A method is presented that allows efficient production of antimicrobial peptides in bacteria by means of fusion to the histone fold domain of the human transcription factor TAF12. This small fusion partner drives high-level expression of peptides and leads to their accumulation in an entirely insoluble form, thereby eliminating toxicity to the host. Using the antimicrobial peptide LAH4 as an example, we demonstrate that neither affinity purification of the TAF12 fusion protein nor initial solubilization of inclusion bodies in denaturing buffers is required. Instead, crude insoluble material from bacteria is directly dissolved in formic acid for immediate release of the peptide through chemical cleavage at a unique Asp-Pro site. This is followed by purification to homogeneity in a single chromatographic step. Because of the elevated expression levels of the histone fold domain and its small size (8 kDa), this straightforward purification scheme produces yields in excess of 10 mg active peptide per liter of culture. We demonstrate that TAF12 fusion allows expression of a wide range of antimicrobial peptides as well as efficient isotope labeling for NMR studies.
"The peptide changes from a transmembrane to an in-plane alignment in a pH-dependent manner  and the spectrum shows the situation at pH 6. The peptide was prepared labeled uniformly with 15 N according to . A comparison of the effects of different peptide topologies on lipid order parameters is shown in . "
[Show abstract][Hide abstract] ABSTRACT: Biological membranes are characterized by a high degree of dynamics. In order to understand the function of membrane proteins and even more of membrane-associated peptides, these motional aspects have to be taken into consideration. Solid-state NMR spectroscopy is a method of choice when characterizing topological equilibria, molecular motions, lateral and rotational diffusion as well as dynamic oligomerization equilibria within fluid phase lipid bilayers. Here we show and review examples where the (15)N chemical shift anisotropy, dipolar interactions and the deuterium quadrupolar splittings have been used to analyze motions of peptides such as peptaibols, antimicrobial sequences, Vpu, phospholamban or other channel domains. In particular, simulations of (15)N and (2)H-solid-state NMR spectra are shown of helical domains in uniaxially oriented membranes when rotation around the membrane normal or the helix long axis occurs.
[Show abstract][Hide abstract] ABSTRACT: The infrared optical transmission and photosensitivity of doped, compensated, narrow-gap semiconductor material Cd<sub>x</sub>Hg<sub>1-x </sub>Te have been studied as functions of temperature under the same experimental conditions in the range from 5 K to 400 K. The strong anomalous changes in the position of the fundamental absorption edge and the maximum and relative photosensitivity of the material in the range 50 K to 65 K are accounted for by collective effects in disordered semiconductors. Potential-contrast scanning electron microscopy has been used to estimate the size of the p-type regions formed in the crystal at liquid nitrogen temperature
[Show abstract][Hide abstract] ABSTRACT: Antimicrobial peptides are an essential component of innate immunity and play an important role in host defence against microbial pathogens. They have received increasing attention recently as potential novel pharmaceutical agents. To meet the requirement for necessary basic science studies and clinical trials, large quantities of these peptides are needed. In general, isolation from natural sources and chemical synthesis are not cost-effective. The relatively low cost and easy scale-up of the recombinant approach renders it the most attractive means for large-scale production of antimicrobial peptides. Among the many systems available for protein expression, Escherichia coli remains the most widely used host. Antimicrobial peptides produced in E. coli are often expressed as fusion proteins, which effectively masks these peptides' potential lethal effect towards the bacterial host and protects the peptides from proteolytic degradation. Although some carriers confer peptide solubility, others promote the formation of inclusion bodies. The present minireview considers the most commonly used carrier proteins for fusion expression of antimicrobial peptides in E. coli. The favourable properties of SUMO (small ubiquitin-related modifier) as a novel fusion partner are also discussed.
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