Production and isotope labeling of antimicrobial peptides in Escherichia coli by means of a novel fusion partner that enables high-yield insoluble expression and fast purification.
ABSTRACT 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.
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ABSTRACT: Peptides confer interesting properties to materials, supramolecular assemblies and to lipid membranes and are used in analytical devices or within delivery vehicles. Their relative ease of production combined with a high degree of versatility make them attractive candidates to design new such products. Here, we review and demonstrate how CD- and solid-state NMR spectroscopic approaches can be used to follow the reconstitution of peptides into membranes and to describe some of their fundamental characteristics. Whereas CD spectroscopy is used to monitor secondary structure in different solvent systems and thereby aggregation properties of the highly hydrophobic domain of p24, a protein involved in vesicle trafficking, solid-state NMR spectroscopy was used to deduce structural information and the membrane topology of a variety of peptide sequences found in nature or designed. (15) N chemical shift solid-state NMR spectroscopy indicates that the hydrophobic domain of p24 as well as a designed sequence of 19 hydrophobic amino acid residues adopt transmembrane alignments in phosphatidylcholine membranes. In contrast, the amphipathic antimicrobial peptide magainin 2 and the designed sequence LK15 align parallel to the bilayer surface. Additional angular information is obtained from deuterium solid-state NMR spectra of peptide sites labelled with (2) H3 -alanine, whereas (31) P and (2) H solid-state NMR spectra of the lipids furnish valuable information on the macroscopic order and phase properties of the lipid matrix. Using these approaches, peptides and reconstitution protocols can be elaborated in a rational manner, and the analysis of a great number of peptide sequences is reviewed. Finally, a number of polypeptides with membrane topologies that are sensitive to a variety of environmental conditions such as pH, lipid composition and peptide-to-lipid ratio will be presented. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.Journal of Peptide Science 06/2014; · 2.07 Impact Factor
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ABSTRACT: Antimicrobial agents have eradicated many infectious diseases and significantly improved our living environment. However, abuse of antimicrobial agents has accelerated the emergence of multidrug-resistant microorganisms, and there is an urgent need for novel antibiotics. Antimicrobial peptides (AMPs) have attracted attention as a novel class of antimicrobial agents because AMPs efficiently kill a wide range of species, including bacteria, fungi, and viruses, via a novel mechanism of action. In addition, they are effective against pathogens that are resistant to almost all conventional antibiotics. AMPs have promising properties; they directly disrupt the functions of cellular membranes and nucleic acids, and the rate of appearance of AMP-resistant strains is very low. However, as pharmaceuticals, AMPs exhibit unfavorable properties, such as instability, hemolytic activity, high cost of production, salt sensitivity, and a broad spectrum of activity. Therefore, it is vital to improve these properties to develop novel AMP treatments. Here, we have reviewed the basic biochemical properties of AMPs and the recent strategies used to modulate these properties of AMPs to enhance their safety.Pharmaceuticals 01/2013; 6(8):1055-1081.
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ABSTRACT: (1)H-(15)N HSQC spectroscopy is a workhorse of protein NMR. However, under physiological conditions the quality of HSQC spectra tends to deteriorate due to fast solvent exchange. For globular proteins only a limited number of surface residues are affected, but in the case of intrinsically disordered proteins (IDPs) HSQC spectra are thoroughly degraded, suffering from both peak broadening and loss of intensity. To alleviate this problem, we make use of the following two concepts. (1) Proton-decoupled HSQC. Regular HSQC and its many variants record the evolution of multi-spin modes, 2NxHz or 2NxHx, in indirect dimension. Under the effect of fast solvent exchange these modes undergo rapid decay, which results in severe line-broadening. In contrast, proton-decoupled HSQC relies on Nx coherence which is essentially insensitive to the effects of solvent exchange. Moreover, for measurements involving IDPs at or near physiological temperature, Nx mode offers excellent relaxation properties, leading to very sharp resonances. (2) Cross-polarization (1)H-to-(15)N transfer. If CP element is designed such as to lock both (1)H(N) and water magnetization, the following transfer is effected: [Formula: see text] Thus water magnetization is successfully exploited to boost the amount of signal. In addition, CP element suffers less loss from solvent exchange, conformational exchange, and dipolar relaxation compared to the more popular INEPT element. Combining these two concepts, we have implemented the experiment termed CP-HISQC (cross-polarization assisted heteronuclear in-phase single-quantum correlation). The pulse sequence has been designed such as to preserve water magnetization and therefore can be executed with reasonably short recycling delays. In the presence of fast solvent exchange, kex ~ 100 s(-1), CP-HISQC offers much better spectral resolution than conventional HSQC-type experiments. At the same time it offers up to twofold gain in sensitivity compared to plain proton-decoupled HSQC. The new sequence has been tested on the sample of drkN SH3 domain at pH 7.5, 30 °C. High-quality spectrum has been recorded in less than 1 h, containing resonances from both folded and unfolded species. High-quality spectra have also been obtained for arginine side-chain H(ε)N(ε) groups in the sample of short peptide Sos. For Arg side chains, we have additionally implemented (HE)NE(CD)HD experiment. Using (13)C-labeled sample of Sos, we have demonstrated that proton-to-nitrogen CP transfer remains highly efficient in the presence of solvent exchange as fast as kex = 620 s(-1). In contrast, INEPT transfer completely fails in this regime.Journal of Biomolecular NMR 02/2014; · 2.85 Impact Factor