we show that the eight amino acids, R-E-H-A-Y-S-R-A, from positions 360 through 367 are sufficient to inhibit complex formation by intact Vmw65. We propose that these residues contain sufficient information to selectively bind one of the cellular partners involved in complex assembly and that these residues are located in a physical surface-exposed domain of the protein.