PubMed · 3237088
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M Skinner, A S Cohen. 1988. Amyloid P component.. https://doi.org/10.1016/0076-6879(88)63048-5
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It has been established that many heterologously produced proteins in E. coli accumulate as insoluble inclusion bodies. Methods for protein recovery from inclusion bodies involve solubilization using chemical denaturants such as urea and guanidine hydrochloride, followed by removal of denaturant from the solution to allow the protein to refold. In this work, we applied on-column refolding and purification to the second crystallin domain D2 of Yersinia crystallin isolated from inclusion bodies. We also purified the protein from the soluble fraction (without using any denaturant) to compare the biophysical properties and conformation, although the yield was poor. On-column refolding method allows rapid removal of denaturant and refolding at high protein concentration, which is a limitation in traditionally used methods of dialysis or dilution. We were also able to develop methods to remove the co-eluting nucleic acids during chromatography from the protein preparation. Using this protocol, we were able to rapidly refold and purify the crystallin domain using a two-step process with high yield. We used biophysical techniques to compare the conformation and calcium-binding properties of the protein isolated from the soluble fraction and inclusion bodies.
The milfoil weevil Euhrychiopsis lecontei is a specialist aquatic herbivore that feeds, oviposits, and mates on the invasive freshwater macrophyte Myriophyllum spicatum. We characterized the weevil's preference for M. spicatum, and through bioassay-driven fractionation, isolated and identified two chemicals released by M. spicatum that attract E. lecontei. Mass spectrometry and nuclear magnetic resonance spectroscopy were used to identify the attractive compounds as glycerol and uracil. Dose-response curves for glycerol and uracil indicated that weevil preference increased as sample concentration increased. Weevils were attracted to a crude sample of M. spicatum-released chemicals from 0.17 to 17 mg/l, to glycerol from 18 to 1800 microM (0.0017-0.17 mg/l), and to uracil from 0.015 to 15 microM (0.00014-1.4 mg/l). Although glycerol and uracil are ubiquitous, weevils are likely responding to high concentrations that are released as a result of the rapid growth of M. spicatum. Uracil concentration was greater in the exudates of M. spicatum than other Myriophyllum spp. E. lecontei was attracted to glycerol at a concentration similar to that at which terrestrial insects are attracted to sugar alcohols. This is the first example of a freshwater specialist insect being attracted to chemicals released by its host plant. Analysis of the water milfoil-weevil interaction provides further understanding as to how insects locate their host plants in aquatic systems.
The recently determined structure of plant photosystem I (PSI) provides the first relatively high-resolution structural model of a supercomplex containing a reaction center and its peripheral antenna. Large amounts of highly purified PSI were required to get enough crystals amenable for structural determination by X-ray crystallography. In addition, a deep biochemical understanding of the large supercomplex was vital for achieving the goal. The stability of PSI was analyzed by sucrose gradient centrifugation and gel electrophoresis. Small amounts of LHCI were detached from PSI following a 12 day incubation under crystallization conditions. The interaction between the reaction center and the peripheral antenna of PSI (LHCI) as well as the interactions among the LHCI monomers are flexible. Nevertheless, the pure and homogeneous preparation of PSI allows for relatively tight crystal packing, which holds promise for obtaining atomic resolution in the future.