Expression of N-terminal Cys-protein fragments using an intein refolding strategy Christian P. R. Hackenberger,   Mark M. Chen and Barbara Imperiali * Massachusetts Institute of Technology, Department of Chemistry and Department of Biology, 77 Massachusetts Ave. 18-590, Cambridge, MA 02139, USA Received 31 January 2006; revised 1 March 2006; accepted 2 March 2006 Available online 22 March 2006 Abstract—The inclusion body expression and refolding of a pH-sensitive intein fusion protein (Ssp DnaB intein) delivered sufficient quantities of an N-terminal Cys-polypeptide for native chemical ligations. This strategy circumvents premature intein cleavage under expression conditions and allows the expression and purification of proteins with uncertain solubility properties. The expressed protein resembles the C-terminal portion of the amphiphilic immunity protein Im7, which can be ligated to synthetic thio- esters to yield synthetic protein analogues for protein folding studies. Ó 2006 Elsevier Ltd. All rights reserved. 1. Introduction Expressed protein ligation (EPL) has been established as a powerful tool for accessing quantities of pure proteins for biophysical and biochemical investigations. 1 The strategy employs a combination of chemical (solid-phase peptide synthesis, SPPS) and biochemical (expression) methods to provide protein fragments for connection via native chemical ligation (NCL). 2 NCL is based on a reversible thioester exchange process involving a pro- tein or peptide thioester 1 and an N-terminal (or a-) Cys-protein or -peptide 2, 3 which ultimately leads to irreversible amide bond formation (Scheme 1). EPL has found particular utility for the preparation of semi- synthetic proteins, since it allows introduction of a vari- ety of chemical modifications into the synthetic peptide fragment. Additionally, EPL affords access to mem- brane proteins that show low expression profiles, as shown by the semisynthesis of a potassium ion channel, 4 and it can even be performed in living cells. 5 For access to semisynthetic proteins, both precursors for the NCL step have to be readily available. SPPS meth- ods are well established and afford good quantities of synthetic peptide thioesters 1 or peptides with an N-ter- minal Cys 2 for the NCL step (routes I and III in Scheme 1A). For the expression and purification of analogous protein fragments for NCL, different routes for each type of protein component are available. For the expression of protein thioesters 1 (route II in Scheme 1A) methods have been developed through the intein approach (IMPACT), which utilizes the self- cleaving property of a protein splicing domain or intein, in order to separate a desired polypeptide sequence from an affinity tag (‘C-terminal fusion,’ Scheme 2, left). 6 Pro- tein splicing is then initiated once the fusion protein is loaded onto an affinity column by the addition of thiols to liberate the corresponding thioester in pure form. This protocol has been widely used to access soluble and insoluble protein domains, the latter as recently demonstrated by Muir and co-workers in the semisyn- thesis of a potassium ion channel, in which the C-termi- nal intein fusion protein was expressed as inclusion bodies and refolded before the intein-mediated rear- rangement was initiated. 4 Soluble N-terminal Cys proteins 2 (route IV in Scheme 1A) are commonly expressed with a purification tag and a protease cleavage site for factor Xa 7 or TEV pro- teases 8 at the N-terminus, which allows the liberation of the protein. 1a In order to access hydrophobic a-Cys-pro- teins that are insoluble or aggregate under protease 0968-0896/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmc.2006.03.003 Keywords: Protein semisynthesis; Intein expression; N-terminal Cys- proteins; Native chemical ligation. * Corresponding author. Tel.: +1 617 253 1838; fax: +1 617 452 2419; e-mail: imper@mit.edu   Present address: Freie Universita ¨t Berlin, Department of Chemistry and Biochemistry, Takustr. 3, 14195 Berlin, Germany. Bioorganic & Medicinal Chemistry 14 (2006) 5043–5048