Protein Expression and PuriWcation 47 (2006) 422–426 www.elsevier.com/locate/yprep 1046-5928/$ - see front matter  2006 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2006.01.003 Enzymatic cleavage of fusion proteins using immobilised protease 3C M. Hedhammar a , H.R. Jung b , S. Hober a,¤ a Royal Institute of Technology, AlbaNova University Center, Department of Biotechnology, SE-106 91 Stockholm, Sweden b University of Southern Denmark, Department for Biochemistry and Molecular Biology, Odense M 5230, Denmark Received 29 September 2005, and in revised form 30 December 2005 Available online 26 January 2006 Abstract A strategy for eYcient cleavage of fusion proteins using an immobilised protease has been developed. Protease 3C from coxsackie virus was recombinantly produced in Escherichia coli and covalently immobilised onto a solid support. Thereafter, Z basic tagged fusion proteins, with a speciWc cleavage sequence between the domains, were Xown through the proteolytic column and circulated until complete cleavage. Subsequently, the processed protein solution was applied on a cation exchanger. Thereby, removal of the released, positively charged fusion tag, Z basic , was done by adsorption to the matrix while the target proteins were recovered in the Xow through. Interestingly, the columns were shown to be reusable without any measurable decrease in activity. Moreover, after storage in 4 °C for two months the activity was almost unaVected.  2006 Elsevier Inc. All rights reserved. Keywords: Immobilised protease; Z basic ; Protease 3C; Cleavage The use of puri Wcation tags has become a major break- through for the puriWcation of recombinant proteins. Many diverse proteins can now be puri Wed in a high throughput fashion using general schemes [1]. However, the intended use of the protein may sometimes require the removal of the puri Wcation tag as it can inXuence the expected properties of the target protein, interfere with the three-dimensional structure and mediate unwanted immunogenic responses. Tag removal can either be performed by chemical or enzymatic methods [2]. Chemical hydrolysis requires rather harsh conditions that often denatures the protein and can cause chemical modiWcations on the side chains [3]. Addi- tionally, chemical cleavage sites are only speciWed by one or two residues and are therefore often found within the target protein, which limits chemical methods to the release of peptides or smaller proteins. Even though considerably more expensive, enzymatic proteolysis is usually preferred since it can be performed under physiological conditions and generally gives a more selective cleavage. A large repertoire of proteases with diVerent speciWcity and varying length of the recognition sequence is now available, e.g., Tobacco Etch Virus (TEV) 1 protease [4], Thrombin [5], Factor X a [6], Urokinase [7], and Protease 3C [8]. Even though recombinant production in bacteria and selective recovery through the use of puriWcation tags has improved the availability, the costs of the proteases are still considerable. The subsequent removal of the protease and the released tag is another important issue. However, this can also be facilitated by having a puriWcation tag fused to the protease, preferable the same as the one that is to be released from the target protein [9]. Another elegant strat- egy is to immobilise the protease on a solid support [10–12] and thereby also be able to reuse the same protease several times to reduce the production cost. The coxsackie virus protease 3C is a site speciWc protease that recognises the octapeptide LEALFQ/GP and cleaves between the Gln and Gly residues. Previously, protease 3C has been produced as a fusion to the positively charged puriWcation tag, Z basic [13] and thereafter been successfully used for cleavage of a fusion protein [9]. * Corresponding author. Fax: +46 8 5537 8481. E-mail address: Sophia.Hober@biotech.kth.se (S. Hober). 1 Abbreviations used: TEV, tobacco etch virus; TSB, tryptic soy broth.