We report oriented immobilization of proteins using the standard hexahistidine (His6)-Ni2+:NTA (nitrilotriacetic acid) methodology, which we systematically tuned to give control of surface coverage. Fluorescence microscopy and surface plasmon resonance measurements of self-assembled monolayers (SAMs) of red fluorescent proteins (TagRFP) showed that binding strength increased by 1 order of magni...

The fungal pathogen Colletotrichum lindemuthianum secretes an endo-chitin de-N-acetylase (ClCDA) to modify exposed hyphal chitin during penetration and infection of plants. Although a significant amount of biochemical data is available on fungal chitin de-N-acetylases, no structural data exist. Here we describe the 1.8 A crystal structure of a ClCDA product complex and the analysis of the react...

The development of synthetic, low-molecular-weight ligand receptor systems for the selective control of biomolecular interactions remains a major challenge. Binding of oligohistidine peptides to chelators containing Ni2+-loaded nitrilotriacetic acid (NTA) moieties is one of the most widely used and best-characterised recognition systems. Recognition units containing multiple NTA moieties (multi...

Insolubility of recombinant proteins in Escherichia coli is a major impediment to their production for structural and functional studies. One way to circumvent this problem is to fuse an aggregation-prone protein to a highly soluble partner. E. coli maltose-binding protein (MBP) has emerged as one of the most effective solubilizing agents. In this chapter, we describe how to construct combinato...

To understand better the mechanisms of resistance-nodulation-division (RND)-type multidrug efflux pumps, we examined the Escherichia coli AcrD pump, whose typical substrates, aminoglycosides, are not expected to diffuse spontaneously across the lipid bilayer. The hexahistidine-tagged AcrD protein was purified and reconstituted into unilamellar proteoliposomes. Its activity was measured by the p...

Carbonic anhydrases catalyze the interconversion of carbon dioxide to bicarbonate. Human carbonic anhydrase isozyme III with a C-terminal hexahistidine tag was overexpressed in Eschericha coli, purified and crystallized. Diffraction data (93.4% completeness) were collected to 2.2 A resolution on an in-house R-AXIS IV++ image-plate system with Osmic mirrors and a Rigaku HU-H3R CU rotating-anode ...

Fusion-tag affinity chromatography is a key technique in recombinant protein purification. Current methods for protein recovery from mammalian cells are hampered by the need for feed stream clarification. We have developed a method for direct capture using immobilized metal affinity chromatography (IMAC) of hexahistidine (His6) tagged proteins from unclarified mammalian cell feed streams. The p...

Since immobilized metal ion affinity chromatography (IMAC) was first reported, several modifications have been developed. Among them, Ni(2+) immobilized by chelation with nitrilotriacetic acid (NTA) bound to a solid support has become the most common method for the purification of proteins carrying either a C- or N-terminal histidine (His) tag. Despite its broad application in protein purificat...

In the histories of logic and of computer science which mention precursors and pioneers, we often find mention of Leibniz, the great l7th-century mathematician and philosopher, who was one of the first to try to build a mechanical calculator, and who tried to formulate a Mathesis universalis, a sort of scientific language which would permit any two disputants to settle their differences merely ...

Hemingway has been unquestionably a great master of storytelling with global clout. Through the analysis, he skillfully uses accomplished techniques in "Farewell to Arms". This paper looks at narrative and characteristics his work, which reflects Hemingway's exceptional writing talent. A close examination use strategies sheds light on fictional story provides new appreciation for style that mak...