Use of phage display methods to identify heptapeptide sequences for use as affinity purification 'tags' with novel chelating ligands in immobilized metal ion affinity chromatography

This study describes the screening of a peptide phage display library for amino acid sequences that bind with different affinities to a novel class of chelating ligands complexed with Ni2+ ions. These chelating ligands are based on the 1,4,7-triazacyclononane (TACN) structure and have been chosen to allow enhanced efficiency in protein capture and decreased propensity for metal ion leakage in the immobilized metal ion affinity chromatographic (IMAC) purification of recombinant proteins. Utilising high stringency screening conditions, various peptide sequences containing multiple histidine, tryptophan, and/or tyrosine residues were identified amongst the different phage peptide sequences isolated. The structures, and particularly the conserved locations of these key amino acid residues within the selected heptapeptides, form a basis to design specific peptide tags for use with these novel TACN ligands as a new mode of IMAC purification of recombinant proteins.     

Simultaneous diffusion of calcium and strontium in KCl single crystals

Concentration dependent diffusion coefficients for ⁴⁵Ca²⁺ and ⁸⁵Sr²⁺ in purified KCl were measured using a sectioning method. KCl was purified by an ion exchange — Cl₂?HCl process and the crystals grown under $\text{1}\text{6}$ atmosphere of HCl. The tracers were purified on small disposable ion exchange columns to remove precessor and daughter impurities prior to use in a diffusion anneal. Isothermal diffusion anneals were made in the temperature range from 451% to 669%C. At temperatures above 580%C (the lowest melting eutectic in this system) diffusion was from a vapor source: below 580%C surface depositied sources were used. The saturation diffusion coefficients. enthalpies and entropies of impurity-vacancy associations were calculated using the common ion model for simultaneous diffusion of divalent ions in alkali halides. In KCl the saturation diffusion coefficients D_S(ca) and D_s(Sr) are given by

$\text{D}{}_{\mathrm{s}}\text{(Ca) = 9.93 × 10}{}^{\mathrm{?5}}\text{exp(?0.592}\text{eV}\text{kT}\text{)}\text{cm}{}^2\text{sec}$

(1) and

$\text{D}{}_{\mathrm{s}}\text{(Sr) = 1.20 × 10}{}^{\mathrm{?3}}\text{exp(?0.871}\text{eV}\text{kT}\text{)}\text{cm}{}^2\text{sec}$

(2) for calcium and strontium, respectively. The Gibbs free energy of association of the impurity vacancy complex in KCl for calcium can be represented by

$\text{Δg(Ca) = ?-0.507 eV + (2.25 × 10}{}^{\mathrm{?4}}\text{eV}\text{\%K}\text{)T}$

(3) and that for strontium by

$\text{Δg(Sr) = ?0.575 eV + (2.90 × 10}{}^{\mathrm{?4}}\text{eV}\text{\%K}\text{)T}$

. (4)     

Structure and thermal stability of aliphatic polyoxamides

A homologous series of aliphatic polyoxamides, nylon 122, 102, 82, and 62, were prepared by the bulk polycondensation of dialkyl oxalates with diamines. Infrared spectroscopy, x-ray diffraction, differential thermal analysis, thermogravimetric analysis, and gas chromatography were used to study the structure and thermal stability of these polyoxamides. From the fiber pattern of nylon 122, a structural repeat distance of 19.5 Å was measured. This was in close agreement with the expected value for an extended planar, zigzag chain conformation. With the exception of the expected decrease in the structural repeat distance along the chain axis resulting from the different number of methylene groups, nylons 122, 102, and 82 were isostructural. Nylon 62 appeared to pack in a similar manner to the other materials studied. However, there were differences in the diffraction pattern of this polymer; these may be indicative of a different chain conformation or of strain in the crystalline regions of the polymer. The infrared spectra indicate the presence of strong hydrogen bonding in all the polymers. Thermal analysis and pyrolysis data revealed catastrophic polymer degradation between 400 and 500°C in nitrogen, with appreciable homolytic cleavage of the oxamide group.     

Crystallinity in poly-N-methyllaurolactam and copolymers

Fully methylated nylon 12 was prepared by the hydrolytic polymerization of N-methyllaurolactam. The polymer, with about 20% crystallinity, has a crystal structure different from that of nylon 12. The X-ray diffraction patterns of copolymers of N-methyllaurolactam and laurolactam were shown to be composition-dependent. Copolymers containing up to 15% laurolactam units exhibited a crystal structure essentially the same as poly-N-methyllaurolactam. Copolymers containing from 20 to 50% laurolactam are transition structures, whereas copolymers containing more than 50% laurolactam units exhibit a crystal structure characteristic of polylaurolactam. DSC data on the homopolymers and copolymers are also reported.     

Combined time- and space-resolved Raman spectrometer for the non-invasive depth profiling of chemical hazards

A time-resolved inverse spatially offset Raman spectrometer was constructed for depth profiling of Raman-active substances under both the lab and the field environments. The system operating principles and performance are discussed along with its advantages relative to traditional continuous wave spatially offset Raman spectrometer. The developed spectrometer uses a combination of space- and time-resolved detection in order to obtain high-quality Raman spectra from substances hidden behind coloured opaque surface layers, such as plastic and garments, with a single measurement. The time-gated spatially offset Raman spectrometer was successfully used to detect concealed explosives and drug precursors under incandescent and fluorescent background light as well as under daylight. The average screening time was 50 s per measurement. The excitation energy requirements were relatively low (20 mW) which makes the probe safe for screening hazardous substances. The unit has been designed with nanosecond laser excitation and gated detection, making it of lower cost and complexity than previous picosecond-based systems, to provide a functional platform for in-line or in-field sensing of chemical substances.