278 Research Article Received: 8 September 2008 Revised: 21 November 2008 Accepted: 28 November 2008 Published online in Wiley Interscience: 2 February 2009 (www.interscience.com) DOI 10.1002/psc.1112 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 Verica Vidovic, Lydia Prongidi-Fix, Burkhard Bechinger and Sebastiaan Werten * 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. Copyright c  2009 European Peptide Society and John Wiley & Sons, Ltd. Keywords: antimicrobial peptide; bacterial expression; NMR; high-throughput Introduction Escherichiacoli(E.coli) is arguably the most convenient microorgan- ism available to date for the large-scale production of recombinant polypeptides in vivo [1]. The bacterium is also ideally suited for uniform labeling strategies to enable structural and biophysical studies, as it can be grown in well-defined synthetic media contain- ing appropriately labeled nutrients. Polypeptides labeled with 15 N, 13 C, or seleno-methionine can be produced very cost-effectively in this manner and as a result have become standard tools in NMR and x-ray crystallography [2]. Unfortunately, expression of foreign genes in E. coli often fails due to vector instability, resulting from toxicity of the molecule of interest towards the host. This problem is particularly common in the case of antimicrobial peptides, natural defense molecules involved in the innate immune system of metazoans, which kill bacteria by permeating and disrupting their membranes [3]. Inves- tigation of the structural properties and dynamics of such peptides by heteronuclear NMR is of considerable biomedical interest, as the emergence of resistance traits in pathological bacteria calls for rational design of new antibiotics [4]. Although native antimi- crobial peptides can be produced synthetically, uniform isotope labeling by chemical means would in most cases be prohibitively expensive. Consequently, many laboratories have sought to establish methods for the production of antimicrobial peptides in E. coli, in spite of their inherent toxicity towards bacteria [5]. The lethal effects of antimicrobial peptides on E. coli can often be circumvented by expressing the molecule of interest as part of a fusion protein. Although this approach allows expression of some peptides in a soluble form (e.g. in conjunction with glutathione S-transferase (GST) [6,7] or thioredoxin [8–11]), most successful cases involve fusion proteins that accumulate entirely in inclusion bodies [12 – 19]. This suggests that protein aggregation provides a powerful means of neutralizing toxicity of antimicrobial peptides towards the host. Antimicrobial peptides may spontaneously form inactive ag- gregates if fused to soluble partners such as ubiquitin [12] or if multimerized [13,14]. Nevertheless, it would be advantageous to develop a robust, generally applicable method that allows target- ing of any toxic peptide of interest to inclusion bodies. To this end, several laboratories have identified highly insoluble proteins that can be used as fusion partners, such as ketosteroid isomerase [15], baculoviral polyhedrin [16,17], RepA [18], and an artificial polypep- tide encoded by portions of the E. coli Trp operon [19]. Although expression levels of the corresponding fusion proteins tend to be satisfactory, all the aforementioned partners have the disadvan- tage that they are relatively large (typically 20 kDa or more). This diminishes the efficiency of the expression system, as the peptide ∗ Correspondence to: Sebastiaan Werten, Institut de Chimie Universit´ e Louis Pasteur CNRS UMR 7177 4 rue Blaise Pascal 67070 Strasbourg France. E-mail: s.werten@chimie.u-strasbg.fr Institut de Chimie, Universit´ e Louis Pasteur, CNRS UMR 7177, 4 rue Blaise Pascal, 67070 Strasbourg, France J. Pept. Sci. 2009; 15: 278–284 Copyright c  2009 European Peptide Society and John Wiley & Sons, Ltd.