Journal of Biotechnology 125 (2006) 48–56 Intein-mediated protein purification of fusion proteins expressed under high-cell density conditions in E. coli Shamik S. Sharma a,b , Shaorong Chong b , Sarah W. Harcum c,∗ a Department of Chemical Engineering, 127 Earle Hall, Clemson University, Clemson, SC 29634, United States b New England Biolabs Inc., 240 County Road, Ipswich, MA 01938, United States c Department of Bioengineering, 112 Biosystems Research Complex, Clemson University, Clemson, SC 29634, United States Received 8 September 2005; received in revised form 10 January 2006; accepted 19 January 2006 Abstract The intein-mediated purification system has the potential to significantly reduce the recovery costs of industrial recombinant proteins. The ability of inteins to catalyze a controllable peptide bond cleavage reaction can be used to separate a recombinant protein from its affinity tag during affinity purification. Inteins have been combined with a chitin-binding domain to serve as a self-cleaving affinity tag, facilitating highly selective capture of the fusion protein on an inexpensive substrate—chitin (IMPACT ® system, New England Biolabs, Beverly, MA). This purification system has been used successfully at a lab scale in low cell density cultures, but has not been examined comprehensively under high-cell density conditions in defined medium. In this study, the intein-mediated purification of three commercially relevant proteins expressed under high-cell density conditions in E. coli was studied. Additionally, losses during the purification process were quantified. The data indicate that the intein fusion proteins expressed under high cell density fermentations were stable in vivo after induction for a significant duration, and the intein fusion proteins could undergo thiol or pH and temperature initiated cleavage reaction in vitro. Thus, the intein-mediated protein purification system potentially could be employed for the production of recombinant proteins at the industrial-scale. © 2006 Elsevier B.V. All rights reserved. Keywords: Intein; Chitin; Fusion protein; Antitrypsin; Cre recombinase; Human bFGF; Recombinant 1. Introduction Protein splicing is a post-translational process, in which an internal segment of the protein, named ∗ Corresponding author. Tel.: +1 864 656 6865; fax: +1 864 656 0567. E-mail address: harcum@clemson.edu (S.W. Harcum). “intein”, catalyzes its excision from a protein pre- cursor, and ligation of the flanking regions, named “exteins”, which results in the production of two pro- teins (Derbyshire et al., 1997; Kane et al., 1990; Paulus, 2000; Perler et al., 1994). Since the initial discovery of protein splicing, it has been extensively studied and exploited for the purification of recombinant proteins (Chong et al., 1997; Derbyshire and Belfort, 1998; Guo 0168-1656/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2006.01.018