TABLE 3.

Summary of expression studies

ProteinActivityConstructPromoterTagaDetectionComments
Beta-galactosidase (SSO3019)Cleaves beta-galactosidase analogspMJ05/pSVA5tf55, araSNative stainReporter, cytoplasmic
FlaI (SSO2316) (putative ATPase [secretion] with nucleotide binding domain)pSVA6tf55Six-HisImmunodetectionIn membrane fraction (versus cytoplasmic in E. coli)
ATPase (SSO2680) (type II or IV secretion)ATPase (Mn > Mg)pSVA15araS10-HisImmunodetection, ATPase assayIn membrane fraction (inclusion bodies in E. coli), 5× higher specific activity when produced in Sulfolobus
ABCE1 protein (SSO0287) (two nucleotide binding domains and two Fe-S clusters)ATPasepSVA31araSStrep plus eight-HisImmunodetection (anti-Strep, anti-His) and 280/410 nmIntact Fe-S clusters (410-nm adsorption, not in E. coli), upscaling to 45-liter fermenter
Sor (sulfur oxygenase/reductase from A. ambivalens)Sulfur oxidation/reductionpASK-SOR.05tf55StrepSor activitySpecific activity as high as protein from native host
  • a All tags were positioned at the C terminus.