Expression of proteins using the third domain of the Escherichia coli periplasmic-protein TolA as a fusion partner Gregor Anderluh, a,c Isa G€ okc ße, b,c and Jeremy H. Lakey c, * a Department of Biology, University of Ljubljana, Ve cna pot 111, Ljubljana 1000, Slovenia b Department of Chemistry, Gaziosmanpas ßa University, Tokat, Turkey c School of Cellular and Molecular Biosciences, University of Newcastle upon Tyne, Framlington Place, Newcastle-upon-Tyne NE2 4HH, UK Received 13 September 2002, and in revised form 4 November 2002 Abstract The third domain of the periplasmic protein TolA from Escherichia coli (TolAIII) was used as a fusion partner in the expression of various proteins from bacteria and eukaryotes. TolAIII is small domain, expressed in high yields as a soluble protein in the cytoplasm of E.coli. Proteins were linked to the C-terminus of TolAIII by a short flexible linker containing sites for endopeptidases. Threedifferentvectorswereprepared,containingsitesforenterokinase,thrombinorfactorXa.FusionproteinsalsocontainaHis 6 – Ser 2 tag at their N-terminus for easier purification. Up to 90mg fusion protein per liter bacterial culture was obtained using these vectors. Colicin N R-domain was expressed with this system as a fusion and processed further for functional studies. The yield of final pure R-domain was doubled as compared to the direct expression. The system may prove to be useful in the preparation of other peptides and proteins. Ó 2002 Elsevier Science (USA). All rights reserved. Pure proteins are required when structural and functional questions are addressed but the isolation of pure proteins and their mutant forms in sufficient quantities is sometimes problematic. Various expression systems are used for heterologous production of pro- teins to accomplish these goals. Escherichia coli is still the most common host, despite huge advances in the area of protein expression in the last 10 years, because expressing proteins in E. coli is simple, there is vast ex- perience of it, and finally and sometimes most impor- tantly, because of the low costs associated with production. Proteins can be expressed in E. coli either directly or as fusions [1,2]. The purpose of fusion partners is to provide affinity tags (His n tag, glutathione-S-transfer- ase, cellulose binding domain) [3–6], to make proteins more soluble (thioredoxin) [7], to enable the formation of disulfide bonds (thioredoxin) [8,9], or to export fused proteins to the periplasm where conditions for forma- tion of disulfide bonds are better [10]. The majority of proteins used as fusion partners are small and their ex- pression in E. coli should be substantial. Any novel proteins used as fusion partners should have such characteristics. TolA is a periplasmic protein that is involved in maintaining the integrity of the inner membrane and uptake of colicins and bacteriophage [11–13]. It is composed of three domains. The short N-terminal do- main is composed of a single transmembrane helix, which anchors TolA in the inner membrane. The second and largest domain is polar and predicted to be mainly a-helical. The C-terminal domain III (TolAIII) is small and composed of 92 amino acids. Its 3D structure was recently solved in the complex with the N1 domain of minor coat gene 3 protein of Ff filamentous bacterio- phage [14]. It is folded into a slightly elongated domain with the aid of one disulfide bond (Fig. 1). Here, we report the cloning of pTol vectors, which use TolAIII as a fusion partner at the N-terminal part of the expressed fusion protein. We show that the levels of expression of variousfusionproteinsarearound20%oftotalbacterial proteinsandwewereabletopurify50–90mgfusionsper liter bacterial culture. Protein Expression and Purification 28 (2003) 173–181 www.elsevier.com/locate/yprep * Corresponding author. Fax: +44-191-222-7424. E-mail address: j.h.lakey@ncl.ac.uk (J.H. Lakey). 1046-5928/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. doi:10.1016/S1046-5928(02)00681-2