Adaptation of the Halobacterium salinarum ssp. NRC-1 gene deletion system for modification of chromosomal loci

• We utilized a gene deletion method to introduce a poly-histidine tag.
• This method allows protein levels to be measured by Western blot.
• Rfa3 protein levels in Halobacterium salinarum increase upon UV irradiation.

The model archaeon Halobacterium salinarum ssp. NRC-1 is an excellent system for the study of archaeal molecular biology. Unlike many other archaea, its only special growth requirement is high levels of sodium chloride and other salts; it requires neither high-temperature incubation nor anaerobic environments. Additionally, there are a number of well-developed post-genomic tools available, including whole-genome microarrays and a ura3-based gene deletion system. While some tools are available for protein expression, a system for measurement and purification of protein expressed from native promoters is lacking. We have adapted the established H. salinarum gene deletion system for this purpose, and have used this to place 8 ×-histidine tags on either the carboxyl or amino terminus of the protein encoded by the chromosomal rfa3 gene. To demonstrate the utility of this approach, we used Western blot analysis to determine levels of the Rfa3 protein under different conditions. This system provides another powerful molecular tool for studies of native protein expression and for simple protein purification in H. salinarum.