We have recently developed the HB tag as a useful tool for tandem-affinity purification under native as well as fully denaturing conditions. The HB tag and its derivatives consist of a hexahistidine tag and a bacterially-derived in vivo biotinylation signal peptide, which support sequential purification by Ni2+ -chelate chromatography and binding to immobilized streptavidin. To facilitate taggi...

Pseudomonas aeruginosa PAO1 grows on a variety of polyamines as the sole source of carbon and nitrogen. Catabolism of polyamines is mediated by the γ-glutamylation pathway, which is complicated by the existence of multiple homologous enzymes with redundant specificities toward different polyamines for a more diverse metabolic capacity in this organism. Through a series of markerless gene knocko...

The polyubiquitin receptor Rpn10 targets ubiquitylated Sic1 to the 26S proteasome for degradation. In contrast, turnover of at least one ubiquitin-proteasome system (UPS) substrate, CPY*, is impervious to deletion of RPN10. To distinguish whether RPN10 is involved in the turnover of only a small set of cell cycle regulators that includes Sic1 or plays a more general role in the UPS, we sought t...

Escherichia coli group 1 K antigens form a tightly associated capsule structure on the cell surface. Although the general features of the early steps in capsular polysaccharide biosynthesis have been described, little is known about the later stages that culminate in assembly of a capsular structure on the cell surface. Group 1 capsule biosynthesis gene clusters (cps) in E. coli and Klebsiella ...

Aeromonas hydrophila secretes a number of degradative enzymes and toxins into the external milieu via the type II secretory pathway or secreton. ExeA is an essential component of this system and is necessary for the localization and/or multimerization of the secretin ExeD. ExeA contains two sequence motifs characteristic of the Walker superfamily of ATPases. Previous examination of substitution...

Cell-penetrating peptides and proteins (CPPs) are important tools for the delivery of impermeable molecules into living mammalian cells. To enable these cells to internalize proteins fused to common oligohistidine affinity tags, we synthesized an artificial cell surface receptor comprising an N-alkyl derivative of 3beta-cholesterylamine linked to the metal chelator nitrilotriacetic acid (NTA). ...

EmrE is a member of the small multidrug resistance (SMR) protein family in Escherichia coli. EmrE confers resistance to a wide variety of quaternary cation compounds (QCCs) as an efflux transporter driven by proton motive force. The purification yield of most membrane proteins are challenging because of difficulties in over expressing, isolating and solubilizing them and the addition of an affi...