Self-Activating Factor X Derivative Fused to the C-Terminus of a Cellulose-Binding Module: Production and Properties Emily Kwan, 2 M. Marta Guarna, 1* Alisdair B. Boraston, 1,2 Neil R. Gilkes, 2 Charles A. Haynes, 1,3 Douglas G. Kilburn, 1,2 R. Antony J. Warren 2 1 Biotechnology Laboratory, 2 Department of Microbiology and Immunology, and 3 Department of Chemical Engineering, University of British Columbia, Vancouver BC, Canada; telephone: 604-822-3308; fax: 604-822-6041; e-mail: rajw@interchange.ubc.ca Received 29 August 2001; accepted 21 February 2002 DOI: 10.1002/bit.10312 Abstract:Inthiswork,anewderivativeofFXwasengi- neered. It comprises a cellulose-binding module (CBM) fused to the N-terminus of the truncated light chain (E2FX)ofFXandahexahistidinetag(H6)fusedtotheC- terminus of the heavy chain. The sequence LTR at the site of cleavage of the activation peptide from the N- terminusoftheheavychainischangedtoIEGRtorender thederivativeself-activating.However,N-linkedglycans ontheCBMofthederivativeblockeditsbindingtocel- lulose and those on the activation peptide slowed its activation.Therefore,thesitesofN-linkedglycosylation on the CBM and on the activation peptide were elimi- natedbymutation.The®nalderivativecanbeproduced ingoodyieldbyculturedmammaliancells.Itispuri®ed easily with Ni 2+ -agarose, it is self-activating, and it can be immobilized on cellulose. When immobilized on a column of cellulose beads, the activated derivative re- tains 80% of its initial activity after 30 days of contin- uous hydrolysis of a fusion protein substrate. Under these conditions of operation, the effective sub- strate:enzyme ratio is >10 4 . ã 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 79: 724±732,2002. Keywords: factor X; self-activating; immobilizable; cel- lulose-binding INTRODUCTION DerivativesoffactorX(FX)wereengineeredpreviously (Assouline et al., 1993, 1995; Guarna et al., 2000) in which a cellulose-binding module (CBM) or a hexahis- tidinetag(H6)wasfusedtotheC-terminusoftheheavy chain. The activated derivatives could be used either immobilized on the appropriate anity matrix or in solution and then removed from the products of its action with the anity matrix. The derivatives were intended to facilitate the release and recovery of target proteins from fusion proteins, but they had several dis- advantages.Snakevenomwasrequiredtoactivatethem to the factor Xa (FXa) forms, and they tended to lose the anity tags by self-digestion. FXa is used to remove anity tags from the N-ter- minioffusionproteins,releasingthenativeformofthe target protein (Nagai and Thùgersen, 1987). However, the FXa must then be removed from the product, a process that generally requires additional, often costly, chromatographic steps. Furthermore, the cost of pro- ducingandactivatingFXprohibitstheuseofFXaona large scale. In an attempt to circumvent some of these problems, a CBM or a H6 tag was fused to the C-ter- minusoftheheavychainofFXtofacilitatepuri®cation and to allow the immobilization on cellulose for ex- tended use. The fusion proteins were produced in mammalian cell cultures (Assouline et al., 1993, 1995; Guarna et al., 1996, 2000). The production of the H6- tagged protein was further enhanced by fusing the transferrinleaderpeptide(T¯p)totheN-terminusofthe light chain from which the Gla domain and the ®rst EGF-likedomainweredeleted(E2FXH6;Guarnaetal., 2000;seeFig.1).InclusionoftheT¯pledtosecretionof the fusion protein into the culture supernatant. All of these fusion proteins, however, were activated moreslowlythannativeFXbytheproteaseinRussell's viper venom (RVV). Moreover, the activated form of the CBM fusion was too unstable for prolonged use in the immobilized state because self-digestion near the C-terminus of the heavy chain (Pryzdial and Kessler, 1996) removed the CBM without destroying FXa activity. This article describes the construction and properties of a new derivative of FX (Fig. 1) that overcomes some of these disadvantages. It is self-activating and stable during continuous use for 30 days while immobilized. Correspondence to: R. A. J. Warren, *Present address: Micrologix Biotech Inc., Vancouver BC, Canada Contractgrantsponsors:ProteinEngineeringNetworkofCentresof Excellence; CBD Technologies, Inc. ã 2002WileyPeriodicals,Inc.